1 Key Laboratory of Protein and Peptide Pharmaceutical, CAS Engineering Laboratory for Nanozyme, Institute of Biophysics, Chinese Academy of Sciences,Beijing 100101, China
2 Nanozyme Medical Center, School of Basic Medical Sciences, Zhengzhou University,Zhengzhou 450001, China
3 Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing National Laboratory of Microstructures, Jiangsu Key Laboratory of Artificial Functional Materials, Nanjing University,Nanjing 210023, China
4 Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences,Shenzhen 518055, China
5 School of Basic Medicine Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences,Beijing 100005, China
6 School of Materials Science & Engineering, Beijing Institute of Technology,Beijing 100081, China
7 Key Laboratory of Bioactive Materials for the Ministry of Education, College of Life Sciences, Nankai University,Tianjin 300071, China
8 School of Biological Science and Medical Engineering, Southeast University,Nanjing 210096, China
9 State Key Laboratory of Rare Earth Resource Utilization,Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,Changchun 130022, China
10 University of Science and Technology of China,Hefei 230026, China
11 Laboratory of Theoretical and Computational Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, China
Nanozymes, a new concept first proposed by Chinese scientists, is a class of nanomaterials with biocatalytic functions. Owing to their nanostructures, nanozymes can catalyze the substrates of natural enzymes and serve as enzyme substitutes. Since the first report in 2007, over 420 research groups from 55 countries have validated this phenomenon. The discovery of nanozymes demonstrates for the first time that nanomaterials may have a unique biological effect-enzyme-like catalytic activity. As a new material, nanozyme has both the physicochemical properties of nanomaterials and catalytic function similar to those of natural enzymes, with the benefits of both. Its nanostructure not only endows nanozymes with extremely effective catalytic activity but also renders them more stable and easier to mass production. The study of nanozymes is an example of interdisciplinary cooperation, being named as one of the 2022 top ten chemical emerging technologies by IUPAC.Nanozymes have become an emerging research focus due to the collaboration of experts from diverse fields worldwide such as chemistry, enzymology, materials science, biology, medicine, and theoretical calculations. Chinese scientists lead the way in this emerging field, investigating the structure-effect relationship of nanozymes, increasing their catalytic activity by 10 000 times, realizing rational design even surpassing natural enzymes, and developing the world's first nanozyme products, as well as publishing books on nanozyme science, releasing nanozyme terminology, and establishing Chinese/international standardization. Furthermore, the new field of nanozymes has attracted a substantial number of talented young multidisciplinary and interdisciplinary scientists who are driving its strong growth by discovering more than 1200 types of nanozymes and uncovering their catalytic mechanisms.It has also evolved from the initial application in detection to nanozyme catalysis medicine, sensor detection, green synthesis, new energy, environmental protection, and many other. This article provides readers with an overview of the significant advances in nanozyme research since its discovery, including newly identified natural nanozymes. Ultimately, our goal is to see nanozymes improve human health and inspire the growth of a new field of study as they go from an idea to new materials, to technology and to products.
Biocatalysis is a process of chemical transformation using enzymes as catalysts, so it is also called enzymatic catalysis, which has the characteristics of high catalytic efficiency, strong selectivity and mild conditions. Biocatalysis has been used in human life for a long time, for example, it was used to make wine more than 4000 years ago (Xia Yu era), and it was used to make maltose and sauce more than 3000 years ago (Zhou Dynasty). However, at that time, people did not know what substances triggered biocatalysis, which has become a key issue for scientists in more than 200 years of enzymology research. In the 19th century, with the popularization of microscopy and fermentation technology, it was found that the process of alcoholic fermentation was closely related to yeast. In 1897, German chemist Biechner discovered that only yeast cell extract could cause sucrose fermentation to produce alcohol without intact cells. Since then, biocatalysis has been recognized as a process that can be initiated independently by enzymes. In 1926, with the improvement of protein purification and crystallization technology, Sumner, an American chemist, successfully crystallized urease, pepsin and trypsin, and confirmed that the chemical nature of these enzymes was protein. Since then, more and more studies have confirmed that enzymes are proteins, and this view has become a textbook conclusion. However, in the 1980s, with the progress of RNA research technology, American chemist Cech and biologist Altman found that some RNAs also have biocatalytic functions, and proposed the concept of Ribozyme, which further expanded the chemical connotation of enzymes.
Enzyme engineering is a science and technology born in the 1960s in response to the large demand for enzymes. Its main task is to improve the catalytic performance of enzymes by various methods, so as to solve the problems of high cost, poor stability, low efficiency and limited application conditions in industrial production of enzymes. The research of enzyme engineering includes the preparation of enzyme preparation, the immobilization of enzyme, the modification and modification of enzyme, etc. The enzymes used in the early stage were usually natural enzymes extracted from the tissues or cells of animals, plants and microorganisms. This production method is not only complex in process, but also limited in raw materials, which makes it difficult to carry out large-scale industrial production. In the early 1970s, with the birth of genetic engineering, people can use genetically engineered strains or cells to produce enzymes, which not only overcomes the high cost of natural enzyme extraction, but also improves enzymes through genetic modification. The related enzyme directed evolution technology has won the Nobel Prize in Chemistry in 2018. In addition, the catalytic performance of the enzyme can also be improved by chemical modification. Although enzyme engineering technology has promoted the application of natural enzymes, it is still unable to fundamentally solve the problem that enzymes are difficult to be applied in industry. Among more than 8300 kinds of natural enzymes, only about 2% can be used in large-scale production, which can not meet the growing demand for enzymes.
Artificial enzyme is a concept proposed by chemists in the middle of the 20th century, which mainly uses small organic molecules such as cyclodextrin and host-guest chemistry to study the chemical nature of enzyme catalysis, and then synthesizes mimetic enzymes that mimic the structure and mechanism of natural enzyme catalytic active centers. In 1965, the first mimetic enzyme based on cyclodextrin structure was reported, followed by the discovery of molecularly imprinted polymerase, abzyme and DNase. Since then, biocatalysis research has focused on proteins and small organic molecules mimicking enzymes[2⇓~4]. However, it is difficult to completely simulate the fine structure of natural enzymes due to the limited molecular structure of traditional mimics, and the catalytic activity of most mimics is not comparable to that of natural enzymes. Therefore, it has been the direction of scientists to explore artificial enzymes with high biocatalytic activity.
With the development of nanoscience and nanotechnology, nanoenzyme is a new and unique nanoeffect of substances, that is, nanomaterials contain enzymatic catalytic characteristics. Unlike traditional artificial mimics, the catalysis of nanozymes is based on special nanostructures, so their catalytic activity is high and can be regulated. Many experiments have proved that the catalytic activity can be significantly improved by changing its nanostructure, such as size, morphology and other factors. In addition, the rich nanostructure makes some nanoenzymes have multiple enzymatic activities at the same time. The most important feature of nanoenzyme is that it has both catalytic activity and unique physical and chemical properties of nanomaterials (such as magnetism, photothermal properties), which makes nanoenzyme a bifunctional or multifunctional material.
Since the first nanoenzyme report, more than 400 laboratories in 55 countries have published more than 13,800 nanoenzyme research papers, especially in the past five years, the number of papers and citations have increased exponentially (Figure 1), gradually forming a new field of nanoenzyme research[1]. The discovery of nanoenzymes has broken through the traditional concept that nanomaterials are biologically inert substances, and for the first time, a large class of substances with biocatalytic functions have been discovered besides proteins and nucleic acids, which has built a bridge between nanomaterials, chemistry, enzymology and medicine.Based on this, the concept of nanoenzyme catalytic medicine is expanding from in vitro detection to disease treatment research, such as tumors, infectious diseases, degenerative diseases and stroke, which proves the feasibility of nanoenzyme intervention in pathological processes, which will lay a solid foundation for nanoenzyme to serve human health.
Nanoenzyme is a new field discovered and led by Chinese scientists. The contributions of Chinese scientists in the field of nanoenzyme are as follows: they discovered and named nanoenzyme, established a method to study the catalytic activity of nanoenzyme, and created the terminology and standardization of nanoenzyme.The structure-activity relationship of nanoenzyme was analyzed, the catalytic activity of nanoenzyme was increased by 10000 times, the rational design of nanoenzyme activity exceeding natural enzyme was realized, and the first nanoenzyme product in the world was created and won the medical device certificate and Atlas International Award[1][5][6]. In 2020, Chinese scientists edited the first English monograph on Nanozymology, which defined the academic field of Nanozymology for the first time. The monograph entered the hot search list of Springer Publishing House that year and has been downloaded more than 40,000 times so far. The new concepts, new materials and new technologies formed by nanoenzymes not only inject new vitality into traditional artificial enzymes and expand the innovation and application of enzyme engineering technology, but also organically combine the theories and technologies of nanotechnology, chemistry, biomedicine and other disciplines to become a new position for cross-integration and common innovation.
In this paper, the discovery of nanozymes was extended to the overall development process of the field of nanozymes, covering the activity/mechanism exploration, rational design, engineering, application direction and key issues that still exist. We hope that this review will broaden people's understanding of nanoenzymes, open up application fields, and promote the research and development of new nanoenzymes. However, due to the limitation of space, it is a pity that all the reports can not be covered. Here, only some typical examples are selected to represent the progress in related fields, and the summary and prospect are given.
1 Overview of Nanozyme
1.1 Discovery and development of nanoenzyme
The discovery of nanoenzyme is purely accidental and is the result of cross-border cooperation among Chinese scientists. Initially, Yan Xiyun's team wanted to study a tumor immune probe. However, there was a "strange" phenomenon in the experiment: the magnetic nanoparticles used as a negative control reacted incredibly with the peroxidase substrate.
At first, they thought it might be caused by the contamination of some components in the reaction system, so they changed various reagents and repeated the experiment, but the result was still the same. Does ferroferric oxide nanoparticle itself have the catalytic activity of peroxidase? In order to confirm this guess, they first consulted academician Xie Sishen, a material scientist, and then used the nanomaterials provided by Professor Zhang Yu, an academician of Gu Ning, to design an experimental scheme with Professor Sarah Perrett, an enzymologist. For the first time, the catalytic efficiency, mechanism and reaction kinetics of inorganic nanomaterials were studied from the perspective of enzymology, and compared with natural enzymes systematically. The research results were published in Nature Nanotechnology in 2007, which was praised as the foundation work in the field of nanoenzymes and attracted great attention from domestic and foreign colleagues[1]. At the same time, News & Views said that the discovery revealed for the first time that inorganic nanomaterials contained an unpredictable biocatalytic activity, changing the traditional concept that they were inert substances[7]. The Journal of the Royal Society of Chemistry published a review of this as a milestone in the history of enzymology. In 2008, Academician Wang Erkang's team published a new method for detecting glucose by peroxidase-like activity of ferroferric oxide[8]. Academician Gu Ning's team found that ferroferric oxide nanoparticles showed catalase-like activity under neutral conditions[9]. Subsequently, scientists from all over the world have reported that other nanomaterials also have enzyme-like catalytic activity, confirming that this enzyme-like catalytic property of nanomaterials is a ubiquitous nano-effect.
Yan Xiyun's team named this new and unique nano-effect "nano-enzyme"[10]. Wang Erkang's team gave it the English name "Nanozyme" and proposed that Nanozyme is a new generation of artificial enzyme[11]. Subsequently, nanoenzyme has entered a period of rapid development, which is mainly reflected in the following five aspects.
First, the concept of nanoenzyme is constantly improving[12]. The early description of nanoenzyme is a kind of nanomaterials with enzymatic properties, which emphasizes the catalysis of enzyme substrates under mild conditions and follows the kinetics of enzyme reactions[11,13]. With the deepening of research, it has been found in recent years that nanoenzyme catalysis can be carried out not only under mild physiological conditions, but also under extreme conditions such as low temperature of -20 ℃, which will have more unique application value[14]. In addition, there is a deeper and systematic understanding of the catalytic mechanism of nanozymes. By studying the catalytic mechanism of different materials (metal oxides, precious metals, carbon), it is proved that nanoenzyme catalysis is not only different from traditional ion catalysis (such as Fenton reaction), but also different from small organic molecules or transition metal catalysts. Nanoenzyme catalysis is closely related to its nanostructure and the microenvironment of the active center. These research advances have further enriched the concept of nanozymes[12].
Secondly, new materials of nanoenzymes are constantly enriched, and more than 1000 different materials of nanoenzymes have been reported, ranging from metal oxides, precious metals, carbon materials to metal single-atom-carbon doped materials, metal-organic framework materials, etc. This not only proves that nanomaterials contain the universality of enzymatic properties, but also further proves that nanoenzymes are a new class of materials with dual functions of physics, chemistry and catalysis. This new bifunctional material provides conditions for people to skillfully design various probes, devices and drugs in the future. Interestingly, the catalytic activity of nanozymes is also modulated by their physicochemical properties, such as the ability of magnetic field to modulate the catalytic activity of iron-based nanozymes. It is expected that future research will make good use of these characteristics to create better new technologies, devices and products of nanoenzymes.
Thirdly, the catalytic activity of nanoenzymes has been continuously improved, and the types of catalysis have increased. Nanoenzyme research has evolved from early random synthesis to controlled rational design, which has greatly improved its catalytic activity, approaching or even surpassing that of natural enzymes[15,540]. The catalytic type of nanozyme has also developed from the initial one redox type to four catalytic types, namely hydrolase, isomerase, oxidoreductase, and lyase. There are six types of natural enzymes in nature, and there are still two types of catalytic enzymes, synthetase and transferase, which need to be studied. With the accumulation of research data in the field of nanozymes, it is possible for big data analysis combined with machine learning to predict and design highly active nanozymes, which is expected to accelerate the further development of nanozymes[16,17].
Fourth, establish national and international (ISO) standards for nanoenzyme terminology and activity detection. The nomenclature of nanoenzyme is "material composition-enzyme name + nanoenzyme", for example, ferroferric oxide peroxide nanoenzyme[5]. In addition, in order to facilitate peer academic exchanges and make the catalytic activity of nanoenzyme products quantifiable, the measurement technology standard of nanoenzyme catalytic activity was established, and the catalytic activity unit (U) and specific activity (U/mg) of nanoenzyme were defined[18]. The standardization of nanoenzyme terminology and catalytic activity not only standardizes the understanding and understanding of nanoenzymes in academia, but also greatly promotes the application research and industrialization of nanoenzymes. In 2020, the POCT Branch of China Medical Device Industry Association established a special committee on the application of nanoenzyme and POCT. With the wide application of nanoenzyme products, it is also necessary to develop industry standards for nanoenzyme to guide the standardization of nanoenzyme commodities, so that it can be widely used in catalytic medicine, including not only in vitro detection, but also anti-tumor, neurodegenerative disease, anti-infection, anti-oxidation, anti-aging and so on. In addition, nanoenzymes are also used in environmental governance, energy, chemical industry, national defense, agriculture and other fields.
Fifth, the discovery of natural nanoenzyme. Recently, some natural nanoenzymes have been found in natural organisms, such as ferritin in cells and magnetosomes in magnetic bacteria. Interestingly, these nanostructures, which are themselves used for magnetic navigation (magnetosomes) and for iron storage (ferritin), also have peroxidase-like activity and can play an antioxidant role in bacteria[19,20]. The discovery of natural nanozymes not only expands the research space of nanozymes, but also suggests new functions of natural biominerals. The discovery of natural nanozymes highlights the deeper significance of nanozymes, which are not only a new class of mimetic enzymes, but also may play an important role in physiology and pathology as a natural catalyst in vivo, participating in the metabolism of life. At the same time, the exploration of natural nanoenzymes not only provides a new direction for the design and synthesis of nanoenzymes, but also further broadens the chemical nature of natural enzymes in life.
To sum up, the rapid development of nanoenzymes has given birth to many new concepts, new materials and new technologies, and it is expected that this emerging field will benefit mankind in the near future.
1.2 Nanoenzyme Terminology and Standardization
Standardization work is guiding and forward-looking. Terminology is an indispensable tool for disseminating knowledge and skills and for social, cultural and economic exchanges. With the vigorous development of the nanoenzyme field, the formulation of standardized nanoenzyme terminology can guide the research and development of nanoenzymes, communication and the formulation of nanoenzyme technology standards, provide important support for the sustainable and healthy development of the nanoenzyme field, and also provide an important technical basis for nanoenzyme-related enterprises.
1.2.1 Evolution of the term "nanozyme"
Since the enzyme-like effect of Chemical Society Reviews nanomaterials was first reported in 2007, Chinese scientists first published a long review article with the title of "nanozyme" in Chemical Society Reviews of the Royal Society of Chemistry in 2013, proposing and using the term "nanozyme" to describe this enzyme-like nanomaterial[1][11]. After more than ten years of accumulation and evolution, nanozyme and related terms have been accepted and used by the academic community. So far, "nanozyme" has been widely accepted in many international academic journals. For example, with "nanozyme" as the keyword, more than 2500 relevant papers since 2013 can be retrieved from the web of science database alone. In 2013, Academician Yan Xiyun's team proposed the Chinese term "nanozyme" for "nanozyme", pointing out that nanozyme is a kind of mimetic enzyme with both unique properties of nanomaterials and catalytic functions[10]. The term is now included in Biophysical Terminology (Second Edition)[21].
1.2.2 Formulation of National Standard for the Terminology of "Nanozyme"
The layout of nanoenzyme-related standards at home and abroad can not only establish China's leading position in the field of nanoenzyme research and development and application, but also help standardize the research and development of nanoenzymes and related evaluation, which is of great significance to academic research, technology transformation and product research and development, and the formulation of relevant standards will fill the gaps at home and abroad. The proposal of nanoenzyme terminology system is conducive to standardizing the development and evaluation of nanoenzyme-related technologies and products, so that R & D and production personnel in different disciplines and fields can form a unified understanding and understanding.
In 2018, the Secretariat of the Working Group on Health, Safety and Environment (SAC/TC 279) under the National Technical Committee for Standardization of Nanotechnology (hereinafter referred to as the "National Committee for Standardization of Nanotechnology"), through preliminary research and a number of universities and research institutes in the United Nations.It submitted an application to the Standardization Administration of the People's Republic of China (hereinafter referred to as the "Standardization Administration of the People's Republic of China", SAC) for the formulation of the national standard of "Nanotechnology Terminology Nanoenzyme", which was officially approved on January 13, 2020 (Project No.: 20193987-T-491). This standard defines terms and definitions related to nanozymes. On the basis of the basic terms "nanoscale", "nanomaterials" and "enzymes", 13 terms describing nanoenzymes are defined, including "nanoenzymes" and common terms related to their activities. In addition, according to the nature of the catalytic reaction, six types of NANases with specific activity were defined, including redox NANase, hydrolytic NANase, lytic NANse, isomeric NANse, junctional NANes, and transfer NANes. In addition, the nomenclature of nanozymes is also given for nanozymes that catalyze some group transfer or exchange between substrates, that is, "material composition-enzyme name + nanozymes".
In the process of standard formulation, the drafting group fully communicated and discussed with experts in the field online and offline, and widely solicited opinions from experts in the field such as domestic universities, research institutions, relevant high-tech enterprises and some foreign scholars. After the preliminary review of national standards and the expert review meeting, the whole draft was completed in January 2022, and the draft of national standards for approval was formed. At present, it has been submitted to the National Standards Committee by the Chinese Academy of Sciences and is now in the approval stage. At the same time, some members of the drafting group jointly wrote the article "Standard Terminology of Nanoenzyme", which was published in the core journal "Chinese Science and Technology Terminology"[5].
1.2.3 Establishment of National and International Standards for Measurement of Catalytic Activity of Nanozymes
In addition to the standards related to nanoenzyme terminology, in recent years, important progress has been made in the technical standards for the measurement of catalytic activity of nanoenzymes. Nanozyme began with the study of peroxidase-like activity of iron oxide nanoparticles, but there has been no relevant standard method to guide the detection of peroxidase-like activity of nanoparticles. Because the enzyme-like activity of nanoparticles significantly depends on their species, size, specific surface area, crystal structure and other factors, the measurement and evaluation of their catalytic activity are complex. The Measurement Method of Peroxidase-like Activity of Nanotechnology Iron Oxide Nanoparticles, led by Southeast University, was approved as a national standard in August 2015, and all the formulation work was completed, released and implemented in 2019, with the standard number of GB/T 37966-2019. In response to the call of the National Standards Committee to promote international exchanges and cooperation of national standards and serve the "Belt and Road" initiative, the standard drafting group applied to the National Standards Committee for a foreign language version of the draft standard and was approved (project number: W20190885). In addition, Academician Yan Xiyun and Professor Liang Minmin also published a research paper in Nature Protocols on the measurement of catalytic activity of nanoenzymes, defining the catalytic activity unit of nanoenzymes (Nanozyme unit,U) and the specific activity (U/mg) of the nanoenzyme, so that the enzyme-like activity of the nanoenzyme product can be quantified, which is beneficial to the evaluation and development of nanoenzyme-related products, and meanwhile, the repeatability and accuracy of the nanoenzymatic analysis method are improved, which effectively promotes the development of nanoenzyme-related products[18].
In terms of the formulation of international standards related to nanoenzymes, a team of Chinese experts presented a draft international standard "Nanotechnologies-Assessment of peroxidase-like activity of metal and metal oxide nanoparticles" at the working meeting of the Working Group on Health, Safety and Environment (WG3) at the 21st Plenary meeting of the International Organization for Standardization Committee on Nanotechnology (ISO/TC229) in November 2019.For the first time, the measurement principle and method of peroxidase-like activity of metal and metal oxide nanoparticles were introduced to international standard experts. The standard standardizes the standard measurement scheme, which is of great significance to the development and quality control of peroxidase-like nanomaterials, as well as to the understanding of the biological effects of nanoenzymes. Through the ISO international project application process and the voting of ISO member countries, the project was approved in 2020 (project number: ISO/DTS 5094). This project is the first international standard proposal approved by China in ISO/TC229/WG3. After more than two years of communication and discussion with experts from various countries, the draft international standard has solved all the technical problems raised by experts and is expected to be published in January 2023.
At present, the number of national/international standards related to nanoenzymes is not sufficient (Table 1), and most of them are formulated by Chinese scientists, which not only reflects the leading role of Chinese scientists in this field, but also shows that Chinese scientists should play a greater role in this regard.
1.2.4 Development Status of National Standard Samples and National Standard Substances Related to "Nanozyme"
In recent years, China has also made important progress in the development of nanoenzyme standard samples/substances. The national standard sample (hereinafter referred to as "standard sample") is formulated for the implementation and formulation of standards, and is generally used only within the scope of the standard, which is a kind of physical standard. Standard samples are implemented, supervised and managed by the National Technical Committee for Standard Samples, and are reviewed, approved and issued by the National Standardization Management Committee. So far, there are 10 particle-related standard samples available in the field of nanoenzymes, mainly including nano-titanium oxide, gold-labeled nano-silver and monocrystalline gold nanorods (Table 2)[22].
National standard material (hereinafter referred to as "standard material") is a measurement standard, which can be used as the transmission of measurement to calibrate instruments, evaluate measurement methods, and determine the value of materials. As long as it is appropriate, it can be used instead of standard samples in the formulation and implementation of standards. Reference materials shall be examined by the National Reference Material Management Committee and approved and issued by the metrological administrative department of the State Administration of Quality Supervision, Inspection and Quarantine. At present, there are 21 particle-related standard materials available in the field of nano-enzymes, mainly involving gold nanoparticles, nano-titanium dioxide, nano-silver, nano-γ-Fe2O3, cadmium selenide and zinc sulfide quantum dots (Appendix I)[23]. On the basis of the development of nanometer γ-Fe2O3 reference materials, Zhang Yu's team also successfully prepared Prussian blue nanoparticles with uniform size and good morphology by single-source method, and is developing Prussian blue nanometer enzyme reference materials for comparing the activity of nanometer enzyme peroxidase, which can be used as an important traceability reference material for the measurement of nanometer enzyme activity[24].
Seven Reference materials of nanomaterials that may have the properties of nanoenzymes can be retrieved from the websites of metrology authorities in the United States, Germany, the United Kingdom, Australia, Canada, Japan, South Korea and other countries (Appendix II)[25][26].
Reference sample/reference material is the "ruler" and "weight" of analysis and detection, and its application involves all aspects of economic activities and human life. With the rapid development of global economy, science and technology, measurement technology and social life demand, the demand for the variety, quantity and quality of reference sample/reference material is increasing and increasing. It can be seen that the number of standard samples/substances of nanoenzyme-related materials in China is far more than that in other countries, which is closely related to the rapid development of this field in China.
1.2.5 Brief summary
Standards are the commanding heights for scientific research and business circles at home and abroad. As a Chinese original, the formulation and layout of nanoenzyme terminology, properties and related standard samples/substances are very important. China's progress in the standardization of nanoenzymes will help to establish China's international leading position in the field of nanoenzyme research and development and application, and is of great significance to the implementation of China's strategic layout from a manufacturing power to a manufacturing power.
1.3 Determination of catalytic activity of nanoenzyme
The kinetic parameters and catalytic mechanism of nanoenzyme are important criteria for judging whether nanomaterials belong to nanoenzyme and quantifying its catalytic efficiency. The analysis of the catalytic mechanism and active center site of nanoenzymes can not only reveal the essence of nanomaterials mimicking natural enzymes, but also provide a theoretical basis for improving the activity and selectivity of nanoenzymes and their rational design, so that nanoenzymes can be better applied to biomedicine, agriculture, industry and other fields.
Similar to natural enzymes, the reaction catalyzed by nanozymes is closely related to the pH, temperature and buffer conditions of the reaction system.
1.3.1 Catalytically active species of nanozyme
So far, nanomaterials can mimic dozens of enzyme activities, including peroxidase, catalase, superoxide dismutase, oxidase, glucose oxidase, glutathione peroxidase, protease, esterase, nuclease and so on. Several typical enzyme activities will be briefly introduced as examples below.
1.3.1.1 Peroxidase-like (POD) activity
At present, the most discovered NANase is peroxide-like NANase. Peroxiredoxin-like nanoenzyme is a dual-substrate nanoenzyme, which can catalyze the hydrogen acceptor to produce a large number of oxidative free radicals in the presence of the first substrate hydrogen acceptor (such as H2O2), and then rapidly oxidize the second substrate hydrogen donor. Peroxiredoxin-like nanozymes can mimic many enzymatic activities, such as peroxidase, glutathione peroxidase, lipid peroxidase, depending on the hydrogen donor. The hydrogen acceptors of peroxidases are various, for example, the hydrogen acceptor of horseradish peroxidase is H2O2, and the hydrogen acceptor of lipid peroxidase is lipid peroxide. Free radicals produced by hydrogen acceptors can oxidize a variety of hydrogen donor substrates, including small molecular metabolites (formic acid, methanol, ethanol, etc.) And biological macromolecules (nucleic acids, proteins, polysaccharides, lipids, etc.).
Taking the earliest discovered horseradish peroxidase-like nanoenzyme as an example, its activity detection method is usually based on the activity detection method of natural horseradish peroxidase (HRP), which uses H2O2 as hydrogen acceptor and is catalyzed by peroxidase-like nanoenzyme to produce a large number of intermediate hydroxyl radicals.Hydrogen donors (such as TMB (3,3 ', 5,5' -tetramethylbenzidine), DAB (diaminobenzidine), ABTS (2,2 '-azinobis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt), etc.) Are further oxidized. The reaction formula is as follows:
As shown in the color reaction in fig. 2a, the peroxidase nanoenzyme can catalyze the color development in the figure[18]. The activity of peroxide-like nanoenzyme mainly occurs under acidic conditions, usually using sodium acetate-acetic acid buffer with a concentration of 0.2 mol·L-1, the pH value is between 3 and 6, and the optimum pH value is around 4; The optimum temperature is near the physiological conditions, and the optimum temperature of the natural enzyme is usually 37 ℃ for the convenience of application[1,18].
1.3.1.2 Catalase-like activity
The catalase-like nanoenzyme has the same catalytic reaction as catalase, which can decompose H2O2 into water and oxygen, and bubbles are produced in the reaction, as shown in Figure 2b. The reaction conditions of catalase activity are neutral or alkaline environment, and the nanoenzyme has a wider pH range than the natural catalase, and the commonly used buffers are phosphate buffer solution (PBS) and sodium carbonate-bicarbonate buffer.
1.3.1.3 Oxidase-like enzyme (OXD) activity
Oxidative nanozymes use oxygen as a hydrogen acceptor to directly oxidize hydrogen donor substrates (such as TMB) without the addition of additional compounds such as H2O2. It has a temperature and pH range similar to peroxidases.
1.3.1.4 SOD-like activity
Like SOD, SOD-like nanoenzyme can convert superoxide anion () into O2 and H2O2, and play an anti-inflammatory and antioxidant role. Its reaction conditions are weakly alkaline, usually pH between 8 and 9.
1.3.1.5 Protease-like activity
This kind of nanoenzyme can hydrolyze the peptide bond of protein and decompose protein and polypeptide into polypeptide or amino acid. In 2016, Qu et al. Reported the nanoenzyme AuNPs @ POMD-8pep with protease-like activity, which had an optimum reaction pH of 8.0 and an optimum reaction temperature of 55 ℃[27].
1.3.1.6 Nuclease-like activity
Nuclease can cleave the phosphodiester bond of polynucleotide chain and hydrolyze DNA or RNA into nucleotide. In 2016, Qu et al. Reported nucleoid nanoenzyme :AuNPs@Fe3O4/SiO2 and applied it to the study of biofilm inhibition. The optimum pH value of this nuclease is 7.4, and the optimum temperature is 37 ℃[28].
Nanozyme has some characteristics different from natural enzymes. One nanozyme can have multiple activities of natural enzymes at the same time. For example, both :Fe2O3 and Fe3O4 nanozymes have both peroxidase and catalase activities[9]. Co/PMCS nanoenzyme has three activities of catalase, superoxide dismutase and glutathione peroxidase[29]; The FeN3P monoatomic nanoenzyme has four activities of peroxidase, catalase, superoxide dismutase and oxidase. In addition, different nanozymes can have the same enzyme-like activity, for example, :Fe2O3, Fe3O4, FeN3P and other nanozymes all have peroxidase activity[15][9][9,18][15]; Co/PMCS, Co3O4, Pd@TiO2 and other nanozymes have catalase activity[29][30][31]. In addition, different kinds of nanoenzymes have different atomic composition, crystal form, size and surface modification. This makes the measurement and evaluation of nanoenzyme activity more complex. Therefore, it is important to establish a standard method for the measurement and evaluation of nanoenzyme catalytic activity. As mentioned above, Yan et al. Developed a set of standard methods for detecting the activity and catalytic kinetics of peroxidase nanoenzymes, and then detected and compared the activity of peroxidase nanoenzymes from different sources, different sizes, different modifications and different materials, and calculated their kinetic constants to compare their catalytic efficiency[18].
1.3.2 Typical nanoenzyme reaction kinetics
Generally speaking, nanozymes have the same kinetic mechanism as natural enzymes, and the catalytic kinetic curve conforms to the Michaelis-Menten equation. For example, the kinetic curve of the first nanoenzyme (Fe3O4 nanoenzyme) discovered by Yan et al. In 2007 is similar to that of natural HRP, which conforms to the Michaelis-Menten equation curve, and its catalytic mechanism conforms to the ping-pong mechanism of natural enzyme, as shown in Figure 3[1]. In terms of mechanism, the nanozyme, like the native enzyme (E), first binds to the corresponding substrate (S) to generate an intermediate complex (ES*, EP), which is then converted into a product (P) through a surface-catalyzed reaction that only accelerates the reaction rate without changing the reaction equilibrium. The reaction is represented by the following equation:
Under the condition of constant enzyme concentration, when the substrate concentration is low, the enzyme in the solution is not saturated, and the reaction rate depends on the substrate concentration and increases linearly with the substrate concentration; When the substrate concentration is sufficiently high, the enzyme in solution is completely saturated, and the reaction rate is independent of the substrate concentration and asymptotically approaches a plateau, as shown by the kinetic curves in Fig. 3. The catalytic efficiency of the nanoenzyme and the relationship between the catalytic efficiency and the reaction conditions can be determined by kinetic analysis and measuring the catalytic reaction rate.
In kinetic analysis, the reaction rate can be determined from the consumption of substrate or the formation of product. At present, the detection method of kinetics is mostly colorimetric method, which is operated by spectrophotometer. If the reaction contains only one substrate, the kinetic analysis is performed by varying the substrate concentration; If there are two or more substrates, it is necessary to fix the concentration of one substrate and change the concentration of the other substrate to detect, so as to obtain the kinetic parameters that can characterize the catalytic efficiency of nanoenzyme. The kinetic profiles of most nanozymes reported so far were in accordance with the Michaelis-Menten equation, with a few exceptions.
1.3.2.1 Michaelis-Menten reaction
Michaelis-Menten kinetics is one of the most famous enzyme kinetic models. The Michaelis-Menten equation represents the quantitative relationship between the substrate concentration ( [S]) and the enzyme reaction rate (V), which can be expressed by the following formula:
The Vmax is the maximum reaction rate when the enzyme in the reaction system is saturated. The KM is also known as the Michaelis-Menten constant, which is the substrate concentration when the reaction rate reaches half of the Vmax. The unit is mol/L, which usually indicates the affinity between the enzyme and the substrate. The smaller the KM value, the higher the substrate affinity, which can be used to judge the optimal substrate of the nanoenzyme. When the substrate concentration is much lower than the KM, the reaction rate is linearly related to the substrate concentration, which is also called First-order kinetics. Under this condition, both Vmax and KM are constants, the ratio of which can be expressed by a constant K ', and the reaction rate can be expressed by the following formula:
When the substrate concentration is much higher than the KM, the reaction rate reaches a Vmax and is not affected by the change of substrate concentration. At this time, the reaction reaches a steady state, which is also called Zero-order kinetics. Therefore, when the reaction is of zero order, the reaction rate is not affected by the concentration of the substrate, but only related to the concentration of the enzyme ( [E]), and the enzyme completely forms a complex with the substrate (ES). The Catalytic activity of an enzyme can be expressed by the kcat, also known as the Catalytic constant, which is the number of moles of substrate converted to product per unit enzyme per second when the enzyme is saturated. Assuming that the total enzyme concentration [ET] in the reaction system is known, at saturating [S],
However, in general, most nanozymes are not saturated with substrate, and the ratio of [S]/KM is between 0.01 and 1.0. Substituting the above formula into the Michaelis-Menten equation, the following formula is obtained:
When [S]≪KM, the concentration of free enzyme [E] is roughly close to [ET], thus obtaining:
At this time, the reaction rate V depends on the concentration of enzyme E and substrate S, which is a second-order reaction. Therefore, the order rate constant is also called the Apparent second-order rate constant or Specificity constant of the reaction between E and S to form the product, which can be used to compare the catalytic efficiency of different enzymes or the same enzyme catalyzing different substrates.
Taking peroxin-like nanozymes as an example, there are two substrates in the peroxidase activity reaction: hydrogen acceptor and hydrogen donor, so it is necessary to change the concentration of one substrate while fixing the other substrate when measuring its kinetics. In the color reaction system with H2O2-TMB as the substrate, the kinetic determination of H2O2 needs to fix the concentration of TMB, while the kinetic determination of TMB needs to fix the concentration of H2O2. As shown in fig. 3, the kinetic curve of Fe3O4 peroxidase nanoenzyme completely conforms to the same Michaelis-Menten equation as HRP, and the kinetic data obtained by calculation are shown in Table 3, wherein the Michaelis-Menten constant KM value of Fe3O4 is smaller than that of natural HRP, indicating that the affinity of Fe3O4 nanoenzyme to the substrate TMB is higher than that of HRP[1]; However, the Michaelis constant KM of another substrate, H2O2,Fe3O4, is larger than that of native HRP, which is consistent with the fact that the H2O2 used in the Fe3O4 nanoenzyme experiment is higher than that of HRP. Fe3O4 nanoenzyme has a strong affinity for TMB and a weak affinity for H2O2, which also exists in many other nanoenzymes, and it may be related to the unique surface physicochemical properties of nanomaterials. Nanomaterials have large specific surface area, rich surface charge, complex morphology, and easy adsorption with positively charged TMB, so they have strong affinity. On the other hand, the catalytic constant kcat value of Fe3O4 is larger than that of native HRP, which may be related to the fact that the surface of nanozyme possesses multiple active sites that can function simultaneously. In addition, in terms of catalytic mechanism, the peroxidase activity of Fe3O4 nanoenzyme conforms to the ping-pong mechanism of natural enzyme (as shown in Fig. 3e, f), that is, Fe3O4 reacts with H2O2 first.Catalyze H2O2 to produce hydroxyl radical (·OH), form Fe3O4 nanoenzyme-radical complex, and then catalyze TMB oxidation and color reaction.
For hydrogen peroxide-like nanoenzyme, since it catalyzes H2O2 to generate H2O and O2 when it works, bubbles will be generated in the solution when the O2 reaches a certain concentration, as shown in Figure 1b, which will bring a certain degree of difficulty to the determination of its kinetic data, so it is necessary to control the concentration of nanoenzyme and H2O2 during the detection, and detect without bubbles[9]. The content of O2 in the solution can be detected by an oxygen electrode to monitor the rate of oxygen generation, so as to obtain the kinetic curve of hydrogen peroxide nanoenzyme, as shown in Fig. 4[9,32][32].
1.3.2.2 Non-Michaelis-Menten reaction
In addition to Michaelis-Menten kinetics, catalysis by some nanozymes may also follow other kinetic equations. For example, molybdenum trioxide (MoO3-TPP) nanoparticles with sulfide-like oxidase activity can catalyze sulfite oxidation in the presence of ferricyanide. Under the condition of constant concentration of MoO3-TPP and ferricyanide, the kinetic curve of nanoenzyme on substrate is S-shaped (also known as Sigmoidal behavior), as shown in Figure 5B[33]. The reason for this phenomenon lies in the cooperative binding of the two substrates to the active site, with sulfite anion binding together with the negatively charged ferricyanide at the binding site on the surface of MoO3 nanoparticles. The kinetic parameters of the nanoenzyme can be calculated by the Hill equation. The KM of the was calculated to be the 0.59±0.02 mmol·L-1,Hill coefficient (also known as the cooperative constant) of 2.35 ± 0.15, which proved that the reaction was indeed a positive cooperative reaction. Once one substrate was bound to the surface of the nanoenzyme, the affinity of the nanoenzyme for another substrate would increase. It is worth noting that the same retroflex behavior is also present in the native enzyme, illustrating that it is a general enzyme kinetics.
1.3.3 Calculation of active sites and particle number of nanoenzyme
As a mimic of natural enzymes, it is necessary to determine the active site of nanozymes to reveal their catalytic mechanism. The active site of natural enzyme is a flexible structure composed of amino acids and cofactors, and can complete catalysis together with the substrate binding site, while unlike natural enzyme, the active center of nanoenzyme and the substrate binding site are a unified structure on the surface of rigid nanomaterials. For example, the active center of horseradish peroxidase and catalase is composed of heme molecule and surrounding amino acid residues, in which the Fe atom in the structural center of heme porphyrin plays an important role in the catalytic process, and it interacts with different amino acid residues to produce different catalytic activities. Unlike natural enzymes, nanozymes have multiple active sites on their surface that can play a role at the same time. Taking Fe3O4 nanoenzyme as an example, it has both peroxidase activity and catalase activity, and its activity is accomplished by the change of the valence state of Fe atoms on the surface of nanoparticles, which is similar to that of natural enzyme, but the difference is that Fe atoms on the surface of Fe3O4 nanoenzyme are coordinated with O atoms, while Fe atoms in hematoporphyrin are coordinated with N atoms. In addition, the Fe atom on the surface of Fe3O4 nanoenzyme has two valence States, divalent and trivalent, which has a greater impact on its activity. In 2007, Yan et al. Reported that the activity of Fe3O4 nanoenzyme increased after NaBH4 reduction treatment, but decreased after NaIO4 oxidation, indicating that the active site of Fe3O4 nanoenzyme was Fe2+[1]. At present, although the catalytic mechanism of Fe3O4 nanozymes has made some progress, the specific catalytic mechanism is still not fully understood, and scientists are expected to reveal the relevant mechanism by using density functional theory (DFT) computational simulation analysis and experimental verification[543]. This method is suitable for the analysis of active sites and catalytic mechanisms of other active and species of nanoenzymes.
For the calculation of the number of nanoenzyme particles, the volume Vn of a nanoparticle can be calculated according to the size of the nanoparticle, and then the density ρn of the nanoparticle can be queried to calculate the volume V of nanoenzyme per unit mass (1 mg), so the number of particles N per milligram of nanoenzyme can be expressed by the following formula:
In addition, with the increase of nanoenzyme types, in order to make the activity of various nanoenzymes comparable, the number of active center atoms participating in the reaction can be used as a unit. For example, in 2021, Yan et al. Reported that the specific activity of single-atom FeN3P nanoenzyme was defined by the molar concentration of Fe atoms[15]. In 2022, Liu et al. Mentioned that the number of active sites on the surface of nanoparticles, the number of nanoparticles, the mass of nanoenzymes, and the total metal concentration could be used as units to measure the specific activity of nanoenzymes[34].
1.3.4 Brief summary
Nanozyme has inherent enzyme-like properties. They not only have similar catalytic behavior to the native enzyme, including substrate, optimal pH, temperature, and activator/inhibitor, but also have similar enzymatic kinetics and catalytic mechanism. The typical processes catalyzed by nanozyme include substrate adsorption, electron transfer, and product release, which are the same as those catalyzed by native enzyme. However, nanoenzymes have amorphous rigid nanostructures, while the protein scaffold of enzymes is a flexible and precise three-dimensional structure, which follows the lock-and-key theory (also known as the "induced fit" hypothesis) in substrate binding and product separation, so the active center will undergo corresponding structural changes, which is also the reason why the activity and selectivity of nanoenzymes are often lower than those of natural enzymes. Therefore, the kinetic calculation enables researchers to have a good understanding of the mechanism and properties of nanoenzymes, which lays a good theoretical foundation for the design of nanoenzymes and the prediction of their activities. On the other hand, the activity of nanozymes can also be improved by mimicking the structure and catalytic properties of natural enzymes. We hope that the introduction of computational methods for nanozyme kinetics in this section will be helpful in recognizing and understanding nanozymes.
1.4 Catalytic mechanism of nanoenzyme
The essence of catalysis is that the catalyst changes the reaction pathway, guides the reaction along the pathway with lower activation energy, and accelerates the reaction rate. Therefore, the important goal of nanoenzyme catalytic mechanism research is to reveal the process of atomic rearrangement of reactants on the surface of nanoenzyme materials, identify the reaction pathway and kinetics with the lowest activation energy, and establish the law of chemical composition and structure affecting catalytic efficiency, so as to provide a basis for nanoenzyme research and design. Because it is difficult to detect the atomic process of nanosurface in situ and dynamically by current experimental methods, the combination of DFT calculation and experiment is the main means to characterize the microscopic catalytic mechanism of nanoenzymes.
The catalytic mechanism of nanoenzymes has been comprehensively and thoroughly discussed in previous reviews, so this section mainly reviews the catalytic mechanism of nanoenzymes from a microscopic perspective and describes the theory and law of catalytic activity of nanoenzymes based on the principle of mechanism development[11,13,35⇓ ~37].
Recent studies have shown that the catalytic mechanism of nanoenzymes can vary with the type of enzyme-like reaction, the type of substrate and the type of nanomaterials. At present, when the two are the same and the types of nanomaterials are similar, they follow similar mechanisms. Therefore, there are two key points worthy of attention in the study of nanoenzyme catalytic mechanism: (1) nanoenzyme catalytic mechanism and its variation; (2) When the catalytic mechanism is determined, the composition and structure of nanomaterials affect the catalytic efficiency. In the following, the catalytic reactions of nanomaterials mimicking peroxidase, oxidase, superoxide dismutase and catalase will be taken as examples to briefly describe the new progress in the catalytic mechanism of nanomaterials in the above two aspects.
1.4.1 Peroxidase nanozyme
The existing DFT calculation methods are difficult to accurately simulate the solution environment, so the calculation study of nanoenzyme mechanism usually adopts the approximation of gas phase conditions. Computational studies in the gas-phase approximation show that nanomaterial-like peroxidase catalysis follows a reaction mechanism similar to that of natural peroxidases. Different research groups have studied the catalytic mechanism of the oxidation of TMB molecules by nano-surface catalyzed H2O2, including precious metals, metal compounds, carbon materials and composite materials, and the reaction equation is as follows:[38][39⇓⇓~42][43⇓~45][15,46⇓⇓ ~49]
In the above formula, oxTMB is the oxidation product of TMB. It is found that the catalytic mechanism of different nanomaterials is similar, which can be roughly divided into three steps: in the first step, one molecule of H2O2 decomposes at the catalytic center of the nanosurface to form one molecule of O (or two molecules of OH) adsorption intermediate; In the second and third steps, two molecules of TMB approach the catalytic center in turn, and each transfers one H atom to the above intermediate to form a H2O and desorb from the nanosurface to complete the catalytic cycle (Figure 6A). H2O2 and TMB sequentially bind and react with the nanosurface, which fits with previous reports that this catalysis follows a ping-pong mechanism[1].
The similar peroxidase-like catalytic mechanism followed by different nanosurfaces lays the foundation for the development of a general activity prediction theory. Based on the peroxidase-like catalytic reaction of iron oxide, Shen et al. Proposed that the hydroxyl radical ·OH adsorption energy (Eads,OH) could be used as a theoretical descriptor to predict the peroxidase-like catalytic activity of nanosurfaces[41]. When the adsorption force of the nano surface to ·OH is too weak (Eads,OH≫-2.6 eV), the electron-donating ability (i.e., reducibility) of the iron oxide surface is too weak, which will make it difficult for H2O2 to decompose to form an active intermediate and limit the catalytic activity; However, when the adsorption of ·OH on the nanosurface is too strong (Eads,OH≪-2.6 eV), the electron-donating ability of the nanosurface is too strong, which will lead to the strong adsorption of active intermediates on the surface and make it difficult to be reduced, and also limit the catalytic activity. The peroxidase-like catalytic activity of the nanosurface is the strongest when Eads,OH is neither too strong nor too weak (Eads,OH≈-2.6 eV)[41]. Fig. 6 B shows the Eads,OH values of different nanosurfaces, which is helpful to systematically understand the law of peroxidase-like activity of nanomaterials. The catalytic activity is the strongest when the adsorption force of the active intermediate on the catalyst surface is neither too strong nor too weak, which is in line with the Sabatier principle of heterogeneous catalysis[50].
The prediction of enzyme-like activity using Eads,OH requires DFT calculations, but most nanomaterials are intangible, and the calculated Edas,OH are difficult to reflect the true statistical average of the material, so it is particularly important to develop more easily characterized activity descriptors for specific types of materials in practical applications. Wei et al. Systematically studied the peroxidase-like activity rule of different perovskite materials (ABO3,A and B represent metal), and found that metal B is the most important catalytic active center[51][51]. Metal B is coordinated by six O atoms, and its five d-orbital levels are split to form two eg and three t2g orbitals (Fig. 7 a). Where the average electron occupation number of the eg orbital is a descriptor of peroxidase-like activity: activity is maximal when the eg occupation number is close to unity (Fig. 7 B). Because the average eg occupation number can be obtained by measuring the magnetic moment of materials, this descriptor provides a feasible method for screening peroxidase nanozymes based on perovskite and related materials in experiments[51].
The catalytic mechanism is often considered to be similar to the Fenton mechanism because the ·OH signal can often be detected by adding a radical trapping agent in the nanomaterial-based peroxidase catalytic reaction system[1]. However, the detection of a radical signal only indicates that the capture agent molecule can also be oxidized by H2O2 as a substrate, and does not prove that free ·OH radicals are generated during the reaction and dominate the subsequent reaction. ·OH radicals have strong oxidizing properties and can oxidize most organic compounds without selection, but nanomaterial peroxidase catalysis does not have this characteristic[52]. For example, He et al. Found that when ascorbic acid and o-phenylenediamine were present in the peroxidase-like alloy reaction system, the former was preferentially oxidized[53]. Examples of nanoenzyme catalysis with certain substrate selectivity also exist in many other nanoenzyme-catalyzed reaction systems, and these results suggest that nanoenzyme catalysis is more consistent with the solid-liquid heterogeneous catalytic mechanism and kinetics rather than the radical-dominated Fenton mechanism[54⇓~56].
1.4.2 Oxidase nanoenzyme
Nanomaterials can catalyze the oxidation of organic substrates by O2 molecules, which is similar to the chemical catalytic function of oxidase. This reaction reduces O2 to H2O2 or H2O with the following equations, respectively:
In the above formula, R and P represent the reactant and product, respectively. The catalytic activity order of the same group of nanomaterials may be different when they catalyze different organic substrates. Recently, Chen et al. Found that the activity order (Au > Pt > Ru > Ir > Pd > Rh) of glucose oxidation and the activity order (Pt > Ir > Pd > Rh > Ru > Au) of TMB oxidation catalyzed by different noble metal nanomaterials with O2 were significantly different[57]. The reduction products are also different, in which O2 is reduced to H2O2 when catalyzed by Au, while O2 is reduced to H2O when catalyzed by other noble metals. This is related to the fact that other noble metals have strong peroxidase-like activity, which can further catalyze H2O2 to oxidize substrates.
Shen et al. Used DFT calculations combined with experiments to study the catalytic mechanism of O2 oxidation of TMB catalyzed by different noble metals, and proposed the catalytic mechanism as shown in Fig. 8A[13]. According to this mechanism, first O2 adsorbs on the noble metal surface and decomposes into O atoms, which are reduced to H2O and desorbed from the metal surface under the action of TMB. In this process, the decomposition of O2 on the surface of noble metal is the key, so the decomposition activation energy of O2 can be used as a descriptor of the catalytic activity. The higher the metal d-band center is, the more easily the metal electron is transferred to the O2 antibonding orbital, and the more easily the O2 decomposes, so the height of the d-band center can also be used as a theoretical descriptor for this catalytic activity.
Cheng et al. Used DFT calculations to study the process of Au-catalyzed O2 oxidation of glucose molecules in the gas phase, and proposed different catalytic mechanisms[58]. As shown in Fig. 8 B, firstly, glucose and O2 molecules are co-adsorbed on the Au surface, and then glucose successively transfers two H atoms to O2 with the assistance of Au to form H2O2, completing the catalytic cycle. Since the Au-catalyzed oxidation of glucose by O2 is generally carried out under alkaline conditions, Chen et al. Proposed a catalytic mechanism similar to that of natural glucose oxidase based on this experimental phenomenon[57]. They believe that under alkaline conditions, the OH- acts as a Brønsted base to remove the hydrogen atom from the C1 OH of glucose (in the native enzyme, the histidine residue functions as a Brønsted base); The hydrogen of glucose C1 is further transferred to Au to form gluconolactone (the ester is further hydrolyzed to form gluconic acid); The hydrogen atom adsorbed on Au further reacts with oxygen to generate hydrogen peroxide, and the catalytic cycle is completed. The catalytic mechanism is different when the substrate is TMB and glucose, which is consistent with the result that the change of the substrate leads to the change of the catalytic activity sequence of the noble metal oxidase mentioned above[57].
Recently, Wu et al. Found that the oxidase-like activity of TMB oxidation by metal-organic frameworks can be precisely regulated by the substituent of the ligand, and the activity is related to the electron-withdrawing effect of the substituent (which can be quantitatively predicted by the Hammett constant of the substituent). The stronger the electron-withdrawing ability of the substituent, the stronger the activity[59]. In order to explain the structure-activity relationship, it is proposed that the electron transfer from TMB to the catalyst is the rate-determining step. It is of great significance for rational design of oxidase-like activity of nanomaterials in the future to systematically study the variation of catalytic mechanism with substrate and the structure-activity relationship and prediction model corresponding to different mechanisms.
1.4.3 Superoxide dismutase nanoenzyme
The SOD catalytic reaction of nanomaterials is as follows:
The reaction is a disproportionation reaction, in which two undergo intermolecular electron transfer in the presence of protons, giving electrons to form O2 and gaining electrons to form H2O2. Osuna et al. Used DFT calculation method to study the course and energy curve of the reaction catalyzed by C60(C(COOH)2)3, and Shen et al. Studied the course and energy change of the reaction on the surface of noble metal under gas phase conditions[60][61]. The above computational studies show that C60(C(COOH)2)3 and noble metals are feasible catalytic materials for this reaction, which explains the experimental results[62].
In order to develop a theoretical model for predicting the SOD-like activity of nanomaterials, Wang et al. Investigated the possible ways of intermolecular electron transfer of nanomaterials in solution conducting , and proposed two SOD-like catalytic mechanisms, the lowest unoccupied molecular orbital (LUMO) mechanism and the highest occupied molecular orbital (HOMO) mechanism, and proposed theoretical criteria respectively[63]. The LUMO mechanism means that the nanomaterial first grabs an electron from a molecular to convert it into a O2, and then transfers the electron to another molecular to convert it into a H2O2, while the nanomaterial returns to its original state. The LUMO (or conduction band bottom) of the nanomaterials plays a key role in the whole process (Fig. 9a). The criteria for nanomaterials to have LUMO-mediated SOD activity are:
φ(O2/) < φNM,LUMO < φ(,H+/H2O2)
φ in the above equation is the reduction potential related to . Correspondingly, the HOMO mechanism means that the nanomaterial first transfers an electron to a molecular to convert it into a H2O2, and then grabs an electron from another molecular to convert it into a O2, while the nanomaterial recovers its original state, and the HOMO (or valence band top) of the nanomaterials plays a key role in the whole process (Fig. 9b). The criteria for nanomaterials to have HOMO-mediated SOD activity are:
φ(O2/) < φNM,HOMO < φ(,H+/H2O2)
Fig. 9c shows the HOMO and LUMO energy levels of several nanomaterials, which can be used to judge whether they may have SOD-like activity and SOD-like mechanism.
Since the numerical values of φ(O2/ ) and φ(O2,H+/H2O2) can be obtained from chemical handbooks, and the HOMO and LUMO values of nanomaterials can be determined by experimental methods such as photoelectron spectroscopy and electrochemical methods,When the structure of the material is determined, the DFT calculation method can also be used, so the above criteria can be used not only to identify which kind of SOD catalytic mechanism the nanomaterial follows, but also to screen the nanomaterial with the corresponding SOD activity. Wang et al. Used this method to screen more than 121 materials from 3814 two-dimensional materials that only meet the condition formula (12) or only meet (13), and made an attempt in the direction of computer-aided high-throughput screening of nanoenzymes[63].
1.4.4 Catalase nanoenzyme
The overall reaction catalyzed by nanomaterials catalase is as follows:
This reaction is also a disproportionation reaction. DFT computational studies under gas-phase conditions show that H2O2 molecules can undergo atomic rearrangement reactions on CeO2, noble metal, and ferrihydrite nanosurfaces to form H2O and O2, completing the catalase-like catalytic cycle[64][38][65]. Among them, CeO2 follows the mechanism shown in Fig. 10 a, while noble metals and ferrihydrite follow the mechanism of Fig. 10 B.
Zhang et al. Found that ferrihydrite has specific catalase-like catalytic activity, which is different from most nanomaterials with both peroxidase-like and catalase-like catalytic activity.This is related to the fact that there are a large number of hydroxyl structures on the surface of ferrihydrite, and the catalase-like activity of ferrihydrite can be regularly regulated by regulating the surface hydroxyl abundance of ferrihydrate during synthesis[65]. DFT calculations show that during a complete catalase-like catalytic cycle, the iron hydroxyl group on the ferrihydrite surface promotes the cleavage of the oxygen-oxygen bond of a H2O2 molecule (basic decomposition) by creating H vacancies.When the second H2O2 molecule is close to the catalytic center, the hydrogen vacancy can easily abstract the hydrogen atom in hydrogen peroxide, thus promoting its breaking of the oxygen-hydrogen bond (acid decomposition) to produce O2[65].
While practical applications of nanozymes occur in aqueous solutions, accurate experimental characterization of the electronic structure of nanomaterials is often only possible in the gas phase or under vacuum conditions. For this reason, Wang et al. Proposed a theoretical model of CeO2 mimicking catalase catalysis under gas-phase conditions[64]. In aqueous solution, the reaction intermediate forms a polar chemical bond with the CeO2 and is easily ionized into the solution. However, the gas phase environmental conditions are different, and the lifetime of the intermediate product adsorbed on the surface of the material is longer, which leads to the formation of new energy levels in the forbidden band of the material. This level energy (EDS) can be used to predict CeO2 catalase-like activity: when φ(O2,H+/H2O2)<EDS<φ(H2O2,H+/H2O), CeO2 has catalase-like activity; The closer the height of EDS is to the middle of the two potentials, the stronger the activity is (Figure 10C). This model is also applicable to other semiconductor-type metal oxide materials. Liu et al. Experimentally characterized the electronic structure of metal-doped MnO2 nanocoatings, and the experimental results support the theoretical model[66].
1.4.5 Brief summary
Although the detection of free radicals by radical trapping agents is often used to study whether homogeneous catalysis is mediated by free radicals, in nanoenzyme catalysis, this method cannot identify whether the trapped free radicals are "free radicals" or reaction intermediates adsorbed on the nanosurface. Therefore, detection of free radicals with a radical trap cannot be used to determine that nanozyme catalysis is radical-mediated. Due to the lack of experimental methods to characterize the dynamic catalytic process in solution in situ, it is still difficult to directly characterize the structural information of the intermediates in the catalytic process. At present, the catalytic mechanism of nanoenzymes mainly relies on DFT calculations. Although computational studies have revealed a variety of nanoenzymatic catalytic mechanisms and successfully explained the corresponding experimental results, the validity of the proposed mechanisms still needs to be tested by extensive experiments due to the lack of direct experimental evidence of the mechanisms. In particular, experiments have proved that the catalytic mechanism of nanoenzymes can change with the change of nanomaterials and reaction substrates, and the change of reaction mechanism still needs further study. Improving the selectivity of nanoenzymes to substrates and reaction directions has important application prospects. Experimental studies have found that many nanoenzymes have certain specificity, and the competitive mechanism and rules of nanoenzymes in catalyzing different reactions need further study[48,65,67]. In addition to mimicking oxidoreductase, nanomaterials mimicking the functions of other types of biological enzymes have also attracted much attention, and the related catalytic mechanisms and activity rules should also be further studied[13,36].
In addition, the relationship between the microscopic catalytic mechanism and the apparent kinetics needs to be further established. For example, experimental studies have shown that most peroxidase nanozymes, like native peroxidases, follow the Michaelis-Menten equation for catalytic kinetics. Among them, Vmax in the Michaelis-Menten equation is related to the catalytic rate constant kcat of the nanoenzyme, and KM is related to the affinity of the catalyst for the substrate, both of which can be used to regulate the nanoenzyme catalytic activity. However, the intrinsic relationship between the macroscopically measured Vmax and KM and the microstructure of nanomaterials, such as specific surface area and size, as well as the microscopic catalytic mechanism described above, still needs to be further studied. The development of nanoenzyme catalytic kinetic equations based on the intrinsic characteristics of nanomaterials is of great significance for the optimization and design of nanoenzymes, and is a topic worthy of attention in the future.
Although DFT calculations have certain advantages in studying the catalytic mechanism of nanoenzymes, there are also the following problems. (1) DFT calculations often use single crystal materials as models, which is not consistent with the amorphous structure of nanoenzymes in most cases. Whether the calculation model fully represents the structure of the active site of real nanoenzymes is a problem. (2) Due to the limitation of computing power, the widely used generalized density approximation or local density approximation functionals are often used to select the calculation method for the unit cell, which produces errors because the electronic self-interaction is not taken into account. (3) Due to the limitation of calculation power, the contribution of solvent effect and entropy change to the reaction energy is often ignored in the calculation process, and the influence of reactant and product diffusion process on the catalytic efficiency is ignored. The above problems may have an impact on the final calculation results, so it is still necessary to combine computational simulation and experimental research to study the microscopic mechanism of nanoenzyme catalysis in the future. In addition, the future development of computational methods for larger nano-enzyme systems is also a direction worthy of attention, such as machine learning potential, QM/MM methods, which can extend the high accuracy of quantum mechanics to the molecular dynamics scale.The effects of larger scale system, complex surface structure, multiple catalytic centers, and solvent effect on the catalytic mechanism of nanoenzymes were comprehensively considered.
2 Design of nanoenzyme
As an interdisciplinary field oriented to practical applications, the research of nanozymes has always been carried out around their catalytic activity and physicochemical properties. Unlike enzymes produced by gene expression, nanozymes can be synthesized de novo and can be designed and modified. Therefore, how to develop effective strategies to achieve high-performance nanoenzyme design has been a core issue in the field of nanoenzyme research. In the early research of nanoenzyme, the strategy of random synthesis and batch-by-batch verification of activity was used to screen nanoenzymes with higher activity. However, due to the lack of clear theoretical guidance and high-throughput screening methods, this strategy is often time-consuming and laborious with little effect.
There are many kinds of nanoenzymes with diverse structures, and how to deeply analyze their structure-activity relationship is of great significance for the realization of evidence-based nanoenzyme design. In recent years, with the increasing amount of nanoenzyme research, some elements that have an important impact on the performance of nanoenzymes have been discovered and used in the rational design of nanoenzymes. In addition, based on the general structure-activity relationship between nanozymes and enzymes, the development of nanozymes with similar structural characteristics to the catalytic microenvironment and active site of enzymes has proved to be an effective strategy to improve the activity and selectivity of nanozymes. In particular, in recent years, with the development of single-atom material development technology, a series of single-atom nanoenzymes with unique catalytic sites have been designed and developed, and the catalytic performance of some single-atom nanoenzymes even surpasses that of natural enzymes with the same activity. In addition, with the progress of big data analysis, machine learning and theoretical computing, it is possible to design and optimize nanoenzymes by high-throughput simulation, which provides a new means for direct design and synthesis of available nanoenzymes.
In this section, the main strategies of nanoenzyme design are introduced, including the optimization of catalytic activity and selectivity, functional modification, and regulation of multi-enzyme activity, focusing on the relationship between key structural elements and nanoenzyme performance, as well as the proposed nanoenzyme design ideas, and looking forward to the feasible nanoenzyme design strategies in the future.
2.1 Regulation of catalytic activity of nanoenzyme
The catalytic activity of nanoenzymes is affected by many factors, among which the composition and structure of materials play a decisive role in the regulation of the activity of nanoenzymes. Different size, morphology, composition and surface modification will endow nanoenzymes with different enzyme-like activities. External factors in the catalytic system, such as solution pH, ions or molecules, light and heat, can also regulate the activity of nanoenzymes in the catalytic process. The influence of different factors on the activity of nanozymes essentially comes from two aspects: one is the influence on the number of active sites of nanozymes, and the other is the influence on the activity of a single active site. In the following, we will introduce how the composition, structure and external factors affect the number of active sites or the activity of a single active site of a nanoenzyme.
2.1.1 Influence of composition and structure
2.1.1.1 Size
The catalytic site of NANase needs to contact with the reaction substrate to play its role, so the number of active sites that can contact with the reaction substrate will directly affect the catalytic activity of NANase. The smaller the size of nanoenzyme, the larger the specific surface area, the more the number of active sites, and the higher the catalytic activity. In 2010, Fan et al. Used the glucose oxidase (GOx) activity of gold particles to catalyze the oxidation of glucose to produce H2O2, which can reduce chloroauric acid to achieve the seed growth of gold particles[71]. Due to the decrease of GOx activity of large-sized gold, the growth of gold particles was stopped, and the self-limiting growth of gold particles was ingeniously realized.
2.1.1.2 Morphology
In addition to the size, the morphology of the nanoenzyme also affects the number of active sites. In 2017, Mugesh et al. Showed that although the size was the largest, the flower-like Mn3O4 nanoenzyme showed the highest enzyme-like activity among five Mn3O4 nanoenzymes with different morphologies due to its high specific surface area and pore size[72].
The active sites on different crystal faces of the nanozyme exhibit different activities. In 2018, Mugesh et al. Prepared four V2O5 nanoenzymes with different morphologies, and confirmed that the (010) crystal face has higher glutathione peroxidase (GPx) activity combined with DFT calculation (Fig. 11A, B)[68]. Chen et al. Also explained the difference of OXD and POD activities of different crystal faces of Pd nanoenzyme through theory and experiment. The Pd cube with the main crystal face of Pd (100) showed better OXD and POD activities than the Pd octahedron with the main crystal face of Pd (111), and showed more efficient antibacterial performance when treated with Gram-positive bacteria[39].
2.1.1.3 Constituent
At present, according to the composition of materials, nanoenzymes can be divided into carbon nanoenzymes, metal oxide nanoenzymes, precious metal nanoenzymes and other types of nanoenzymes. Different kinds of nanozymes can change the activity of their active sites through specific composition regulation, thus realizing the regulation of catalytic activity.
Heteroatom doping of carbon nanoscale enzymes and simulation of the active site of native enzymes are common ways to improve the activity of enzymes. In 2018, Wei et al. Found that when heteroatom N was doped into reduced graphene oxide, H2O2 easily formed oxygen radicals near the N atom and was stabilized by the lone pair electrons of N, thus specifically improving the POD activity[67]. N-doping can increase the POD activity of reduced graphene oxide and mesoporous carbon by more than 100 and 60 times, respectively. Many carbon-based monoatomic nanoenzymes have achieved enzyme-like activity promotion by mimicking the active site of natural enzymes[73]. In 2019, the research of Academician Dong Shaojun's team found that the catalytic behavior of monatomic nanoenzyme with FeN5 active center constrained by carbon nanoframe is similar to that of heme coordinated by axial ligand of cytochrome P450, and the additional axial ligand N makes Fe-N5 have stronger electron push effect than Fe-N4, showing better OXD activity[74]. In 2020, Academician Yan Xiyun's team learned the role of cofactors in natural enzymes and designed peroxisome-like bodies co-acted by Fe-N4 and iron atom clusters[75]. The peroxisome has many kinds of enzyme activities, such as SOD, CAT, urea oxidase (UOD), POD and OXD.
For metal oxide nanoenzymes, element doping is also an effective way to regulate enzyme activity. In 2017, Chen et al. Doped different proportions of praseodymium into cerium dioxide. Because praseodymium has a lower conduction band position than cerium, praseodium-doped cerium dioxide is more conducive to accepting electrons from the reducing agent TMB, showing higher oxidase activity[76]. Hyeon et al. Also found that the doping of zirconium ions can change the proportion and conversion speed of trivalent cerium and tetravalent cerium in cerium dioxide nanoenzyme, thus obtaining cerium zirconium nanoenzyme with higher SOD activity[77].
Alloying is a common way to adjust the composition of precious metal nanoenzymes. The activity of the active site will be affected by the change of the geometric morphology of the active site of the raw material and the charge transfer between the original metal and the metal after the incorporation of different metals[78]. In 2010, Wu et al. Doped different metals in the preparation process of silver nanoparticles to form AgM (M = Au/Pd/Pt) alloy, and the AgM alloy showed composition- and ratio-dependent POD activity, confirming that the way of alloying can achieve effective regulation of catalytic activity[53]. Another alloying method of noble metal nanoenzyme is to form a core-shell structure. There are surface stress and ligand effect between the outer metal and the inner metal, which affect the central position of the d-band of the whole material, and then affect the activity of the active site[79]. In 2015, Xia et al. studied the growth of Ir outside Pd nanocubes. The expansion of Ir lattice caused by Pd significantly increased the POD activity of Pd @ Ir, and this change was related to the thickness of the outer metal (Figure 11c)[69].
2.1.1.4 Surface modification
In the working system, nanoenzyme is prone to aggregation due to the influence of salt ions and small molecules in the system, so it is necessary to enhance the stability of the colloid by surface ligand modification[80]. The modification of surface ligands can be performed either at the time of material synthesis or after the material synthesis[81]. In addition to stabilizing the particle, surface ligands can also change the number of active sites or the activity of a single active site, which is mainly achieved by changing the interaction with the substrate or adjusting the electron transfer of the nanoenzyme catalytic process. In this section, we will introduce the effect of surface modification on the catalytic activity of nanozyme.
If the coverage of the surface ligand only reduces the number of active sites of the nanoenzyme, the catalytic activity of the nanoenzyme will be significantly inhibited. In 2014, Bansal et al. Showed that the POD activity of gold nanoparticles was significantly reduced due to the shielding of the active site after the surface modification of nucleic acid aptamers, and this reduction in activity would be restored after the addition of aptamer-responsive molecules[82].
The effect of surface modification on the activity of single active site of nanoenzyme, on the one hand, comes from the regulation of substrate adsorption. In 2017, Yan et al. increased the affinity of Fe3O4 for substrate H2O2 by 10 times and the Kcat/KM by 20 times by introducing histidine residues into the surface of Fe3O4 (Fig. 11d)[70]. In 2021, Wu et al. Modified cysteine on the surface of gold nanorods, and the electrostatic attraction between the carboxyl group of the cysteine side chain and the protonated amino group of the substrate TMB could enhance the POD activity of gold nanorods, and this effect only occurred in the oxidation of the TMB substrate[83].
Surface ligands can also regulate the activity of the nanoenzyme active site by affecting electron transfer during catalysis. In 2016, Liu et al. Modified the surface of CeO2 with F-. F- can not only regulate the adsorption of chromogenic substrates through electrostatic interaction, but also promote the charge transfer in the catalytic process, thus enhancing the OXD activity of CeO2 by 100 times[84]. In 2020, Berret et al. Used polymers with different groups and side chain lengths to modify CeO2 after synthesis, and found that the effects of polymers on the activities of several oxidoreductases of CeO2 were not consistent[85]. The polymer may change the oxidation state of CeO2 through the interaction with the metal center, thus inhibiting its CAT and SOD activities, while the synergistic effect between the polymer and the particle can enhance its POD activity.
The composition and structure of nanoenzyme fundamentally determine its catalytic performance, and the size, morphology, composition and surface modification influence each other to regulate the catalytic type and efficiency of nanoenzyme. At present, the calculation of Gibbs free energy required for the reaction based on density functional theory can explain how the composition and structure of nanoenzymes regulate the catalytic activity in many cases, and also provide a reference for the rational regulation of nanoenzyme activity. The details have been introduced in Section 1.4.
2.1.2 Influence of external factors
External factors can act on nanozymes to change the number of active sites or the activity of active sites, and this effect is sometimes reversible. Common external factors include pH, ions, or molecules in the solution environment. In addition, combined with the physical and chemical properties of nanoenzymes, energy conversion can be achieved through external energy such as light energy, heat energy, sound energy and so on, which has an impact on the activity of active sites. The regulation of the catalytic activity of nanoenzyme by the above factors will be introduced below.
2.1.2.1 pH
The activity of the active site of nanozymes can be affected by pH. Generally, the POD activity of nanozymes is more efficient in acidic environment, and the CAT activity is more efficient in alkaline environment. In 2015, Gao et al. Used noble metal nanoenzyme as a model to analyze the mechanism of pH affecting the activity of nanoenzyme[38]. The results show that pH can change the decomposition process of reactants on the surface of precious metals. Under acidic conditions, H2O2 tends to undergo base-like decomposition on the metal surface, and the pre-adsorbed hydrogen will combine with the obtained OH to form products H2O* and OH *; However, under alkaline conditions, the pre-adsorption of OH makes H2O2 tend to undergo acid-like decomposition, and finally H2O* and O2* are obtained. At different pH, the noble metal nanoenzyme achieved the change between POD and CAT activity through different decomposition pathways (Fig. 12A).
2.1.2.2 Ion or molecule
In addition to the common H+ or OH- ions in solution, other ions or molecules artificially added can bind to the surface of the nanoenzyme, affecting the stability of the nanoenzyme itself or the binding between the substrate and nanoenzyme, thus affecting the number of active sites or the activity of a single active site. The binding of ions or molecules is affected by the properties of ions, molecules and the charge on the surface of nanoenzymes. For example, Guo et al. Have shown that different halide ions have different binding strength on the surface of gold nanoenzyme particles, which has different effects on their POD activity[88]. The inhibitory effect of F-, Cl-, Br-, I- on Au NANase increased in turn, similar to the reversible and irreversible inhibition of the native enzyme. They suggested that I- has a strong adsorption with the surface of Au nanozyme, which may block the binding sites on the surface of nanozyme. Binding to the active site is a common principle of inhibition, but it does not apply to all ions, and the specific principle needs to be analyzed in detail. For example, in 2011, Self's team found that phosphate ions could affect the catalytic activity by changing the valence of Ce on the surface of cerium nanoenzyme[89]. Phosphate ions make the surface of cerium nanoenzyme tend to + 3 valence, thus inhibiting the SOD activity of cerium nanoenzyme and increasing the CAT activity. In 2017, the research group selected four ions similar to phosphate ions, including tungstate, molybdate, sulfate and selenate, to explore their effects on the activity of cerium nanoenzyme[90]. Their results showed that these four ions had no effect on the surface valence, tungstate and molybdate could inhibit the phosphatase activity by inhibiting the interaction between ATP substrate and nanoenzyme, but did not affect the CAT activity, while sulfate and selenate had no effect on the activity.
Free small molecules in solution, such as ATP, ADP, AMP and other adenosine molecules, can also bind to the surface of the nanoenzyme[86,91,92]. Such small molecules can enhance the activity of nanoenzyme POD by promoting the activation of H2O2 and enhancing the interaction between hydroxyl radicals and nanomaterials (Fig. 12b). When macromolecular substances (such as proteins) are bound to the surface of nanoenzymes, the binding between substrates and nanoenzymes is usually affected by large steric hindrance, which inhibits the activity of nanoenzymes. In 2013, Self et al. Found that bovine serum albumin, which is commonly used in cell culture, can inhibit the phosphatase activity of CeO2 nanozyme (Fig. 12C)[87]. When nanozyme is injected into the blood for in vivo therapy, it will face a large number of proteins, which will form a protein crown on the surface of nanozyme, which may lead to irreversible activity inhibition and seriously hinder the application of nanozyme in vivo. Therefore, the effect of protein corona on the activity of nanozyme in vivo is a concern. In response to this phenomenon, Rotello et al. Developed a nanoenzyme activation strategy with protein corona in 2020, which uses intracellular proteases to hydrolyze protein corona to activate the activity of nanoenzyme in cells[93].
2.1.2.3 External energy
The ion or molecular activity regulation method described above is mainly through direct contact with the nanoenzyme to produce the effect, and by using the physical and chemical characteristics of the nanoenzyme itself, the activity of the active site can be remotely regulated by energy conversion. For example, many metals have the characteristics of surface plasmon resonance, which can convert light energy by electronic oscillation. At present, a variety of noble metal nanoenzymes such as Au nanoenzyme and Pd @ Au nanoenzyme have been synthesized, and the activity of the active site is improved through the surface plasmon resonance effect under the irradiation of near-infrared light[94,95]. In addition, materials with photoresponsiveness can also be modified on nanozymes to achieve remote photoregulation. For example, when the nanoenzyme is modified with an azobenzene material responsive to ultraviolet light, the cyclodextrin can be connected to the surface of the nanoenzyme through the azobenzene to block the active site on the surface of the nanoenzyme, and the allosteric effect of the azobenzene after ultraviolet irradiation promotes the release of the cyclodextrin, so that the activity of the nanolase is restored[96].
Other energies, such as sound energy and thermal energy, can also affect the activity of nanoenzymes. This remote regulation may enhance the activity of a single active site by increasing the thermal motion of reactants near the active site, or affect the number of active sites by regulating the coverage of molecules at the active site. In 2020, Lin et al. Synthesized a disordered graphite-structured nanoenzyme by carbonization, which can exert the highest enzyme activity when the temperature rises to 60 ℃. With the help of the photothermal conversion ability of graphene itself, the specific activation of nanoenzyme in tumor therapy can be achieved by near-infrared light[97]. In 2019, Yang et al. Synthesized PtCu3 nanoenzyme, whose metal atoms are highly ordered and have the characteristics of sonosensitizer, which can enhance the activity of POD under ultrasonic stimulation[98]. In 2020, Zheng et al. Attached a sonosensitizer T790 to the surface of Pd @ Pt nanoenzyme. T790 will hinder the CAT activity of Pd @ Pt, and the enzyme activity can be restored after ultrasound stimulation, so as to achieve controllable targeted therapy at the tumor site. The stronger penetration ability of ultrasound makes up for the lack of penetration of light energy regulation in vivo[99].
The regulation of nanoenzyme activity by external factors creates conditions for a variety of applications of nanoenzymes. The regulation of enzyme activity by ions or molecules can realize the high activity detection of these ions or molecules, control the activity of nanoenzymes under different conditions, and realize the controllable treatment of diseases. Specific applications will be described in detail in Section 4.
The catalytic activity of nanoenzyme is affected by many factors in the material itself and the external solution, which regulate the activity of nanoenzyme by affecting the number of active sites or the activity of a single active site, so these two aspects are the fundamental factors affecting the activity of nanoenzymes. In the study of nanoenzyme activity, how to determine the number of active sites is very important for the objective evaluation of nanoenzyme activity and the analysis of the source of activity change. Recently, Liu Juewen's research group put forward the view that it is more scientific to use surface atoms as active sites when comparing the activity of nanoenzymes, and also suggested that the author should provide as comprehensive as possible when comparing the activity of nanoenzymes[100][34].
The specific process of activity regulation is specific to the type of nanoenzyme, and a systematic understanding of how these factors regulate the catalysis of nanoenzymes and appropriate optimization according to different types of nanoenzymes can provide ideas for improving the catalytic efficiency of nanoenzymes. Different from the current preparation of nanoenzymes mainly by trial and error method, through the statistics and analysis of previous research experience, using the existing nanoenzymes to establish a database, combined with a variety of data analysis methods in machine learning to provide guidance for the design of nanoenzymes, it is expected to promote the research of nanoenzymes to further develop in the direction of rational design[16].
2.2 Selective regulation of nanozyme
Strict selectivity for substrates and reaction paths is one of the important features of enzymes. The vast majority of enzymes act only on a certain class of substrates and catalyze their specific chemical reactions. In contrast, nanozymes lack catalytic specificity. At present, no nanoenzyme that strictly catalyzes a specific reaction with a specific substrate has been synthesized or discovered. On the one hand, the substrate selectivity of nanozyme is poor. For example, peroxide nanozymes are often able to catalyze the oxidation of a variety of organic molecular substrates, such as TMB, OPD, ABTS, and luminol. On the other hand, the nanozyme reaction path selectivity is poor. For example, some nanozymes have both peroxidase-like and catalase-like catalytic activities, which can simultaneously catalyze the decomposition of H2O2 to produce ·OH or O2. Therefore, most nanozymes exhibit pan-catalytic properties with multiple substrates and multiple activities.
The broad substrate spectrum makes nanozymes have certain advantages in wastewater treatment, antibacterial, tumor inhibition and other applications with broad-spectrum catalytic requirements. It is also possible that the multiple activities of nanozymes cooperate with each other through cascades to perform diverse catalytic functions. However, disordered catalysis, which lacks selectivity, may produce unhelpful by-products during the reaction. In addition, the competitive inhibition caused by multi-substrate and multi-enzyme activity also makes the single activity of nanoenzyme not high. Therefore, it is difficult for nanoenzymes to make a difference in the application field requiring high selectivity for catalysis. Especially in industrial catalysis, the selectivity of the catalyst is often more important than the activity. Therefore, it is necessary to study and improve the catalytic selectivity of nanozymes so that they can meet the needs of multiple applications.
The main reason for the poor catalytic specificity is that nanozymes lack domains to specifically recognize and bind substrates, as well as catalytic sites that are finely regulated by the catalytic microenvironment. The reaction catalyzed by an enzyme generally involves three steps: substrate binding, substrate conversion, and product release. In the substrate-binding stage, the structural complementarity of the enzyme to the substrate forms the basis of substrate selectivity. In the process of binding with the substrate, the conformation of the enzyme is induced by the substrate to change, so that the active center structure fits the substrate structure, thus specifically catalyzing the substrate reaction. In addition, the active center of the enzyme is finely regulated by the microenvironment, which enables the substrate bound to the enzyme to be converted into a specific product through a specific reaction path. For example, peroxidase, oxidase and catalase, which also use iron porphyrin as a catalytic group, show completely different specific catalytic effects due to the differences in the microenvironment of the substrate binding site and the active center. The reaction steps catalyzed by nanozymes are similar to those of enzymes. The difference is that the catalytic site of most nanozymes is directly exposed on the rough surface, but lacks the binding pocket that can specifically recognize and bind to the substrate and the catalytic microenvironment that can precisely regulate the electron/group transfer. Therefore, although many nanoenzymes have porphyrin-like structures, they are difficult to show the same catalytic specificity as porphyrin-containing enzymes.
In view of this, the selective study of nanoenzymes involves the use of molecular imprinting, physical and chemical adsorption, surface modification and other strategies to create binding sites.A series of secondary interactions (hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals forces, etc.) Are introduced to establish structural complementarity between the nanoenzyme and the substrate, so as to realize the selective recognition and binding of the nanoenzyme to the substrate. On the other hand, through the biomimetic design strategy and the creation of catalytic microenvironment based on nanostructure-activity relationship, the integrated effect and cascade reaction are used to amplify specific catalytic reactions to optimize the catalytic reaction path of nanoenzymes. In this section, the main strategies and progress of selective regulation of nanoenzymes are introduced.
2.2.1 Molecular imprinting
In order to establish structural complementarity between nanoenzyme and substrate, many techniques have been applied in the field of nanoenzyme synthesis. Among them, molecular imprinting is considered to be one of the most potential and convenient methods, which can create three-dimensional network template molecules with specific shapes and functional group positions to achieve the recognition of specific substrates without the introduction of other substances. The synthesis of molecularly imprinted nanoenzyme generally includes the following three steps: (1) adsorption of substrate template and functionalized monomer on the surface of nanoenzyme; (2) polymerize that functionalized monome around the nanoenzyme to produce a cross-linked matrix; (3) The substrate template is removed, leaving a binding pocket complementary to the substrate structure. For example, nanogels with high selectivity can be constructed by adsorbing TMB or ABTS on the surface of Fe3O4 nanoenzyme, which can be used as a template for crosslinking, precipitation and polymerization with acrylamide monomer. The nanogel after substrate elution retained the Fe3O4 catalytic center and the binding pocket and steric hindrance matching the size and shape of the substrate molecule. Therefore, this molecularly imprinted nanozyme exhibits excellent affinity and high selectivity. The catalytic specificity of the molecularly imprinted Fe3O4 peroxiredoxin nanozymes was increased nearly 100-fold compared with that of the Fe3O4 nanozymes. Correspondingly, its peroxidase-like activity was also significantly increased[101]. Studies have shown that this increase in activity may be attributed to the local substrate concentration enrichment and the lower activation energy of the imprinted polymer[102]. Using similar strategies, PtPd peroxiredoxin and g-C3N4 peroxiredoxin have been reported to achieve specific catalysis of TMB substrates[103][104]. Diversified molecularly imprinted NANases can be designed by varying the substrate template, functionalized monomer, and type of NANase. For example, the Fe3O4 peroxide nanoenzyme developed by using methyl blue as a substrate template and polypyrrole as a molecularly imprinted polymer has excellent catalytic selectivity for methyl blue[105]; MIL-53 (Fe) peroxide nanoenzyme, developed with metronidazole as the substrate template and dopamine as the functionalized monomer, can specifically recognize and catalyze metronidazole[106]. With the advantages of variable template molecule, strong substrate specificity and improved catalytic performance, various types of molecularly imprinted polymer nanoenzymes have been widely designed and applied in the fields of biosensing and molecular detection. For example, heme as the catalytic center and different substrates as the template can be used for the specific determination of 2,4,6-trichlorophenol and serotonin[107][108]; In combination with metals, researchers have developed a biomimetic enzyme-linked immunosorbent assay platform based on molecularly imprinted polymers for the detection of serum samples and the determination of histamine in food[109,110][111]. In recent studies, molecularly imprinted polymer nanoenzymes based on Co and Fe bimetallic active sites have been used for dual-mode detection, which has better peroxidase-like activity than single-metal nanoenzymes. Vanillin was used as a template molecule to synthesize molecularly imprinted polymers, which showed excellent substrate specificity and selective catalytic ability[112]. The combination of molecular imprinting and nanozyme solves the defect of lack of specificity of nanozyme and correspondingly improves its catalytic activity, which opens up a new horizon in the field of nanozyme. Although a considerable number of molecularly imprinted polymer nanoenzymes have been successfully designed, the existing research on their binding efficiency and related thermodynamics and kinetics is still shallow, and how to better regulate the selectivity of nanoenzymes and carry out practical applications needs more in-depth and lasting research.
2.2.2 Hydrophilic-hydrophobic interaction
Enzymes achieve high catalytic activity and selectivity by creating favorable microenvironments around the active center, such as hydrophobic pockets, which may interact with substrates or products. Therefore, people began to pay attention to the influence of hydrophilic/hydrophobic environment on the catalytic activity and substrate selectivity of nanozyme. A series of oxidized nanoenzymes with different physical characteristics were constructed by using hexagonal mesoporous silica as the framework, Co3O4 nanoparticles as the active center, and phenyl as the organic ligand. The increase of phenyl content can change the hydrophilic/hydrophobic microenvironment of the active center. The experimental data showed that the catalytic performance of the nanoenzyme for ethylbenzene oxidation was significantly improved with the increase of the hydrophobicity of the microenvironment, which was attributed to the enhanced ethylbenzene adsorption by the porous hydrophobic microenvironment[113]. Up to now, many studies have shown that providing a hydrophobic microenvironment for NANases can enhance the recognition and binding ability of substrates. In the field of inorganic catalysis, hydrophilic/hydrophobic interactions are often used in phase-selective catalysis. Therefore, introducing interactions between NANases and substrates is an effective strategy to achieve selective regulation of NANases[114⇓~116][117,118].
2.2.3 Physical and chemical adsorption
Physical/chemical adsorption is an inevitable step for the substrate to contact with the nanoenzyme and start the catalytic reaction, so using or enhancing this process is a simple and effective strategy to regulate the selectivity of nanoenzymes. For example, a biosensing platform can be constructed based on the difference in affinity of nanozymes for single-stranded DNA and double-stranded DNA[119,120]; Efficient enzyme-like catalytic activity and substrate selectivity are achieved based on the coordination structure between the transition metal and the nitrogen atom, which can selectively interact with the substrate and form a critical bound oxygen intermediate[121]. Similarly, electrostatic interaction as a stronger interaction force was applied to the adsorption process of NANase with substrate. For example, a series of nanosystems with excellent enzyme-like catalytic properties can be prepared by crosslinking gold clusters encapsulated by cationic cages inside porous polyionic liquids.The external polyionic liquid is negatively charged, so the formed supramolecular complex can mimic the substrate sorting behavior of natural enzymes through electrostatic interaction, and can also significantly improve the catalytic performance[122]. Selective regulation of nanoenzymes based on physical/chemical adsorption has the characteristics of coordination and high applicability, but its weak binding force and low sensitivity limit its further application to a certain extent.
2.2.4 Surface modification
The catalytic reaction usually occurs on the surface, involving three steps of substrate adsorption, conversion and release, so the surface groups and composition of nanoenzymes will affect the catalytic process, and the regulation of nanoenzyme activity and selectivity is also crucial. The selective catalysis of the nanoenzyme can be realized by surface modification of an antibody, for example, the Fe3O4 nanoenzyme is coupled with an antibody against rotavirus and human epidermal growth factor receptor 2 (EGFR2) to realize the specific detection of a corresponding antigen[123]; Co3O4 nanozyme with peroxidase-like activity and catalase-like activity was conjugated with EGFR single domain antibody for immunohistochemical analysis[124]. The coupling of the nanoenzyme to the antibody described above can be achieved simply by chelation or a coupling agent. Therefore, selective recognition and catalysis of different substrates by nanozymes can be achieved only by changing the antibody species. However, antibody preparation is cumbersome and expensive, which correspondingly increases the cost of nanoenzyme industrialization. Aptamers developed in recent years are a hot topic in nanoenzyme surface modification. Compared with traditional antibodies, they not only have high affinity for target molecules, but also have the advantages of low cost, high stability, easy modification and strong immunogenicity. Huang Pengcheng and Wu Fangying combined aptamer with gold nanozyme with peroxidase-like activity to construct a tetracycline detection platform with high specificity and sensitivity[125]. Specific binding of the aptamer to tetracycline alters the catalytic behavior of this nanozyme, thereby enabling recognition and detection of specific substrates. The same strategy has also been used for the detection of exosome proteins and kanamycin, that is, the surface modification of NANase can selectively bind to a variety of substrates by simply replacing the antibody species or the target aptamer, which has broad application prospects[126,127].
2.2.5 Chiral recognition
Chiral discrimination of substrate enantiomers is an important aspect of natural enzyme specificity, yet only a very small number of nanozymes exhibit selectivity and stereospecificity. Therefore, it is an effective strategy to regulate the selectivity of nanozymes by introducing chiral molecules. Most biomolecules in life exhibit chirality, such as the left-handed property of amino acids, the right-handed structure of DNA, etc., and in fact, these molecules have been widely used in the synthesis of chiral nanozymes. For example, gold nanoparticles with glucose oxidase-like activity can be synthesized by using DNA as an environmentally responsive chiral ligand. DNA in the random coil state has a higher affinity for L-glucose, while DNA in the stacked state (duplex, G-quadruplex, etc.) preferentially recognizes D-glucose, and this structural change can be triggered by metal ions to achieve stereoselective catalysis regulated by NANase[128]. The L-phenylalanine modified cerium oxide nanoparticles showed high enzyme-like activity and more effective stereoselectivity for D-DOPA[129]. Amino acid-modified NANase has been reported to exhibit catalytic activity and stereoselectivity beyond the native enzyme, demonstrating the feasibility of amino acid ligands to endow NANase with selective catalytic ability and improve catalytic activity[130][131]. Considering the importance of chiral selective catalysis in biomedicine, various chiral nanozymes have been developed and applied in fields such as asymmetric catalysis and molecular detection[132⇓⇓~135]. Recent studies have also paid more and more attention to the optical and biological effects of chiral ligands, and it can be predicted that the application fields of chiral nanoenzymes with stereoselectivity will continue to be expanded.
2.2.6 Bionic design
Over a long period of evolution, enzymes have developed active center structures that are optimal for substrate binding and specific catalysis. In particular, the catalytic microenvironment of the enzyme active center can precisely regulate the activity and selectivity of the catalytic reaction. Inspired by this, some studies have used rational design strategies to simulate the active center structure of enzymes to create nanoenzymes and achieve the optimization of nanoenzyme selectivity. In 2017, by simulating the amino acid microenvironment of HRP, Van Kron and others first introduced the concept of biomimetic design into the activity and selectivity optimization of nanoenzymes. At the structural center of HRP, a histidine (His42) located axially distal to the iron porphyrin plane can assist the positioning of H2O2 into the active center pocket through hydrogen bonding and promote the generation of the enzyme-substrate initiation intermediate state (complex I). Inspired by this specific structure, researchers found that the introduction of histidine on the surface of Fe3O4 nanozymes could significantly improve their affinity for H2O2 and enhance their peroxidase-like catalytic activity[70]. In addition, histidine contains an imidazole group, which plays an important role in the nucleophilic attack of the substrate and usually acts as a catalytic group in the active center of hydrolases. Based on this, Lou Wenyong et al. Successfully constructed a series of biological nanoenzymes with intrinsic hydrolase activity, which can catalyze the degradation of p-nitrophenylacetate, by incorporating histidine into the engineered peptide self-assembly system driven by zinc ions[136]. In addition to providing amino acid microenvironment, mimicking the coordination structure of the active center of natural enzymes is also one of the strategies for biomimetic design of nanozymes. Metal-organic frameworks (MOFs) have independent metal sites and surrounding ligands, which are similar to the structure of metalloproteases, so they have natural advantages in the field of mimetic enzymes. Based on the tetrahedral coordination structure of the active center of human carbonic anhydrase II and the double copper ion center of the active site of catechol oxidase, Dong Shaojun et al. Constructed ZIF-8 nanoenzyme that can effectively promote the hydration of CO2 and MOF-818 nanoenzyme that selectively catalyzes the oxidation of o-diphenol, respectively, which has higher catalytic activity and specificity than the same type of nanoenzyme[137,138]. Based on the multi-copper coordination structure of natural laccase, Cu-MOF synthesized with nucleotide as ligand can show excellent laccase-like activity and specific catalysis for phenol-containing substrates[139]. It can be seen that based on the general structure-activity relationship between enzymes and nanoenzymes, exploring and simulating the structural characteristics of enzymes is a feasible means to optimize nanoenzymes.
2.2.7 Integrated nanoenzyme
Complex but specific enzyme catalytic systems widely exist in living systems, which inspires us to construct similar nanoenzyme catalytic platforms to achieve substrate selectivity. Wei Hui's research group proposed the concept of integrated nanoenzyme (INAzyme), that is, by combining a variety of nanoenzymes in a certain limited space, the overall enzyme-like catalytic efficiency and substrate selectivity can be greatly improved[140]. This improvement is usually considered to be the result of the confinement effect to increase the substrate concentration, the proximity effect to facilitate the mass transfer, and the cascade reaction to accelerate the reaction rate. Zhang yuanjian's research group designed a nanoenzyme catalytic platform based on microfluidic devices, introducing two rationally designed nanoenzymes in a confined space to achieve cascade reactions and substrate selection[141]. In the case of carbon nitrogen nanocages (oxidase-like activity) and Prussian blue nanoparticles (peroxidase-like activity), the first order reaction generates the byproduct H2O2 via a two-electron pathway and serves as a substrate for the second order reaction to initiate a cascade catalytic reaction. The researchers screened a variety of small molecule substrates and found that only ascorbic acid could be oxidized to the end product, reflecting a significant increase in substrate selectivity. Therefore, this reaction platform was applied to the detection of ascorbic acid, and its sensitivity was increased by hundreds of times, which successfully realized the highly selective catalytic reaction based on nanoenzyme. In fact, similar studies have made some progress in the fields of in vitro detection, biosensing, and disease treatment[142]. Although the underlying mechanism and reaction pathway of cascade reactions still need to be further studied, the emergence of integrated nanoenzymes not only broadens the idea of improving substrate selectivity, but also is an effective supplement to nanoenzyme catalytic system, which has broad prospects for development.
2.2.8 Brief summary
In this section, several common strategies for selectivity optimization of nanoenzymes are discussed, mainly including molecular imprinting, surface modification, biomimetic design, and integration effect to achieve structural complementarity or proximity effect between nanoenzymes and substrates. In these strategies, the type of ligand is critical for the substrate selectivity of the nanozyme. By selecting a suitable ligand as the substrate binding domain of the nanoenzyme, the recognition and binding ability of the nanoenzyme to the substrate can be effectively improved, and thus the catalytic activity of the nanoenzymes to a specific substrate is improved. However, it is undeniable that although some progress has been made in the study of the selectivity of nanozymes, they are still far from the strict specificity of enzymes. Similar to enzymes, the catalytic process of nanozymes involves steps such as substrate binding, substrate conversion, and product release. In addition to ligand-mediated substrate binding, the structure of some nanoenzymes also has an important impact on substrate binding and conversion. For example, the unique nanostructure of ferrous disulfide peroxide nanoenzyme has a high affinity for H2O2[143]. The surface iron-associated hydroxyl group of ferrihydrite NANase has an important effect on the cleavage of H2O2 to produce O2[65]. In order to further optimize the selectivity of nanoenzymes, on the one hand, future research should screen new highly selective ligands to mediate the highly selective binding of nanoenzymes to substrates.On the other hand, it is necessary to clarify the key structure-activity relationship of nanoenzymes that affects the substrate conversion path. The former affects the substrate selectivity of nanoenzymes, while the latter determines the specificity of nanoenzyme-catalyzed reactions. In addition to improving the selectivity of nanozymes, how to use the pan-catalytic characteristics of nanozymes to develop unique applications, as well as to study the structure-activity relationship and regulatory means between multi-catalytic activities, are also important directions for future research.
2.3 Multifunctional properties of nanozymes.
Nanoenzyme is a kind of nanomaterials with enzymatic properties. As a new type of mimetic enzyme, nanoenzyme is different from natural enzyme and traditional mimetic enzyme, which has the "dual identity" of enzyme and nanomaterials, and its greatest feature is that it not only has catalytic function, but also has the unique physical and chemical properties of nanomaterials, which makes nanoenzyme a bifunctional or multifunctional molecule. Scientists are skillfully combining the catalytic activity and physicochemical properties of nanoenzymes to create many unique new functions, which can be applied to environmental monitoring, biosensors, disease diagnosis and treatment, and other fields. This section will focus on the study of multifunctional properties of nanozymes and their applications.
2.3.1 Introduction
The rapid rise of the nanoenzyme field is due to its unique advantages. As a new type of mimetic enzyme, compared with natural enzymes, nanozymes have the advantages of economy, stability, easy modification and self-assembly, and activity can be adjusted by size/structure factors[11]. More importantly, nanoenzyme also has the unique physical and chemical properties of nanomaterials, such as Fe3O4 nanoenzyme not only has peroxidase activity, but also has superparamagnetism; Molybdenum disulfide (MoS2) nanoparticles not only have catalytic activity, but also have near-infrared photothermal properties, which makes nanoenzymes a bifunctional or multifunctional molecule. Many scientists have skillfully combined the catalytic activity of nanoenzymes with their physical and chemical properties to create many unique new functions, which have been well applied in the biomedical field.
2.3.2 Magnetic binding of enzyme activity to materials
The superparamagnetism of nanomaterials endows them with two functions: first, they can be used as contrast agents for magnetic resonance imaging (MRI) for in vivo imaging of cells and tissues; second, they can enrich the molecules and cells adsorbed by nanomaterials through an external magnetic field, thereby increasing the concentration of molecules or the density of cells.
Iron oxide is a typical superparamagnetic material, which can be used in magnetic resonance imaging. It has high biosafety and biocompatibility, and has become the first generation of inorganic nanomaterials approved by the Food and Drug Administration of the United States[144][145]. Chen et al. Found that superparamagnetic iron oxide nanomaterial-Ferucarbotran can not only effectively enter mesenchymal stem cells to realize nuclear magnetic imaging of cells, but also exert its nanoenzyme activity to remove intracellular H2O2, thus promoting the growth of stem cells, which not only realizes the tracking of stem cells, but also promotes the growth of cells[146]. Wang et al. Further used Fe3O4 nanomaterials for MRI imaging of tumor tissues, while killing tumors by reactive oxygen species (ROS) generated by their enzymatic activity[147].
Superparamagnetism of Fe3O4 nanozymes has also been widely used for the enrichment of molecules and cells[1,148,149]. For example, in immunoassay, the antigen adsorbed by magnetic nanomaterials is enriched by external magnetic field, so as to improve the sensitivity of detection. Based on this property and the signal amplification catalyzed by reduced graphene oxide/molybdenum disulfide (rGO/MoS2) composite and Fe3O4 nanoenzyme, Tian et al. Developed an ultrasensitive electrochemical detection strategy for circulating tumor cells[150]. Immunomagnetic beads were coated on the surface of a magnetic glassy carbon electrode by inserting a magnet for electrochemical cell sensing. The synergistic catalysis of rGO/MoS2 composite with Fe3O4 nanoenzyme for H2O2 showed good electrocatalytic activity. Utilizing rGO/MoS2 and Fe3O4 nanozymes as electrochemical signal indicators and enhancers to fabricate biosensors would eliminate the need for additional redox mediators to amplify detection sensitivity.
The magnetic effect of nanoenzyme also has very high application value in vivo. Murohara et al. Incubated induced pluripotent stem cells with Fe3O4 nanoenzyme, and formed a multilayer cell plate under the action of an external magnetic field. The mechanical properties of the formed cell plate were enhanced, and the Fe3O4 could also exert its enzyme activity and produce antioxidant function, thus promoting the growth of induced pluripotent stem cells[149]. Shen et al. Reported a mitochondria-targeted magnetocaloric nanoenzyme (Ir@MnFe2O4NPs) for highly effective cancer therapy[151]. Iridium (Ⅲ) complex (Ir) acts as a mitochondrial targeting agent on the surface of MnFe2O4NPs. Upon exposure to an alternating magnetic field, Ir@MnFe2O4NPs causes a local temperature increase that leads to mitochondrial damage (MHT effect). This nanoplatform combines these excellent therapeutic properties with two-photon microscopy (confirmed in vitro) and magnetic resonance imaging (confirmed in vivo), which can precisely and effectively treat cancer.
2.3.3 Enzyme activity combined with metal plasmonic properties
The plasma property of metal means that the freely vibrating electrons existing on the metal surface interact with photons to produce surface electromagnetic waves propagating along the metal surface. This electromagnetic wave makes metals have special optical properties, such as surface-enhanced Raman scattering (SERS) and plasmon resonance (SPR).
SERS effect refers to the phenomenon that the Raman scattering signal of adsorbed molecules on the surface of some special good metal conductors (such as gold, silver, copper) is greatly enhanced compared with the ordinary Raman scattering signal due to the enhancement of the electromagnetic field on or near the surface of the sample. Scientists have built an interesting sensing system for the detection of a variety of substances by using the nanoenzymatic activity and SERS effect of gold and silver nanoparticles. For example, Wei et al. Ingeniously combined the HRP enzyme activity of gold (Au) nanoparticles with the SERS effect to couple glucose oxidase and lactate oxidase to form a cascade reaction.The H2O2 generated by oxidizing the substrate by the oxidase and the nanoenzyme jointly catalyze the TMB to generate an oxidation product as a probe of a Raman signal, and real-time and high-sensitivity detection of the substrate is realized according to the intensity of the Raman signal of the probe[152]. The method can be used for the detection of glucose and lactic acid in brain and tumor tissues, and provides a new method for the medical detection of biological samples. Similarly, silver nanoparticles also have both nanoenzymatic activity and SERS effect, which can be used for highly sensitive detection of H2O2 by catalyzing Raman signal probes[153].
SPR is a special optical physical phenomenon. When the size and shape of a material with this property change, its SPR spectral properties change. Fan et al. Constructed a novel detection system by combining the GOx activity of Au nanoparticles with their SPR properties[154]. The H2O2 generated by the oxidation reaction of GOx and the HAuCl4 form Au particles, which are attached to the surface of Au nanomaterials and change their size and shape, thus changing their SPR optical properties. However, single-stranded and double-stranded DNA have different affinities for Au particles, resulting in different blocking effects on their enzymatic activity. Therefore, the system can specifically detect single-stranded and double-stranded DNA in real time based on the change of SPR properties of individual gold nanoparticles (fig. 14).
2.3.4 Combination of enzyme activity and photothermal properties
In recent years, nanoenzyme-photothermal therapy has attracted great interest because of its enhanced therapeutic efficacy and fewer adverse effects in the treatment of infectious diseases. Near-infrared photothermal properties refer to the properties of materials with high photothermal conversion efficiency, which convert light energy into heat energy under the irradiation of near-infrared light, so that materials can be used to kill cancer cells and bacteria. Molybdenum disulfide (MoS2) and polypyrrole/hemin (PPy/hemin) nanomaterials have good near-infrared photothermal properties, which can be combined with their own enzymatic activities to achieve the synergistic application of multiple functions of nanoenzymes[155,156].
Zhao et al. Developed a synergistic antibacterial system using PEG-MoS2 nanozymes (Fig. 15)[155]. The antibacterial mechanism comes from two aspects: :(1)MoS2 has horseradish peroxidase activity, which can catalyze low concentrations of H2O2 to produce ·OH, avoiding the toxic and side effects of high concentrations of H2O2 on normal tissues, and ·OH has higher antibacterial ability. (2) The excessive heat produced by near-infrared light irradiation can accelerate the oxidation of glutathione in bacteria, thus destroying the protective mechanism of bacteria and accelerating the death of bacteria.
However, due to the limitation of size effect, the development of nanoenzyme-photothermal agents that can rapidly, effectively, and synergistically combat pathogenic bacteria remains a great challenge. Wang et al. Fabricated a novel metal-organic framework material (MOF) -derived hybrid nanoenzyme antibacterial strategy by modifying platinum nanoenzyme on zinc-based photosensitizer for enhanced photothermal therapy[157]. In the presence of low concentration of H2O2, the MOF-derived hybrid nanoenzyme has excellent nanocatalytic activity, which can effectively catalyze the release of H2O2 to generate toxic ·OH, thereby improving bacterial membrane permeability and thermal sensitivity. Once the near-infrared laser is introduced, the nanoenzyme-photothermal antimicrobial platform can play the role of a "nanoknife" to further induce the death of damaged bacteria through physical cleavage.
Li et al. Reported a novel nanozyme (PtFe@Fe3O4) with dual enzymatic activities for efficient tumor-catalyzed therapy[158]. PtFe@Fe3O4 exhibits an intrinsic photothermal effect as well as light-enhanced peroxidase- and catalase-like activity in the acidic tumor microenvironment, thereby effectively killing tumor cells and overcoming tumor hypoxia. Although photothermal therapy is a promising cancer treatment, overheating will inevitably damage the normal tissues near the tumor, and the damage of mild hyperthermia to cancer cells is easily repaired by stress-induced heat shock proteins. Therefore, it is necessary to simultaneously maximize photothermal efficiency and minimize damage to healthy tissue by employing an appropriate treatment temperature. Chang et al. Proposed a new concept of iron death promoting mild temperature photothermal therapy using palladium single-atom nanoenzyme Pd SAzyme as a model[159]. NANase with POD and glutathione oxidase (GSHOx) mimicking activity and photothermal conversion properties, which can cause iron death, is characterized by up-regulation of lipid peroxide (LPO) and reactive oxygen species.
2.3.5 Combination of enzyme activity and photoacoustic phenomena
Some nanoenzymes or catalytic products have a photoacoustic effect, which can generate ultrasonic waves when exposed to a very short duration (about nanoseconds) pulsed laser beam in the near-infrared region (wavelength 700 ~ 1000 nm).
Yan et al. Synthesized an exosome-like graphene quantum dot nanoenzyme vesicle for in vivo H2O2 response photoacoustic imaging (PAI) of nasopharyngeal carcinoma[160]. The intrinsic peroxidase-like activity of the graphene quantum dot nanoenzyme efficiently converts ABTS to its oxidized form in the presence of H2O2. Oxidized ABTS exhibits strong near-infrared absorbance, making it an ideal contrast agent for PAI. This study provides interesting insights into how to design PAI contrast agents based on carbon nanoenzyme, which provides new ideas and techniques for the diagnosis of nasopharyngeal carcinoma.
Wu et al. Reported a ROS-sensitive nanoenzyme-enhanced photoacoustic nanoprobe for in vivo imaging of acute liver failure (ALF)[161]. Due to the synergism between nanoenzyme-catalyzed bubble generation and thermoelastic expansion, the deep penetrating acoustic signal of the nanoprobe can be activated by ROS overexpressed in ALF. Impressively, NANase enhanced ROS imaging is able to diagnose ALF earlier than clinical ALT methods, and the ROS-activated catalytic activity of the nanoprobe can catalyze the treatment of ALF by NANase activity in a timely manner.
2.3.6 Combination of enzyme activity and fluorescence properties
The fluorescence properties of materials have been widely used in the field of imaging and detection. The combination of enzyme activity and fluorescence properties of nanomaterials makes the functions of nanoenzymes more diversified.
Cai's team synthesized a new type of fluorescent and peroxidase-mimetic Au nanocluster, which was conjugated with folic acid, the ligand of tumor marker, on its surface, so that the probe could detect tumors by fluorescence and staining at the same time. The two methods complement each other and improve the specificity and accuracy of diagnosis[162]. In addition, Meng et al. Synthesized a Cu-CDs nanoenzyme, which has laccase activity and fluorescence characteristics[163]. Interestingly, when the laccase mimics the substrate hydroquinone, its fluorescence gradually weakens and is proportional to the amount of the substrate, and the substrate can be detected by using its enzyme activity and fluorescence decay.
Luo et al. Synthesized multifunctional nanoparticles ——Fe3O4@SiO2@Au MNPs w ith fluorescence and peroxidase-like catalytic activity in aqueous phase[164]. Since the fluorescence of Fe3O4@SiO2@Au MNPs was quenched by H2O2, a method for visual detection of glucose was established.
Carbon nanoenzymes with various structures have been widely used for bioimaging and have attracted much attention in recent years due to their unique fluorescence properties. Su et al. Developed a Co and N-doped carbon dot nanoenzyme, which showed peroxidase-like activity toward o-phenylenediamine in a non-acidic environment, and also demonstrated the application of ratiometric fluorescence imaging[165].
2.3.7 Combination of enzyme activity and conductivity of materials
Colorimetry, chemiluminescence and electrochemical methods are commonly used for substance detection. Among them, the electrochemical method has the advantages of rapid reaction, simple operation and high sensitivity, but the natural enzyme, which is commonly used, has poor stability and high cost, which limits its application. Heavy metals, metal compounds and carbon nanotubes in nanoenzyme not only have catalytic activity, but also have good conductivity, which can catalyze the generation of electrons and accelerate the transfer of electrons, thus increasing the sensitivity of detection[166⇓⇓⇓⇓⇓~172]. At present, a variety of nanoenzymes such as Ag, Fe3O4, Fe2O3, Co3O4, CdS, FeS, FeSe, and C have been used for electrochemical detection, mainly using their peroxidase activity to catalyze H2O2 to produce electron transfer, thus realizing the detection of H2O2.
2.3.8 Surface modification enables nanoenzyme to have new functions
The surface modifiability of nanozymes provides a versatile platform for numerous applications in biomedicine. Antibody modification is a common means used in nanozymes. In a pioneering study of nanozymes, Yan et al. Reported two methods for protein detection using nanozymes modified with antibodies[1]. Since then, Yan et al. Have developed a nanoenzyme test strip for Ebola virus diagnosis using Fe3O4 nanoenzyme modified with antibodies[148]. Compared with the standard colloidal gold strip, the nanozyme strip amplifies the signal by catalyzing the oxidation of the peroxidase substrate in the presence of H2O2, significantly improving the detection sensitivity by 100-fold, with a detection limit as low as 1 ng/mL for Ebola virus (EBOV) glycoprotein. A 10-fold enhancement can be achieved if the magnetic separation property of the Fe3O4 nanoenzyme is utilized to enrich the target. In addition, clinical samples can be diagnosed within 30 min, and the detection accuracy is comparable to that of ELISA. These results suggest that nanozyme strips could provide a faster and simpler platform for infection diagnosis in Ebola-affected areas. Xia et al. Used palladium-iridium (Pd-Ir) cubes labeled with antibodies for immunoassay of human prostate specific antigen (PSA)[173]. The Pd-Ir nanozyme-based ELISA has a 110-fold lower detection limit compared to the traditional immunoassay. To further improve the detection sensitivity of the immunoassay, the team developed gold-encapsulated Pd-Ir NPs as peroxidase mimics in the following study (Fig. 16)[174]. Many Pd-Ir NPs can be released from gold vesicles upon heating, thus forming a signal amplification platform for immunoassays. The detection limit of this method for PSA is femtograms per milliliter, which is three orders of magnitude lower than that of the traditional immunoassay.
Specific recognition can come not only from antigens and antibodies, but also from aptamers and their corresponding targets. For example, Yang et al. Developed a label-free colorimetric method for thrombin detection[175]. One antithrombin aptamer was first immobilized on a 96-well microplate via biotin-streptavidin interaction. The other ssDNA contained a portion of the antithrombin aptamer and another portion of the template DNA for Ag/Pt NC preparation. In the presence of the target thrombin, the two aptamers will combine to form a sandwich structure. DNA-templated Ag/Pt bimetallic nanoclusters catalyze the oxidation of TMB to oxTMB for colorimetric signals. The detection limit of thrombin was 2.6 nmol·L-1. In addition, based on the recognition between Zr4+ and phosphate, Song et al. Synthesized Zr4+ functionalized Pt/carbon dots as peroxidase mimics to recognize and detect phosphoproteins[176].
Chen et al. Developed folate-modified platinum nanoparticles/graphene oxide (PtNPs/GO) nanocomposite for specific cancer cell detection[177]. PtNPs/GO exhibited enhanced peroxidase-like activity that could catalyze the oxidation of TMB in the presence of H2O2 for signaling. When modified by folic acid, the nanocomposite can be targeted to cancer cells via folate receptors overexpressed on the cell membrane. The detection platform based on the peroxidase-mimicking PtNPs/GO nanocomposite has high sensitivity to cancer cells. Even 125 cells could be detected by eye. In order to target pyrite nanoenzyme to tumor tissue in mice, Yan et al. Modified the surface of the nanoenzyme with folic acid, a tumor-targeting molecule. As a result, the amount of the surface-modified nanoenzyme taken up by the tumor was greatly increased by binding to the folic acid receptor highly expressed on the surface of CT26 tumor, which produced better tumor therapeutic effect than the unmodified material[143].
Ferritin has recently emerged as an important modification that confers nanozyme targeting. Yan et al. Prepared magnetic ferritin nanoenzyme (M-HFn) by encapsulating the nanoenzyme in a recombinant human heavy chain ferritin shell[178]. The HFn shell can be targeted to tumor tissue by the transferrin receptor overexpressed on the surface of tumor cells without additional recognition ligands. At the same time, the iron oxide core catalyzes the oxidation of the peroxidase substrate, resulting in a colored product that is used to visualize tumor tissue. In the presence of H2O2 and DAB, M-HFn showed an intense brown color for visualization of tumor tissue (Fig. 17). To demonstrate the high specificity, sensitivity, and accuracy of the M-HFn-based staining platform, 474 clinical specimens from patients with nine types of cancer were tested. Their results showed that the nanozyme imaging platform was able to distinguish cancer cells from normal cells with 98% sensitivity and 95% specificity. Ferritin can also play a targeting role on the surface of nanozyme through modification. Yan et al. Prepared nitrogen-doped porous carbon nanospheres (N-PCNs), which can exhibit four kinds of enzymatic activities, including oxidase, peroxidase, catalase and superoxide dismutase, and can regulate reactive oxygen species in vivo[32]. Further modification of N-PCN with ferritin facilitates targeted delivery into lysosomes via receptor-mediated endocytosis. The acidic environment of the lysosome then promotes N-PCN to carry out peroxidase- and oxidase-mimicking activities to produce ROS and consume oxygen, resulting in toxic effects on tumor cells and hypoxia. In addition, their animal experimental results showed that N-PCNs could effectively reduce tumor volume and improve the survival rate of tumor-bearing mice.
2.3.9 Brief summary
As a new type of mimetic enzyme, nanoenzyme is different from natural enzyme and traditional mimetic enzyme. It has the "dual identity" of enzyme and nanomaterial. In addition to its catalytic function,It also has the unique physical and chemical properties of nanomaterials in the fields of light, electricity, magnetism and sound, so it can be used as a bifunctional or multifunctional molecule in the fields of medicine, environment and chemical industry. Although there have been some studies and applications on the multifunctional properties of nanoenzymes, in order to make them better used for human health and environmental improvement, the research and application of multifunctional properties of nanoenzymes are still the direction of future efforts.
For the current applications of multifunctional nanozymes, most studies are based on peroxidase mimics, or in combination with natural oxidases for the detection of H2O2 and glucose, and the degradation of organic pollutants. Given that six major types of natural enzymes currently exist, we expect to see more multifunctional NANases with novel catalytic activities beyond redox reactions. In addition, the problems of various nanomaterials, including clinical toxicity, stability and catalytic mechanism, need to be carefully studied and tested before the clinical use of multifunctional nanoenzymes.
For MRI applications, in addition to magnetic iron oxide T2 agents, gadolinium (Gd) -based nanomaterials are another type of MR contrast agents that exhibit an increase in signal intensity and appear bright in T1 weighted images. T1 weighted imaging can be used to identify adipose tissue, while T2 weighted imaging can be used to detect inflammation. Therefore, Gd-based nanomaterials as peroxidase mimics should be considered and explored to broaden the MRI applications of multifunctional nanozymes. For SERS-based active nanoenzymes, the catalytic activity is usually relatively low due to the limitation of metal nanoparticles (Au, Ag) themselves. To solve this problem, a nanomaterial with high catalytic activity can be deposited on the surface of Au/Ag. In addition to the functions mentioned in this paper, other functions should be investigated to broaden the research and application of multifunctional nanozymes. This aspect has not been explored to a great extent and may be a new and important direction for future research.
2.4 Nanoenzyme multienzyme activity
As a kind of nanomaterials with catalytic activity and enzymatic reaction kinetic characteristics similar to natural enzymes, nanoenzymes have attracted wide attention in recent years. Compared with natural enzymes, nanoenzymes have higher stability, simpler storage conditions, lower cost and adjustable catalytic activity. Since Gu Ning's team first discovered that Fe3O4 nanoenzyme has dual enzymatic activity in 2012, more and more researchers have found that nanoenzyme can simultaneously show a variety of enzymatic activities similar to natural enzymes, which enables nanoenzyme to be widely used in sensor detection, biomedicine and environmental engineering[9]. With the in-depth study of nanoenzymes, scientists have found that some nanoenzymes not only have multi-enzyme activity, but also can skillfully form a cascade reaction system between the enzyme activities, realize the self-sufficiency of intermediates and form a closed loop of the reaction, which greatly improves the reaction efficiency. This section will introduce the multi-enzyme activity contained in the nanoenzyme itself and its cascade catalysis (Fig. 18), and look forward to its application prospects.
The design of next-generation artificial enzyme cascade systems using nanoenzymes has attracted more and more attention in the field of catalysis, and the construction of enzyme cascade catalytic systems based on the multi-enzyme activities contained in nanoenzymes is full of opportunities and challenges. In recent research work, it has been found that some nanoenzymes can show different catalytic properties under specific conditions, and the discovery of multi-enzyme activity of nanoenzymes enriches the application scope of nanoenzymes. POD-CAT, POD-OXD and SOD-CAT are the main multi-enzyme activities contained in the nanoenzyme itself. In addition, there are OXD-CAT and three or more enzyme-like activities (Table 4). Next, we will introduce the characteristics and applications of the multi-enzyme activities contained in the nanoenzyme itself from the above five multi-enzyme activity systems.
2.4.1 POD-CAT
PODs are generally able to catalyze substrate oxidation in the presence of H2O2, consuming H2O2 and producing ·OH. Native POD is a hemopexin that catalyzes substrate oxidation with iron porphyrin as the prosthetic group and H2O2 as the electron acceptor. CAT normally catalyzes the decomposition of H2O2 into H2O and O2. Natural CAT is also a conjugated enzyme with iron porphyrin as a prosthetic group. Therefore, a large number of iron-based nanomaterials have been found to have POD-like or CAT-like catalytic properties. The first discovered nanoenzyme with POD-like and CAT-like enzymatic activity is also the Fe3O4 of iron-based nanomaterials. In 2012, Gu Ning and Zhang Yu first found that Fe3O4 nanoenzyme has pH-dependent POD-like and CAT-like enzymatic activity, and Fe3O4 nanoenzyme can exert POD-like activity under acidic conditions.It catalyzes H2O2 to produce ·OH, while under neutral conditions, Fe3O4 nanoenzyme exerts CAT-like activity.Decompose H2O2 directly into H2O and O2 (Figure 19 A)[9]. According to the author's analysis, in acidic environment, Fe3O4 nanoenzyme undergoes Fenton-like catalytic reaction, produces intermediate products FeOOH2+ and H through reactions (1) ~ (3), and oxidizes H2O2 to produce ·OH through the transformation between ferric and ferrous iron. When the pH increases, reaction (1) accelerates, the products FeOOH2+ and H accumulate in excess, and H is further ionized to (reaction 4).In turn, the ·OH produced by reaction (3) reacts rapidly with H / to produce O2 (reaction 5) (fig. 19B). This characteristic of Fe3O4 nanoenzyme enables it to show different functions under different pH conditions. Under acidic conditions, its POD-like activity can induce cell damage, while under neutral conditions, its CAT-like activity can play an antioxidant role and protect cells.
Subsequently, pH-dependent POD-like and CAT-like activities were also found on other iron-based nanoenzymes, such as Fe-N4 monoatomic nanoenzymes and manganese-based nanoenzymes, such as Mn monoatomic nanoenzymes[75][179]. Nanomaterials based on CeO2 were also confirmed to have POD-like and CAT-like activities, and similar to iron-based and manganese-based nanoenzymes, CeO2 has mixed valence States of Ce3+ and Ce4+ as well as oxygen vacancies, which endows it with POD-like and CAT-like catalytic activities[180].
In addition, nanoenzymes based on noble metals Pt and Pd have also been found to have pH-dependent POD-like and CAT-like enzymatic activities, and the excellent electron transfer function of Pt and Pd endows them with redox enzymatic activities[181,182]. For example, Huang et al. Reported a two-dimensional PdMo bimetallic nanoenzyme based on transition metal Pd, which exerts POD-like activity under acidic conditions and catalyzes H2O2 to produce ·OH to induce apoptosis of tumor cells[181,182]. Under neutral conditions, it exerts CAT-like activity, directly decomposes H2O2 into H2O and O2, down-regulates the expression of hypoxia-inducible factor-1α, and then reduces the uptake of glucose by tumor cells. The activities of the two kinds of enzymes of PdMo bimetallic nanoenzyme have a synergistic effect and jointly promote the apoptosis of tumor cells. Moreover, the design of PdMo bimetallic nanoenzyme greatly improves the enzyme-like catalytic activity of Pd through transition metal doping and alloying effect.
In addition to metal and metal oxide nanoenzymes, metal sulfide CuS nanoclusters and inorganic non-metallic carbon-based materials such as nanodiamond have also been found to have pH-dependent POD-like and CAT-like enzymatic activities[183][184]. From iron-based nanoenzymes similar to natural enzymes to transition metal oxide nanoenzymes with mixed metal valence, to noble metal nanoenzymes, to carbon-based nanoenzymes,Both of them were found to have pH-dependent POD-like and CAT-like enzymatic activities, indicating that POD-like activity under acidic conditions and CAT-like activity under neutral conditions may be a common phenomenon in the existence of nanozymes. POD-like and CAT-like catalytic activities play opposite roles in affecting cell function, so the rational use of pH-dependent POD-like and CAT-like enzymatic activities of NANase is essential for the biomedical application of NANase.
2.4.2 POD-OXD
Since Gu Ning and Zhang Yu's team first discovered that Fe3O4 nanoenzyme can show double enzyme activity under different pH conditions, the research on multi-enzyme activity of nanoenzyme has become more in-depth, and more and more nanoenzymes with multi-enzyme activity have been found. In the course of the study, people were surprised to find that some nanoenzymes can simultaneously show multi-enzyme activity under specific conditions, forming a cascade system, producing a cascade effect, and amplifying the reaction signal, among which the nanoenzymes with POD-OXD-like dual-enzyme activity are more important.
Natural OXDs can be divided into different types according to their substrates, such as glucose oxidase (GOx), laccase (LAC), ascorbate oxidase (AAO), etc. Their common feature is that they oxidize substrates while reducing O2 to H2O2 or H2O. Therefore, the nanoenzyme with POD-OXD-like bienzymatic activity can combine the ability of OXD to reduce O2 to H2O2 with the ability of POD to consume H2O2 to produce ·OH, forming a cascade enzyme catalytic system, which is essential for improving the catalytic performance of nanoenzyme and expanding its application scenarios. According to the types of catalytic substrates of OXD activity, the reported nanoenzymes with POD-OXD-like bienzymatic activity can be divided into POD-GOx, POD-AAO, POD-LAC, POD-GSH oxidase, and POD-cysteine oxidase.
2.4.2.1 POD-GOx
Gold nanoparticles (AuNPs) have been found to have the ability to oxidize glucose to gluconic acid, and later it was confirmed that AuNPs have the catalytic activity similar to the natural enzyme GOx, which can consume oxygen and oxidize glucose to gluconic acid and H2O2[185][186]. Subsequently, it was found that Au nanoenzymes could show GOx-like or POD-like activity by different surface modifications. For example, mercaptoethylamine-modified Au nanozyme exhibited POD-like activity, whereas citrate-modified Au nanozyme exhibited GOx-like activity[187]. The authors further found that by loading Au nanoenzyme without surface modification on mesoporous silica materials, Au nanoenzyme could not only avoid heterogeneous distribution and aggregation and improve its stability, but also show GOx-like and POD-like activities at the same time. The GOx-like activity of the Au nanoenzyme catalyzes glucose to generate gluconic acid and H2O2, and the product gluconic acid reduces the reaction pH environment and further activates the POD-like activity of the Au nanoenzyme,The intermediate H2O2 can also catalyze the color reaction of TMB. The self-cascade catalytic reaction system greatly improves the detection efficiency of glucose, and has a wide range of applications in the fields of biocatalysis, bioassay, nano-biomedicine and nanotechnology.
Similar to Au nanoenzyme, Pt nanoenzyme, which is also a noble metal, has also been found to have GOx-like and POD-like enzymatic activities. Zhang and Gu et al. Used PLGA to encapsulate perfluorooctane bromide (PFOB) and then modified Pt nanoenzyme to form an enzyme cascade catalytic system for cancer therapy[182]. PFOB can release O2 to provide a source of O2 for the GOx-like activity of Pt NANase to oxidize glucose, and the H2O2 generated by catalysis is consumed by the POD-like activity of Pt NANase to generate ·OH for killing cells (fig. 20).
Based on this, Dong et al. Systematically studied the mechanism of GOx mimicking by a variety of noble metal nanoenzymes including Au[57]. The authors found that Au nanoenzyme could produce H2O2 when O2 was used as electron acceptor in the process of glucose oxidation. At the same time, 2,2 '-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS+·) can also act as an electron acceptor to catalyze the oxidation of glucose. However, for other noble metal nanoenzymes (such as Pt, Pd, Ru, Rh and Ir),O2), they are reduced to H2O instead of H2O2 as electron acceptors (fig. 21).
2.4.2.2 POD-AAO
Natural AAO is a copper-containing enzyme, which can oxidize ascorbic acid to water and dehydroascorbic acid, so there is a theoretical basis for the design of nanoenzymes that mimic AAO activity around copper-containing nanomaterials. On the basis that CuS nanoparticles have been found to have POD-like activity, Liu et al. Designed a CuS nanocluster nanoenzyme by improving the synthesis method. The CuS nanocluster not only has POD-like activity, but also shows AAO-like catalytic activity, which can catalyze the oxidation of ascorbic acid to dehydroascorbic acid[183]. In this work, the size and morphology of CuS nanoenzyme were changed to make it have multi-enzyme catalytic activity.
2.4.2.3 POD-LAC
LAC is a kind of polyphenol oxidase with four copper ions in the catalytic active center, which belongs to copper blue oxidase. It can oxidize polyphenol compounds and produce H2O, and has attracted much attention in green chemistry. Inspired by the active center of natural LAC enzyme, Pan Haibo's team designed a Cu metal-organic framework material nanoenzyme synthesized by Cu2+ and fumaric acid (FMA) to simulate the catalytic activity of LAC. The nanoenzyme exerts LAC-like activity at pH = 8.0, reducing Cu2+ to promote the dissociation of hydrogen-oxygen bonds in phenolic compounds, which can be used in green chemistry[188]. When pH = 4.0, the activity of POD-like is stronger, which promotes the cleavage of O — O bond in H2O2 to produce ·OH, and produces color reaction for the detection of glucose and adrenaline in human body.
Subsequently, Liang et al. Synthesized hematin @ Cu/GMP nanoenzyme with both LAC-like and POD-like catalytic properties by adding hematin to the Cu/GMP system and co-assembling with Cu2+ and GMP on the basis that the amorphous metal-organic framework material Cu/GMP assembled by Cu2+ and GMP was found to be able to mimic the catalytic activity of LAC[189]. By simulating the catalytic active center of LAC and POD, the nanoenzyme simulates the catalytic activity of similar LAC and POD, and can efficiently catalyze the degradation of harmful dyes. This work shows that the simulation of catalytic active centers of multiple enzymes in material design can provide a way to achieve multi-enzyme activity design.
2.4.2.4 POD-GSH oxidase
In 2021, Yan et al. Reported that a pyrite (FeS2) nanoenzyme had POD-like and GSH-oxidase-like catalytic activities[143]. On the one hand, the FeS2 nanoenzyme has an ultrahigh affinity for the substrate H2O2, so that the FeS2 nanoenzyme can use the low concentration of H2O2 in the tumor microenvironment to exert POD-like activity to produce ·OH, and on the other hand, the FeS2nanoenzyme can bind the substrate GSH,In the presence of O2, GSH is oxidized to produce GSSG and H2O2, and the produced H2O2 is further used by POD-like activity to enhance the production of ·OH, which together catalyze apoptosis and iron death of tumor cells. This multi-enzyme cascade strategy not only effectively consumes GSH in the tumor microenvironment and destroys the antioxidant system in tumor cells, but also increases the oxidative damage of tumor cells. But also catalyze that intermediate product of the reaction, H2O2, to generate ROS, and amplify the anti-tumor effect. This work provides a new idea for nanoenzyme to exert multi-enzyme cascade catalytic activity to treat tumors.
2.4.2.5 POD-cysteine oxidase
A CuO nanoenzyme-based bifunctional self-cascade catalytic system simulates the aerobic oxidation of cysteine by virtue of the OXD-like activity thereof to generate cystine and H2O2, and catalyzes the H2O2 to generate ·OH by combining with the POD-like activity of the CuO nanoenzyme to oxidize terephthalic acid to generate a fluorescence signal, thereby constructing a detection platform for cysteine in drugs and human plasma[190].
2.4.2.6 POD-substrate diverse oxidase
The OXD-like activity of several POD-OXD-like nanoenzymes Au, Pt, Cu metal organic framework, CuS, FeS2, and CuO we introduced earlier can catalyze the oxidation of specific OXD substrates, but there are also some reports that the POD-OXD dual-active nanoenzymes do not have a clear OXD substrate, but these nanoenzymes can reduce O2 to or H2O2 while showing POD-like activity.
Noble metal nanoenzymes, such as AuPt, PtRu and AuPtPd, have been reported to show POD-OXD-like activity similar to Au and Pt nanoenzymes[191][192][193]. Moreover, the POD-OXD-like catalytic activity of the AuPt nanoenzyme is positively correlated with the Pt/Au ratio in the alloy material, and the catalytic activity of the nanoenzyme can be adjusted by adjusting the alloy ratio.
Because the active center of many natural oxidases contains copper ions, some copper-based nanozymes can often mimic OXD-like catalytic activity. In addition to the Cu metal organic framework, CuS and CuO nanoenzyme described above, Cu2WS4 nanocrystal nanoenzyme has also been reported to have POD-OXD-like bienzyme activity. In the presence of O2, reducing substances in bacteria are oxidized to generate H2O2,H2O2, which is further used by its POD-like activity to generate ·OH to play an antibacterial role[194]. In addition, the Fe-N-S-doped porous carbon nanoenzyme has the Fe-N coordination structure of natural POD or OXD, so it can also simulate the activity of POD-OXD-like double enzymes, and the nanoenzyme can catalyze the reduction of O2 to produce , singlet oxygen (1O2) and H2O2, so it can cooperate with its POD-like activity to produce ·OH to achieve synergistic and efficient sterilization[195]. Similar enzymes such as vanadium oxide (VOx) nanodot nanoenzyme also use this feature to achieve cascade synergistic sterilization, and the multiple oxidation valence States of VOx provide the basis for its multi-enzyme activity[196]. Compared with single enzyme catalysis, the efficiency of sterilization by using the POD-OXD-like double enzyme cascade catalysis of nanoenzyme to generate a variety of free radicals is greatly improved.
Mn-based nanoenzyme has also been reported to have POD-OXD-like bienzyme activity, for example, Li et al reported that two-dimensional MnO2 nanosheet nanoenzyme can oxidize substrate TMB to produce color reaction in the presence or absence of H2O2[197]. Moreover, only the nanosheet MnO2 nanoenzyme showed obvious POD-OXD bienzyme activity, while the activity of the bulk and spherical MnO2 nanoenzyme decreased significantly, which may be related to the large specific surface area, more active centers, ability to bind more TMB, O2 and H2O2 substrates, and higher electron transfer efficiency of the two-dimensional MnO2 nanosheet.
2.4.3 SOD-CAT
In addition to the multi-enzyme activity of POD-OXD, which combines the ability of OXD to reduce O2 to H2O2 with the ability of POD to consume H2O2 to produce ·OH, forming a cascade enzyme catalytic system to produce free radicals for sensing detection, antibacterial, tumor therapy and other aspects, the research of nanoenzyme based on SOD-CAT cascade system is also fruitful. In the SOD-CAT dual enzyme activity cascade system, SOD activity scavenges to produce H2O2, and then CAT activity can decompose the H2O2 produced in the previous step into O2.This enzyme activity-mimicking cascade strategy not only reflects the high efficiency of integrating multiple enzyme activities, but also reduces the production of harmful intermediate reaction product H2O2 while scavenging ROS at the site of inflammatory injury, amplifies the antioxidant effect, and improves the therapeutic effect. In general, the cascade catalytic reaction on a single nano-enzyme material has the advantages of high local reactant concentration, reduced intermediate decomposition and high mass transfer efficiency, which can greatly improve the enzyme catalytic efficiency compared with single enzyme catalysis or two enzymes used alone, and has great potential in alleviating inflammatory diseases and fighting against oxidative damage[142].
Natural SOD belongs to metalloenzyme, which can be divided into three types according to the type of metal ion in the active center: CuZnSOD, MnSOD and FeSOD. The three SOD enzymes can catalyze the disproportionation of free radicals to produce H2O2 and O2, thus eliminating the peroxide free radicals produced in the process of inflammation and producing a powerful anti-inflammatory effect. Many nanomaterials containing the above metal elements of Cu, Zn, Fe, and Mn have been used to design and synthesize SOD nanoenzymes, and their catalytic mechanisms mostly involve the protonation of and the adsorption and rearrangement of . As shown in Fig. 22, the adsorption of metal SOD nanoenzyme represented by Au and Pt is a highly exothermic process, which indicates that once is adsorbed on the surface, it can be easily converted into O2 and H2O[61].
As we have mentioned above, natural catalase is a conjugated enzyme with iron porphyrin as the prosthetic group, which can promote the decomposition of H2O2 into molecular oxygen and water, and remove hydrogen peroxide in the body, so that cells can be protected from the toxicity of H2O2, and it is one of the key enzymes in the biological defense system. To mimic the SOD-CAT bienzyme activity, a number of metal-containing NANases have been designed.
Wei Hui's team has been focusing on the design of antioxidant cascade catalytic nanoenzyme for a long time. In 2018, the team reported that Mn-based nanoenzyme Mn3O4 could mimic highly active SOD and CAT enzymes through the conversion between Mn2+ and Mn3+ oxidation States, and anti-inflammatory through SOD-like and CAT-like catalytic cascades[198]. Similarly, the team also used the SOD-like and CAT-like catalytic cascade of cerium oxide (CeO2) nanoenzyme to treat enteritis, and the conversion between the Ce3+ and Ce4+ oxidation States of CeO2 nanoenzyme provided the source of its catalytic activity[199]. Recently, the team also synthesized Pt @ PCN222-Mn nanoenzyme by co-assembly of zirconium (Zr), Mn porphyrin and Pt. In the nanoenzyme, trivalent Mn porphyrin provides SOD activity source by mimicking natural enzyme, and Pt nanoparticles provide CAT-like activity source, so Pt @ PCN222-Mn nanoenzyme forms SOD-CAT cascade catalysis, which is efficiently used for ROS scavenging in vivo (Fig. 23)[142].
In addition, Pd nanocrystal nanoenzyme has been reported to have SOD-like and CAT-like cascade catalytic activity against oxidative damage, and this study found that,The catalytic activity of (111) Pd octahedral nanoenzyme with lower surface energy is significantly higher than that of (100) Pd cubic nanoenzyme with higher surface energy, which indicates that besides the size, structure and elemental composition of nanoenzyme, surface energy is also an important factor affecting the catalytic performance of nanoenzyme cascade[200].
2.4.4 OXD-CAT
Wei et al. Reported that a Pt/CeO2 nanoenzyme synthesized by in situ growth of Pt nanoparticles on CeO2 nanorods had OXD-CAT-like bienzyme activity[201]. The authors screened potential urate oxidase-like (UOD) nanoenzymes from platinum group element (Pt, Ir, Rh, Pd) nanoenzymes with CAT-like catalytic activity, and found that Pt nanoenzymes had the highest UOD-like catalytic activity while having CAT-like catalytic activity. Pt nanoenzyme is loaded on the CeO2 nanorod to improve the stability and biocompatibility of the nanoenzyme, and the Pt/CeO2 nanoenzyme can catalyze the decomposition of uric acid by utilizing the activity similar to UOD-CAT,The O2 is reduced to H2O2, and then the CAT-like activity is cascaded to decompose the H2O2 to O2, so that cyclic catalysis is realized, the uric acid is efficiently decomposed, and the accumulation of the intermediate reaction product H2O2 is avoided. The advantages of multi-enzyme activity and cascade reaction simulated by nanozyme were effectively verified in the treatment of acute gout (Fig. 24).
2.4.5 Nanoenzyme with three or more enzyme-like activities
2.4.5.1 SOD-CAT-UOD
Recently, an arginine polypeptide-Pt nanoparticle cluster hybrid nanoenzyme was found to have three catalytic activities of UOD-like, SOD-like and CAT-like at the same time, in which two cascade catalytic reactions of UOD-CAT and SOD-CAT were formed, which could mimic natural peroxisomes, decompose uric acid and scavenge free radicals, and alleviate inflammation or oxidative damage[202]. The Pt nanoenzyme is modified by arginine to simulate the amino acid residue microenvironment of the active center of the natural enzyme, and the arginine contains a guanidine functional group with a nitrogen lone pair, which can be combined with a variety of substrates through hydrogen bonds or electrostatic interaction, so it can simulate natural peroxisomes and has a variety of oxidoreductase catalytic activities.
2.4.5.2 SOD-CAT-POD
In 2016, Gu and Zhang et al. Reported Prussian blue (PB) nanoenzyme with POD-like, SOD-like, and CAT-like catalytic properties[203][203]. PB nanoenzyme has Fe with multiple oxidation valence States, and PB material has abundant redox potential, which can transfer electrons efficiently, thus endowing it with a variety of enzyme-like catalytic activities. The authors also proposed that, unlike the previous Fe-based nanoenzyme, Fenton-like reaction under acidic conditions produces ·OH,PB nanoenzyme exerts POD-like catalytic activity to consume H2O2, oxidize reducing substances in cells, and inhibit the production of ·OH. Synergistic SOD-CAT cascade catalysis can efficiently scavenge ROS and protect cells from oxidative damage.
Liu et al. Designed a Fe2NC using selenium (Se) MOF materials to encapsulate double iron atoms to synthesize Fe2NC@Se nanoenzyme, which has SOD-like, CAT-like and GPx-like catalytic activities at the same time, and can efficiently scavenge free radicals through cascade catalysis[205]. Among them, Se-MOF can mimic the active center of natural selenium-containing GPx enzyme, produce GPx-like activity, oxidize GSH to GSSG, and catalyze the decomposition of H2O2 to H2O. The diiron atom Fe2NC nanoenzyme has an excellent SOD-CAT-like cascade catalytic function, and the two functions cooperate to achieve free radical scavenging against and H2O2, which is used to protect cerebral ischemia-reperfusion injury.
In addition, Wei et al. found that ZnMn2O4 nanoenzyme only has SOD-like activity, while LiMn2O4 nanoenzyme has SOD-like, CAT-like, and GPx catalytic activity (Fig. 25). Further mechanism analysis found that by doping metal elements and increasing the proportion of Mn4+/Mn3+, not only the SOD-like activity of Mn-based nanoenzyme can be improved, but also the Mn-based nanoenzyme can have multi-enzyme activity and play an antioxidant cascade catalytic reaction[204]. SOD scavenging is the first step in the antioxidant cascade, and the decomposition of H2O2 is the second step in the antioxidant cascade, which can be achieved by both CAT and GPx activities, so LiMn2O4 NANase shows better anti-inflammatory effect in vivo.
2.4.5.3 POD-OXD-CAT
Recently, a Mn monoatomic nanoenzyme has been reported to have three catalytic activities: POD-like, OXD-like, and CAT-like[179]. Compared with the MnO2 nanoenzyme with POD-OXD-like activity described above, the Mn monoatomic nanoenzyme has more active centers, so its catalytic activity is higher. After entering the cell, the nanoenzyme uses its POD-like and CAT-like activities to catalyze intracellular H2O2 to generate ·OH and O2,O2, respectively, which are further used by its OXD-like activity to generate radicals. The ·OH and generated by the catalytic cascade system synergistically induce tumor cell death and can be used for tumor catalytic therapy.
2.4.5.4 POD-OXD-SOD-CAT
In addition, nanozymes with four oxidoreductase catalytic activities (POD, OXD, SOD, CAT) have also been reported. For example, Gao et al. Reported a biomimetic synthetic Fe-N4 monatomic nanoenzyme, which can mimic the Fe-N coordination form of the active center of natural oxidoreductases such as SOD, CAT, UOD and POD, so it has the catalytic activities of SOD, CAT, UOD and POD at the same time as natural peroxisomes[75]. Among them, the nanozyme is used to treat hyperuricemia and ischemic stroke by decomposing uric acid and scavenging free radicals through two sets of cascade catalytic reactions of UOD-CAT and SOD-CAT (Fig. 26).
Recently, the CoFe2O4/H2PPOP nanoenzyme synthesized by depositing CoFe2O4 on porphyrin porous organic polymer materials in situ has been endowed with efficient electron transfer function through the valence conversion between its Fe3+/Fe2+ and Co3+/Co2+, so that it has four kinds of enzyme activities, namely POD, OXD, SOD and CAT, and can produce sensitive color reaction for heavy metal detection through the synergistic catalysis of its four enzyme activities[206].
2.4.6 Brief summary
In a word, this section reviews the nanozymes with multi-enzyme activities discovered by researchers in the past decade, and classifies the multi-enzyme activities of nanozymes according to the types of multi-enzyme activities (POD-CAT, POD-OXD, SOD-CAT, OXD-CAT, and nanozymes with three or more enzyme-like activities).
Since Yan Xiyun's team first proposed that Fe3O4 nanoparticles have POD-like catalytic activity in 2007, the research work of nanozymes has attracted the attention of scientists from different disciplines[1]. A large number of different types of nanoenzymes have been discovered or designed, from metal to non-metal, from inorganic to organic, from single metal to multi-metal, and different types of nanomaterials have been designed to explore enzyme-like catalytic properties. In the course of the study, it was found that the catalytic reaction of nanoenzyme has two characteristics, one is the broad substrate adaptability, that is, for a specific catalytic reaction, nanoenzyme can catalyze different substrates to react; The second is multi-enzyme activity, that is, for a specific substrate, nanoenzymes can catalyze different reactions to produce different products. It is these two characteristics that make the substrates of nanoenzymes very broad, and a nanoenzyme can often catalyze different substrates to play the same catalytic reaction in different application scenarios.At the same time, the multi-enzyme activity of nanoenzyme has also been excavated. Nanoenzyme can catalyze the same substrate to play different reactions under specific conditions, showing different catalytic activities.
In 2012, Gu Ning's team first discovered that the Fe3O4 nanoenzyme has pH-dependent POD-like and CAT-like enzyme activities, and at different pH conditions, the nanoenzyme can catalyze different reactions of the same substrate H2O2[9]. In subsequent studies, more and more nanozymes were found to have pH-dependent multienzyme activity similar to that of Fe3O4, indicating that the POD-like activity under acidic conditions to promote free radical generation and the CAT-like activity under neutral conditions to scavenge free radicals may be a common phenomenon of nanozymes. Therefore, in the application of nanozymes, the pH environment has an important impact on their catalytic activity, especially in vivo applications, through rational design, nanozymes can be captured in subcellular organelles with different pH environments, which can achieve precise regulation of nanozymes function.
Later, it was found that some nanoenzymes can simultaneously show multi-enzyme activity under specific conditions, and enzyme-catalyzed reactions can form a cascade system, resulting in a cascade effect, which can not only improve the catalytic efficiency, but also reduce the formation of reaction intermediates. For example, the combination of the ability of OXD to reduce O2 to H2O2 and the ability of POD to consume H2O2 to produce ·OH through the POD-like OXD dual-enzyme activity cascade can accelerate the production of ·OH, which makes nanoenzymes have great application potential in the fields of sensing detection, antibacterial and anti-infection, and tumor catalytic therapy. In addition, through the SOD-CAT double enzyme activity cascade, SOD activity scavenges to produce H2O2, and then CAT activity can decompose the H2O2 produced in the previous step into O2, thus realizing the complete scavenging of free radicals, which doubles the application potential of nanoenzyme in the field of anti-inflammatory and anti-oxidative damage. In addition, UOD-CAT cascade and SOD-GPx cascade can achieve efficient removal of free radicals. The cascade catalytic reaction on a single nano-enzyme material has the advantages of high local reactant concentration, reduced intermediate decomposition and high mass transfer efficiency, and compared with single enzyme catalysis or single use of two enzymes,It can greatly improve the catalytic efficiency of enzymes, so the multi-enzyme cascade catalytic activity of nanoenzymes brings new opportunities for the efficient catalytic application of nanoenzymes.
The mechanism of multienzyme catalytic activity of nanozymes has not been thoroughly studied, but some efforts have been made to explain the mechanism. For example, Fe ions are essential for the active center of many oxidoreductases (such as POD, CAT, OXD, SOD), so some iron-based nanoenzymes (such as Fe-N4 monoatomic nanoenzymes) designed by mimicking the Fe-N coordination characteristics of the active center of natural enzymes can simultaneously mimic the catalytic activities of the above four enzymes[75]. There are also some copper-based nanoenzymes that can mimic the catalytic activity of some copper-containing natural oxidases (such as AAO, LAC, etc.), and then add POD-mimicking functional elements to the nanoenzyme system, such as hematin @ Cu/GMP nanoenzyme, which can mimic the catalytic active center of LAC and POD, thus obtaining POD-OXD catalytic activity[189]. Furthermore, it was found that the nanoenzymes synthesized by coordination assembly of other metal elements and amino acids with electron transfer function could also mimic the catalytic activity of various oxidoreductases. These works enlighten us to rationally design nanoenzymes by referring to the active centers of specific natural enzymes, and to fuse the active centers of different nanoenzymes to the design of the same nanoenzyme, thus providing the possibility of obtaining nanoenzymes with specific cascade catalytic properties.
The oxidation valence of metal elements can also affect the multi-enzyme activity of nanoenzyme. For example, the proportion of Mn4+/Mn3+ reported by Wei Hui's team affects the catalytic activity of nanoenzyme. ZnMn2O4 nanoenzyme only has SOD-like activity, while LiMn2O4 nanoenzyme has SOD-like, CAT-like and GPx-like activity. Increasing the proportion of high-valence Mn can enhance the CAT activity of Mn-based SOD nanoenzyme and cause multi-enzyme activity[204]. The same phenomenon was also found in cerium oxide nanoenzyme. Increasing the proportion of Ce4+/Ce3+ can also increase the CAT activity of cerium oxide SOD nanoenzyme, resulting in multi-enzyme activity[199]. In addition, the appearance of multienzyme activity of PB nanoenzyme and CoFe2O4 nanoenzyme is related to the multiple oxidation valence States and abundant redox potentials of Fe and Co metal elements[203][206]. Therefore, there is a theoretical basis for the design of nanoenzymes with multi-enzyme activities based on variable valence metal elements.
In addition, the size, morphology and surface energy of nanozymes can also affect the multienzyme activity of nanozymes, but the specific mechanism needs further exploration[183][200]. How to further reveal the multi-enzyme activity mechanism of nanozymes requires the addition of new research models or theoretical calculation systems, and the participation of interdisciplinary teams. An in-depth understanding of the catalytic mechanism of multi-enzyme activity of nanozymes will guide us to design cascaded nanozymes with higher activity and catalytic efficiency, which will provide theoretical support for the realization of efficient catalytic applications of nanozymes.
3 Nanoenzyme engineering
Over the past few decades, the commercial applications of enzymes in different fields such as food, agriculture, biofuels, pharmaceuticals, leather, cosmetics, waste management and so on have increased dramatically, and the global demand and trade of industrial enzymes are steadily increasing, which is expected to increase to $8.7 billion by 2026[207]. Although the enzyme industry has a huge market demand, the limitations of natural enzymes, such as fragility, high cost and high cost, have accelerated the research and development of artificial enzymes. At present, the industrial production of natural enzymes is mainly realized by microorganisms, which often involves genomics technology, strain improvement, medium optimization, fermentation method improvement, screening and identification, separation and purification, etc., with cumbersome steps and high cost[208]. In addition, the instability of natural enzymes requires strict conditions such as preparation environment, operation process, storage and transportation, which further increases the process and cost. As a new generation of artificial enzymes, nanoenzymes not only have catalytic properties similar to natural enzymes, but also have unique physical and chemical properties, which provide unlimited possibilities for regulating their catalytic performance. In addition, as an inorganic nanomaterial, the production and processing steps of nanoenzymes are simpler and more convenient, with high yield and purity, and their high stability also greatly reduces the requirements and costs of production environment and storage. Therefore, the engineering of nanoenzymes has attracted much attention in recent years[209]. Since its first discovery by academician Yan Xiyun in 2007, the research of nanoenzymes has made rapid and brilliant progress, and a large number of papers have reported on nanomaterials that mimic enzyme activity, including doped carbon, metal oxides, precious metals and metal organic frameworks.It has been used in disease treatment, antibacterial, biosensing and pollutant degradation, and these research results have promoted the systematization, modularization and standardization of basic and applied research of nanoenzymes[1][32][30][38][139][75][210][211][212].
Nanomaterials with different physical and chemical properties show different catalytic activities of natural enzymes, showing the complexity and diversity of nanoenzymes in enzyme-like catalysis. In order to meet the needs of practical applications, the catalytic performance of nanoenzymes can be regulated by precise control of their composition, size, surface modification, encapsulation carrier and other strategies[213]. At present, the synthesis of nanozymes is usually through experience and trial and error to obtain the desired catalytic properties, which is a time-consuming and laborious strategy. In order to break these limitations, it is necessary to further explore the mechanism of nanoenzyme action, accurately construct nanoenzyme models with the help of computer-aided design, and use their structure-activity rules to guide and predict the synthesis of nanoenzymes, so as to achieve the purpose of precise customization of nanoenzymes.
This section will focus on reviewing the latest progress in nanoenzyme engineering, first introducing the establishment of nanoenzyme database, and using machine learning and other methods to establish models to reveal the effects of different preparation conditions on nanoenzyme structure and enzyme mimetic properties, and then summarizing the latest progress in nanoenzyme preparation and characterization methods.Finally, the effects of different modification methods on the structure of nanoenzyme in the synthesis process were emphasized, and the relationship between the structure and performance of nanoenzyme was further discussed, and the structure-activity relationship of nanoenzyme was established, which provided theoretical guidance for the design and preparation of nanoenzymes.
3.1 Nanozyme and Machine Learning
Machine learning (ML) is a subfield of artificial intelligence (AI) that has attracted increasing attention due to its tremendous success in big data processing, computer vision, speech recognition, and natural language processing[214]. In some fields that were considered to be only mastered by human beings in the past, such as complex strategy games and medical image diagnosis, machine learning has shown a level close to or even exceeding that of experts[215][216]. New things based on machine learning methods are rapidly becoming popular in our lives, and the emergence of Alpha Go has made the world marvel at the potential value of artificial intelligence[215,217]. In the field of scientific research, the concept of big data is changing the mode of scientific researchers' exploration of unknown areas[218].
In previous research on nanomaterials, experimental and computational simulation methods are the two mainstream ways to explore nanomaterials, which are essentially limited by the threshold of expertise, experimental conditions or existing theoretical basis. However, with the increasing complexity of materials, the use of these two traditional methods has been unable to meet the needs of people for new materials or higher performance. Machine learning is a powerful tool capable of finding regularities in complex data, which can learn from empirical data by modeling linear or nonlinear relationships between material properties and related factors[219]. Data mining and performance prediction based on machine learning has become an important way to evaluate the performance of materials and develop new materials. This data-driven machine learning method usually requires a large amount of relevant data as a basis, and the launch of the Materials Genome Project (MGI) in 2011 also provides a basis for the development of machine learning in the field of materials. The research on materials using machine learning is mainly divided into three steps (Figure 27): Database establishment, Model establishment, and Model Output. (1) Establishment of database. Data is fundamental, and the establishment of new databases or the use of existing databases is a prerequisite. At present, the database of materials science is still very limited, and the data is mainly collected by ourselves, and the data collection is mainly from literature reports, so there will be great heterogeneity in the data, so it is necessary to further "clean" the data, and finally select the appropriate features for the training of machine learning models. (2) Model establishment. Building an accurate machine learning model is the key to this work, and the selection of appropriate algorithms plays a decisive role. The selected material characteristics and/or properties are used to train the model to establish a linear or nonlinear relationship, which is then verified to prevent overfitting. (3) Model application. Whether the established model can be used is the embodiment of its value or significance. At present, the main applications include the prediction of material properties, the design/screening of required materials, and the quantification of some material characteristics.
The basic principle of machine learning method determines that it can find the hidden law between materials and properties according to the existing data when the structure-activity relationship of materials is not clear. At present, the research of machine learning in materials mainly includes two aspects: the prediction of material properties and the discovery of new materials. In terms of material property prediction, Ruoff et al. Used a simple regression method to predict the solubility of C60 in different solvents, which opened the door to data-driven materials research methods[220]. Gu et al. Predicted the mechanical properties (toughness and strength) of two-dimensional composites by using 35960 pieces of data related to two-dimensional composites generated by the FEM (Finite element model) model constructed by previous work, combined with convolutional neural network, and predicted the properties of more than one million target materials by computer high-throughput screening[221][222]. Sun et al. used a data set containing 425 silver nanoparticles with diameters ranging from 0.5 to 4.9 nm (13 to 2947 atoms), and used different machine learning analysis algorithms (including k-means, logistic regression, random forest, and artificial neural network) combined with electronic structure simulation to study the relationship between the Fermi level of silver nanoparticles and various structural and morphological characteristics[223]. Rajan et al. Generated 23,870 possible MXene structures, randomly selected 7200 MXenes from them to create a data-optimized database, and developed a machine learning model that can be used to accurately predict the band gap of MXene, which can accurately predict the band gap of functionalized MXene to study its electrical properties[224]. In the discovery of new materials, Lu et al. Used the data of 2569 two-dimensional materials in the two-dimensional materials database (C2DB) to calculate the thermodynamic stability, magnetic ground state and band gap as output targets.A gradient enhanced separation model was established, and an intelligent design platform for two-dimensional ferromagnetic functional materials was built, and 93 two-dimensional materials with ferromagnetic stability were successfully screened out by using the model[225]. Lu et al. Used data from 212 organic-inorganic perovskite materials (HOIPs) for training, and selected six solar cell materials with thermal stability and reasonable band gap from 5158 lead-free perovskite materials by machine learning method[226]. Guo et al. Used machine learning methods to analyze the self-built catalyst additive database data (including 12 metal salts and 200 water-soluble organic molecules), and used the continuous cycle of "experimental testing-machine learning-prediction and redesign".It was determined that the regulator concentration and ligand solubility were the key factors in the synthesis of zirconium hafnium oxygen cluster nano-MOFs, and a series of zirconium hafnium oxygen cluster nano-MOFs with different morphologies were precisely prepared[227]. Erps and others combined machine learning methods with 3D printing technology to find 3D printing materials with the best mechanical properties[228]. This semi-automated data-driven workflow greatly reduces the trial-and-error process and reduces the burden on developers through the optimization of the algorithm.
Similar to other nanomaterials, data-driven approaches are beginning to penetrate the nanoenzyme research field. Wang et al. Proposed that the number of electrons in the eg orbital can be used to predict the activity of ABO3-type perovskite-like peroxidases[51]. This study found that the peroxidase-like activity of this kind of nanoenzyme changed with the number of electrons in the eg of B, and thus realized the activity prediction of ABO3 type perovskite peroxidase-like enzyme. Li et al. Systematically analyzed 105 published papers to discover high-performance hydrolytic nanozymes to screen and predict the catalytic active sites of MOF-based hydrolytic nanozymes[17]. Wang et al. Used DFT calculations to study the thermodynamics and kinetics of the catalytic process of superoxide dismutase-like, and proposed the energy level principle and the adsorption energy principle.The ability of this principle to predict partial superoxide dismutase-like activity was experimentally verified, and they could be used to computationally screen nanozymes with intrinsic superoxide dismutase-like activity from the C2DB database (Fig. 28)[63].
At present, the process of discovering and testing new nanoenzymes is still based on the traditional trial-and-error method and chemical theoretical calculation, which has high knowledge threshold, long development cycle, high cost and cumbersome evaluation process, seriously limiting the development speed and rapid application of nanoenzymes. Therefore, the efficient design and development of nanoenzymes has become a new scientific research hotspot. However, due to the lack of theoretical guidance, the discovery of nanoenzymes mainly depends on the limited fine-tuning of their chemical composition. How to use the existing technology to achieve the rapid design and discovery of nanoenzymes is the focus of current research on nanoenzymes. Therefore, the development of nanozymes by rational design employing automated methods is highly anticipated to accelerate the discovery of novel nanozymes with enhanced activity. In recent years, with the improvement of computer computing power and algorithm optimization, machine learning methods are accelerating the discovery, design and research of nanomaterials and nanoenzymes. Razlivina et al. Created DiZyme, an open-access nanoenzyme database from more than 100 published research papers, which contains more than 300 existing inorganic nanoenzyme materials[229]. The authors used the database to develop and optimize a random forest regression (RFR) model for evaluating the activity of NANase, which achieved the activity prediction of peroxidase-like NANase. Recently, Huang Xinglu's team at Nankai University collected 920 nanoenzyme data from existing published papers and constructed a transition metal nanoenzyme database[16]. The database contains physicochemical characterization data for a variety of enzyme mimics, including peroxidase-like nanozymes, oxidase-like nanozymes, catalase-like Nanozymes, superoxide dismutase-like Nanozymes and hydrolase-like Nanozymes. The database divides the characteristics of nanozymes into two categories: intrinsic characteristics and external conditions. The intrinsic characteristics include the element composition and its proportion, the size, morphology, zeta potential and other properties of the nanoenzyme, while the external conditions refer to the external reaction system conditions in which the nanoenzyme exerts its enzyme-like activity, including the temperature, pH and the type of catalytic substrate of the system. Using the data in the database, the author combined the artificial neural network algorithm in machine learning to realize the selection of characteristic descriptors for the catalytic activity of nano-enzymes and the prediction of the catalytic activity of nano-enzymes.Peroxidase-like nanozymes and oxidase-like nanozymes were predicted with R2 as high as 0.66 and 0.80, respectively, and the selective synthesis of highly active transition metal nanozymes was successfully realized by using the artificial neural network model, which fully demonstrated the potential of the combination of nanoenzymes and machine learning methods (Fig. 29).
At present, machine learning has just started in the research of nanoenzymes, and there are very limited reports on the prediction of nanoenzyme performance, and there is no report on the discovery of new nanoenzyme materials. In order to make better use of machine learning to make substantial breakthroughs in nano-enzyme research methods, it is urgent to solve the following problems, such as database, training data, machine learning itself and so on.
(1) Incomplete features covered by the database. At present, the data samples collected by the database related to nano-enzyme materials are not systematic and comprehensive enough to meet the increasing research and development needs in the field of nano-enzyme. Generally speaking, the sample data in the database comes from published experimental data, but only a small number of "successful" results have been published, and a large number of "failed" experimental data have not been published. Harvard University researchers proposed "learning from failure" in Nature 2016.In this study, failed or unsuccessful experimental data were used to predict the synthesis of new materials, and it was found that the accuracy of machine learning model prediction exceeded that of experienced chemists, which means that machine learning will change the way traditional materials are discovered, and the possibility of inventing new materials will be greatly increased[230]. Therefore, the establishment of a nanozyme database containing "successes" and "failures" is equally important for information mining using machine learning. In addition, the acquisition of some important characteristic parameters of nanoenzymes, such as the size and morphology of nanoenzymes, mainly depends on image information, and this kind of unstructured data acquisition mainly depends on the subjective judgment and measurement of database builders.It is difficult to ensure its accuracy, so it is necessary to establish a public material image database to promote the development and application of this kind of unstructured data-driven nanoenzyme materials. For a specific class of nanoenzymes, a small database of specific nanomaterials can be established, so that the class of nanoenzymes can be explored more pertinently.
(2) Heterogeneity of training data. Data-driven machine learning methods largely depend on the quality of data. However, there is no uniform standard for the new material concept of nanoenzyme and its characterization methods in various research institutions around the world. The data obtained from experimental measurements will have different material defects caused by different synthesis methods, as well as systematic errors caused by the uncertainty of equipment and experimental conditions in different laboratories. These errors bring about great differences in experimental data, which brings great trouble to data-driven nanoenzyme research. Therefore, at this stage, there is an urgent need for a standardized general method for nanoenzyme research, which can clarify the characterization of nanoenzymes and the evaluation index of enzyme catalytic activity. In addition, there are also great difficulties in the controlled synthesis of nano-enzyme materials, which leads to the heterogeneity of data. In 2020, Wang Cheng et al. Used machine learning to guide the morphology control of nano-metal-organic framework nanomaterials, and they used "decision tree", "random forest" algorithm and image recognition method to find the conditions and rules for the synthesis of "metal-organic monolayer".Metal-organic framework materials with more morphologies (such as hexagonal nanosheets, octahedra, concave octahedra, hollow octahedra and truncated octahedrons) have been successfully obtained[231]. Therefore, using machine learning to improve the controllability of nano-enzyme materials is also the key to effectively solve the data heterogeneity of nano-enzyme materials.
(3) Defects of machine learning methods. Machine learning models can provide some guidance for the study of the catalytic mechanism of nanozymes, but their interpretation is limited. It only cares about the results but not the process, which is contrary to theoretical research such as computational chemistry, which is the defect of machine learning method itself. Therefore, machine learning methods can also be combined with computational chemistry methods. For example, researchers from Carnegie Mellon University in the United States have proposed a fully automated screening method, which uses machine learning methods to guide density functional theory calculations.The researchers proved the feasibility of this method by screening 31 alloys of different elements, thus realizing the screening of some d-block elements and p-block elements[232]. In addition, the whole process of machine learning only focuses on the sole goal of improving the accuracy of model prediction, which usually ignores the importance of expertise in the corresponding field.Combining the results of machine learning with expertise in the field will help to improve the understanding of model results and further optimize the feature selection of related machine learning models.
In short, despite the many challenges, the combination of materials science and information technology is still an important innovation in approach. Machine learning models can help researchers fundamentally understand the relationship between the structural properties of nanomaterials and enzyme-like activity, and have guiding significance for the development and design of nanoenzymes and even the machine learning technology related to nanoenzymes. Many successful application examples of machine learning methods in the field of nanomaterials make it gradually become a new research direction in the field of nanoenzymes, which opens up a new way to accelerate the research of nanoenzymes and is expected to make a major breakthrough in this field. One of the future development directions of nanoenzymes will be to accumulate raw data through process-oriented, automated high-throughput computing and experiments in the form of big data such as machine learning. Through systematic and automated extraction of implicit links between material characteristics and enzyme-like properties, a material screening and prediction mechanism is formed to accelerate the research and development of nano-enzyme materials.
3.2 Preparation and characterization of nanoenzyme
3.2.1 Preparation of nanoenzyme
In this part, the preparation methods of different types of nanoenzymes are introduced in detail, including metal nanoenzymes, metal compound nanoenzymes, carbon-based nanoenzymes, metal-organic framework nanoenzyme, single-atom nanoenzyme and composite nanoenzyme, which are summarized in Table 5.
Metal nanoenzymes have been widely studied in the fields of chemical analysis, biological detection, antibacterial, disease treatment and so on because of their excellent optical, electrical, thermal and surface chemical properties. Metal nanoenzymes can be roughly divided into single metal and metal alloy nanoenzymes, and the main synthesis methods include general reduction, co-reduction, replacement reaction, etc[233][250][251].
The general reduction method refers to the use of reducing agents to reduce metals from their compounds at a certain temperature to synthesize metal nanoenzymes, which belongs to redox reaction. Das et al. Heated a solution of chloroauric acid and sodium citrate at 100 ° C and stirred vigorously, thereby reducing the Au3+ to Au atoms, which then formed gold nanoparticles (GNP) through a nucleation-growth process[233]. The size, morphology and structure of nanoparticles can be effectively adjusted by changing the experimental parameters and improving the experimental methods, which is of great significance to the study of the structure-activity relationship of nanoenzyme catalytic properties. At present, a variety of macromolecules (such as dendrimers, proteins, DNA, etc.) And small molecules (such as citric acid, thiols, etc.) Have been widely used as stabilizers and surface coating agents to synthesize monometallic nanoenzymes, and the adsorption characteristics of these molecules on the surface of nanoenzymes will affect the catalytic performance of nanoenzymes. For example, Liu et al. Prepared amino-terminated PAMAM dendrimer-encapsulated gold nanoclusters (AuNCs-NH2)[252]. The tertiary amine on the surface of AuNCs-NH2 can inhibit the production of hydroxyl radicals, thereby inhibiting its POD-like enzyme activity. Meanwhile, tertiary amines are easily protonated to be positively charged in acidic environment, which promotes the pre-adsorption and decomposition of H2O2 on the particle surface, thus broadening the pH range for AuNCs-NH2 to exert its catalase-like (CAT) activity.
The co-reduction method refers to the simultaneous reduction of two metal precursors into zero-valent atoms, which nucleate and co-grow to generate A-B bimetallic nanocrystals. The structure and size of the bimetallic nanoenzyme can be controlled by changing experimental parameters such as the reduction potential of the metal ion, the reducibility of the reducing agent, the property of the ligand, and the reaction temperature. He et al. Prepared Au/Pt alloy nanoparticles by co-reduction of Au3+ and Pt2+ in aqueous solution, and found that the morphology and alloy composition of the materials could be controlled by adjusting the molar ratio of added Pt2+ to Au3+[191]. When the Au/Pt molar ratio is 1/1, the prepared nanoparticles have a spherical dendritic and porous structure, as shown in fig. 30, and have good dispersion in water. The peroxidase-like and oxidase-like activities of Au/Pt alloy nanoparticles are highly dependent on the composition and composition of the alloy, and the co-reduction method provides an effective means to regulate the catalytic performance of the alloy.
The replacement reaction method is to add the metal nanoparticles with strong reducibility to another metal salt solution with weak reducibility, and replace the metal with weak reducibility according to the principle of replacement reaction. Yang et al. Prepared Ag/Au bimetallic nanoparticles with core-shell structure by displacement reaction between Ag nanoparticles and HAuCl4 in toluene using Ag nanoparticles as template[235]. The results show that the prepared Ag/Au bimetallic nanoparticles have a smaller particle size than the Ag template nanoparticles. The replacement reaction is a complex process controlled by both kinetics and thermodynamics. The experimental parameters, such as the structure and size of the template nanoparticles, the valence of the metal ion, the reaction temperature and time, and the amount and type of the protective agent, are important factors affecting the geometric structure and catalytic performance of the prepared metal nanoparticles.
In addition, electrochemical deposition and solvothermal methods are often used to prepare metal nanoenzymes[234][200]. The size and morphology of nanoenzyme can be regulated by controlling the electrodeposition parameters in the deposition process. Solvothermal method refers to the preparation of metal nanoenzyme by reducing metal ions at high temperature and high pressure in a closed reactor with water, ethylene glycol and other solvents as reaction media. The method has the main characteristics of simple operation and low cost, and can synthesize the metal nanoenzyme with special morphology and excellent performance. Researchers have found that the composition and structure of the particles are important factors affecting the enzyme-like properties of metal alloy nanomaterials, so adjusting the proportion of various metals and expanding the porosity and specific surface area of alloy nanomaterials have been considered as an effective way to regulate the catalytic activity of enzyme-like materials[253,254][78].
Metal compound nanoenzymes mainly include metal oxide nanoenzymes, metal sulfide nanoenzymes, metal selenide nanoenzymes and metal phosphide nanoenzymes. The main synthesis methods include solvothermal method, liquid precipitation method and thermal decomposition method.
Solvothermal method refers to the method of preparing metal compound nanoenzyme by reacting raw materials under high temperature and high pressure. Based on the solvothermal method, Liu et al. Dissolved ferric chloride (FeCl3) and sodium methoxide (CH3ONa) in propylene glycol, and reacted at 200 ° C for 10 H under high temperature and high pressure environment to prepare ferroferric oxide (Fe3O4) nanoenzyme, which can control the morphology (spherical, octahedral, triangular) of the obtained Fe3O4 nanoenzyme by adjusting the feeding amount of CH3ONa, and then adjust its peroxidase activity[236]. Based on the solvothermal method, Wu et al. Obtained a series of molybdenum diselenide (MoSe2) nanoenzymes exposing different crystal faces by adjusting the ratio of N, N-dimethylformamide (DMF) and water, and found that the increase of the amount of DMF was beneficial to the increase of the number of (002) crystal faces of MoSe2 nanoenzymes, and the Tafel slope of hydrogen evolution reaction (HER) catalyzed by MoSe2 nanoenzymes rich in (002) crystal faces decreased from 100 to 62 mV·dec-1, showing stronger HER electrocatalytic activity[255].
The liquid phase precipitation method is a preparation method for directly generating a metal compound nanoenzyme by mixing a metal salt solution and a precipitant. Ayodhya et al. prepared copper sulfide (CuS), zinc sulfide (ZnS) and cadmium sulfide (CdS) nanoenzymes by adding metal acetate dropwise to an equal volume of sodium sulfide solution based on a liquid precipitation method, and found that CdS nanoenzyme had stronger photocatalytic activity among the three nanoenzymes due to its larger specific surface area, and its catalytic degradation of BTB dye was as high as 83.42% within 60 min[237]. Zhang et al. Prepared polyhedral, cubic, sheet-like and rod-like cobaltosic oxide (Co3O4) nanoenzymes by liquid precipitation method and solvothermal method, respectively (Fig. 31),The results showed that the peroxidase-like activity and catalase-like activity of the sheet-like Co3O4 nanoenzyme with (112) exposed crystal face were as high as 0. 031 and 3. 23 U/μg, respectively, and the higher surface Co3+ ratio made it show stronger peroxidase-like and catalase-like activities in various morphologies of Co3O4 nanoenzyme[124].
The thermal decomposition method refers to a preparation method for obtaining the corresponding metal compound nanoenzyme by pyrolyzing a metal compound precursor in an oxygen-free organic solvent. Gaur et al. Synthesized CdS nanoenzymes based on thermal decomposition method, and a series of CdS nanoenzymes were obtained by mixing cadmium salts containing different anions with thiourea to form cadmium thiourea complexes, which were then treated at high temperature[238]. The authors discussed in detail the differences in physicochemical properties of CdS nanoenzymes prepared under two different conditions of solid phase thermal decomposition and liquid phase thermal decomposition of cadmium thiourea complex, and pointed out that the photocatalytic activity of CdS nanoenzymes prepared by solid phase thermal decomposition was three times higher than that prepared by liquid phase thermal degradation in the degradation of violet.
Carbon-based nanoenzyme materials mainly include fullerenes (C60), carbon nanotubes (CNTs), graphene oxide (GO), carbon dots (CDs) and their doped derivatives or composites, which have attracted wide attention in the field of nanoenzyme research due to their excellent physicochemical properties. The preparation methods of carbon nanoenzyme, metal/metal compound composite carbon-based nanoenzyme and atom doped carbon-based nanoenzyme are introduced below.
The enzyme-like catalytic properties of carbon nanomaterials have been widely reported, which are composed of carbon atoms tightly bound in the honeycomb lattice sp2 bonding, and have the characteristics of good conductivity, high specific surface area, and good chemical stability. At present, the main preparation methods of carbon nanomaterials include chemical vapor deposition, microwave-assisted synthesis, hydrothermal method and exfoliation (liquid phase exfoliation, electrochemical exfoliation and intercalation exfoliation)[256⇓⇓~259]. For example, Nghia et al. Synthesized GO with excellent peroxidase-like activity based on the classical Hummers method and ultrasonic stripping method, which can be used for sensitive detection of Cr6+[239].
Composite carbon-based nanoenzyme is prepared by loading metal or metal compounds with certain catalytic activity on carbon nanomaterials in a coating or deposition manner to prepare composite nanomaterials with more excellent catalytic performance. At present, impregnation method, sol-gel method and high temperature calcination method have been developed to realize the construction of carbon-based supported metal compound nanoenzymes[240,260,261]. For example, Wahab et al. Used impregnation method and high temperature calcination method to integrate Fe3+ as the catalytic active center and three-dimensional mesoporous carbon (3D-MC) as the carrier and substrate binding site to prepare biomimetic iron oxide-composite mesoporous carbon nanoenzyme (IO-MC) for colorimetric detection of glucose (Fig. 32A)[241].
Atom-doped carbon-based nanoenzymes refer to the preparation of carbon-based nanoenzymes by doping one or more metals/heteroatoms into carbon nanomaterials. After doping with metal or heteroatom, the element composition and surface electronic environment of carbon materials will change, thus effectively improving the catalytic performance of materials[262]. The preparation methods of the nanoenzyme include high-temperature carbonization method, solvothermal method, high-temperature pyrolysis method, chemical vapor deposition method and the like[263,264]. For example, Lou et al. Synthesized a high nitrogen-containing carbon-based nanoenzyme by high temperature pyrolysis using a negatively charged natural clay mineral (i.e., montmorillonite, MMT) as a hard template and a positively charged polyethyleneimine (PEI) with a high nitrogen-to-carbon ratio as N and C sources (Fig. 32B)[242]. The results showed that the N-doped CNase had good peroxidase-like activity and could be used for the determination of hydrogen peroxide, glucose and ascorbic acid.
In addition to the above preparation methods, carbon-based nanoenzymes can also be prepared by post-functional modification, template-assisted method and adjusting the geometric structure of carbon-based materials[265][266][267]. In a word, carbon-based nanoenzyme not only has the excellent performance of nanomaterials, but also has enzyme-like catalytic function, which has potential applications in biocatalysis, environmental governance, chemical industry and other fields.
MOFs are unique organic-inorganic hybrid porous materials with periodic network structure, which are self-assembled by inorganic metal centers (metal ions or metal clusters) and organic ligands. MOFs materials have unique chemical composition, ordered porous structure, large specific surface area, and tunable structure. In addition, the high content of metal centers and easy modification also endow MOFs with intrinsic enzyme-like activity. MOFs can be used as precursors or templates to synthesize nanoenzymes with high stability and catalytic activity, and can be widely used in catalysis, sensing, detection and imaging.
At present, the main preparation methods of MOFs nanoenzyme in the laboratory are as follows: direct synthesis method, in which metal ion salts are mixed with specific ligands to synthesize MOFs under certain conditions, such as Fe-MOFs, Zn-MOFs, Cu-MOFs, Co-MOFs and Ce-MOFs. For example, Li et al. Prepared MOF-818 by direct synthesis (fig. 33a), which showed high catechol oxidase activity and good specificity[138]. The post-synthesis modification method is to introduce new metal ions or ligands into MOFs which do not have enzyme-like catalytic activity or have weak activity, so that they have enzyme-like catalytic activity. For example, Zhang et al. Used a wet chemical method to prepare ZIF-8-graphene oxide (ZIF-8-GO), further introduced and , and obtained AuPt nanoparticle-composite ZIF-8-rGO through a reduction method, which showed enhanced peroxidase activity and was used for the detection of H2O2 in human serum samples with ideal performance (Fig. 33 B)[243]. The composite preparation method is to use MOFs as a carrier to load other nanoenzymes to prepare composite materials. Wang et al. used ZIF-8 as a carrier to integrate GOx and gold (Au) nanoenzyme to prepare ZIF8/Au-GOx NPs (Fig. 33c), which showed a multimodal antibacterial therapeutic effect[244].
Different metal centers and organic ligands can endow MOFs materials with different space types and crystal structures, enabling MOFs nanoenzymes to form not only three-dimensional crystal structures, but also two-dimensional sheet structures. Compared with the three-dimensional structure, two-dimensional ultrathin MOFs nanosheets (2D MOFs) show more excellent catalytic efficiency due to more abundant active sites and larger specific surface area. For example, 2D MOFs of iron porphyrin [Cu-TCPP (Fe)] have peroxidase-like activity and higher fluorescence quenching efficiency than graphene oxide and bulk MOFs[268].
Although great progress has been made in the research and application of nanoenzymes, there are still two challenges. Firstly, the low density distribution of nanoenzyme active sites leads to the fact that the activity of nanoenzymes is much lower than that of natural enzymes[269]. Secondly, the catalytic mechanism of nanoenzymes is complicated due to the differences in element composition, size structure, and crystal face exposure[61]. Monoatomic nanoenzymes mimic the M-NX of natural enzymes with transition metal-derived macrocyclic structures (M-N-C, M refers to transition metal), thus having a clear electronic and geometric structure, which is expected to become an alternative to traditional enzymes. More importantly, the enzyme-like activity and catalytic mechanism of monoatomic nanozymes mainly depend on the spatial configuration of the active center, which will facilitate the unified study of the enzyme-like activity and catalytic mechanism of different monoatomic nanozymes[74]. The synthesis of monoatomic nanoenzymes is mainly based on the two principles of preventing atom aggregation and enhancing metal-support interaction, and there are some main synthetic strategies such as steric constraint and metal exchange[270].
The steric constraint strategy is to obtain the nanoenzyme with good monoatomic dispersion by inhibiting the aggregation and migration of atoms during pyrolysis. Huang et al. Prepared N-coordinated Fe monoatomic nanoenzyme (Fe-N5SA/CNF) constrained by carbon nanoframes, and calcined and pyrolyzed MOF-encapsulated iron phthalocyanine at 900 ° C in nitrogen atmosphere. During the carbonization process, the isolated FeN4 sites were constrained by carbon nanoframes, forming thermodynamically more stable FeN5/C sites, which are similar to the active sites of natural cytochrome P450 (Figure 34 A)[74]. Experiments show that the monoatomic nanoenzyme has good oxidase-like catalytic activity.
MOF material is a new type of porous material, which can produce a large number of coordination sites after treatment, and can be coordinated and anchored with metal atoms. By doping other metal ions, the single-atom nanoenzyme to be doped with metal can be obtained. Cao et al. Used a controllable metal-organic coordination strategy to prepare more than 20 kinds of single-atom artificial metal nanoenzymes (AMEs) doped with different metal atoms by reacting zinc ions (major ions), heteroions (minor ions) and 2-methylimidazole to form metal precursors, and then calcining and pyrolyzing them.The characterization results show that this series of monoatomic nanoenzymes are similar in terms of structural morphology, size, elemental composition, and crystal face exposure (Fig. 34b), which will be helpful for the comparison of nanoenzyme activities and the study of catalytic mechanism[245].
In addition to the above preparation methods of monoatomic nanoenzymes, researchers have developed some methods to synthesize monoatomic nanoenzymes, such as self-assembly method, sacrificial template method, electrochemical corrosion method, low temperature (< 0 ℃) photocatalytic reduction method, etc[271][272][273][274].
In order to further improve the catalytic efficiency of nanoenzymes and make them have multi-enzyme catalytic activity, it is a common preparation strategy to assemble different kinds of nanoenzymes to form composite nanoenzymes[275]. For example, Huang et al. Used V2O5 nanowires to mimic GPx,MnO2 nanoparticles to mimic SOD and perCAT, and dopamine as a linker to prepare a V2O5@pDA@MnO2 nanocomposite. This multi-nanoenzyme synergistic platform can effectively mimic the antioxidant enzyme defense system in cells, and has potential applications in the treatment of inflammation (Fig. 35 A – C)[246]. Wang et al. First synthesized PtCo nanoparticles, and then used the KMnO4 reduction method to control the growth of MnO2. By adjusting the ratio of reactants, they prepared MnO2@PtCo biomimetic nanoflowers with good catalytic efficiency (Fig. 35d, e)[276]. Yang et al. First prepared ZIF-8 nanocarriers using a two-solvent synthesis strategy, and SiO2 was encapsulated on the surface of ZIF-8 particles according to the Stober method[247]. ZIF-8-derived hollow porous carbon nanoenzyme was obtained by chemical etching and high temperature carbonization. Finally, the ultra-small Pt nanoenzyme was immobilized into the MOF-derived carbon nanoenzyme by in situ reduction method, and the Pt-C integrated nanoenzyme with good catalase-like activity and photosensitivity was successfully constructed. Kong et al. First prepared magnetic Fe3O4 nanoparticles with uniform particle size distribution by high temperature pyrolysis, then modified Fe3O4 nanoparticles with PEG with functional groups at the end, and then used the functional groups on the particle surface to adsorb Pt ions and prepared Pt nanoparticles by in situ reduction to form Fe3O4@Pt binary composite nanoenzyme (Fig. 35 f, G)[248]. Sun et al. Assembled magnetic Fe3O4 nanoparticles prepared by high temperature pyrolysis and ultra-small CeO2 nanoparticles prepared by hydrothermal method by chemical coupling to prepare Fe3O4@CeO2 composite nanoenzyme with coronal morphology, and the assembled nanoenzyme could play a short-range relay catalytic role of multienzyme-like activity (Fig. 35 H)[249];
In addition, they also prepared a Au@CeO2 composite nanoenzyme assembled by Au nanoparticles and CeO2 nanoparticles through a one-pot method. The assembly prepared by this method also has a coronal morphology (Figure 35i), which can play a relay catalytic role in various enzyme-like activities[277].
3.2.2 Characterization of nanozyme
The size, morphology, exposed crystal face, chemical composition and surface modification of nanoenzyme are the key factors to determine its catalytic performance and application[278]. In order to explore the complex relationship between the structure and properties of nanoenzymes, it is necessary to characterize and analyze them at the nanoscale and atomic scale. The common characterization methods of nanoenzymes are summarized from three aspects: morphology analysis, composition and structure analysis, and surface and interface analysis.
The morphology analysis of nanoenzyme mainly uses electron microscopy, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to analyze and measure the morphology, size, size distribution and aggregation state of particles. By combining with other techniques, such as selected area electron diffraction (SAED), high resolution TEM, and energy dispersive X-ray spectroscopy (EDS), reliable information about the crystal structure, atomic arrangement, and elemental composition and distribution of materials can be further obtained.
Scanning tunneling microscope (STM) can observe conductive samples with atomic resolution by using the tunneling effect in quantum theory. For the observation of the surface of an insulator material, an atomic force microscope (AFM) can be selected. Higher angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) allows observation of finer structures and lower concentrations of components. Due to its excellent single-atom imaging capability, HAADF-STEM has been widely used in the characterization of single-atom nanoenzymes in recent years. For porous materials, BET (Brunner-Emmet-Teller measurements) can be used to measure and calculate the specific surface area and pore size of particles. In addition, the hydrodynamic size and surface potential of nanoparticle solution can be measured by dynamic light scattering (DLS), which reflects the real state of nanoparticles in solution.
The composition and structure analysis of nanoenzyme mainly includes the characterization of its phase composition, elemental composition, crystal structure and electronic structure. X-ray diffraction (XRD) is an effective method to identify the crystalline phase and crystallinity of a substance. Through the analysis of the position and intensity of the diffraction peak, the information of the phase, structure and intermolecular interaction of the material can be obtained. In addition, the grain size of the particle can be calculated by using the full width at half maximum and the position of the diffraction peak (Scherrer formula). Atomic absorption/emission spectrometer, inductively coupled plasma mass spectrometer (ICP-MS) or X-ray fluorescence spectrometer (XFS) can be used to measure the bulk phase elemental composition and doped elements of nanoenzymes. Modern synchrotron radiation light sources have important applications in many fields, such as materials science, chemistry, environmental science, life science, medicine and so on, because of their high brightness, high polarization, broadband, narrow pulse and other characteristics, which can characterize the research objects with high spatial and temporal resolution. The combination of conventional laboratory characterization methods and synchrotron radiation technology is the "key" to open the wonderful nano-world. For example, X-ray absorption fine structure spectroscopy (XAFS) based on synchrotron radiation X-ray technology can directly provide atomic electronic structure information (such as valence, band structure, electronic orbital hybridization, charge transfer, electronic orbital spin, etc.) And coordination environment information (coordination atom, coordination number, etc.).It can provide important information for the study of catalytic mechanism of nanoenzyme.
For the surface and interface analysis of nanoenzymes, there are many methods that can be used, such as scanning probe microscopy, spectral analysis, and thermal analysis. Among them, X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) can be used as qualitative and semi-quantitative analysis of surface elements of nanoenzyme, as well as chemical state analysis of surface elements. Electron energy loss spectroscopy (EELS) is also an analytical method to obtain physical and chemical information of surface atoms and molecules. It has the ability of micro-area analysis that XPS does not have. Compared with AES, EELS has more sensitive characteristics and can obtain more surface information. The surface defect structure was proved to play an important role in improving the catalytic performance of nanoenzyme[213]. These defect structures can be characterized by aberration-corrected HAADF-STEM, STM, electron paramagnetic resonance (EPR), XRD, XPS, XAFS, Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy (Raman), positron annihilation spectroscopy (PALS), solid-state nuclear magnetic resonance spectroscopy, etc. In addition, DFT is also a common means to study the properties of vacancies through theoretical calculations. Nuclear magnetic resonance spectroscopy (NMR) can show the Lewis acid strength of the material surface through the chemical shift of the probe molecule combined with the surface of different nanoenzymes, and then qualitatively analyze the electron density of the material surface, which is used to explore the relationship between the crystal face with different electron density and the enzyme activity. Thermal analysis methods mainly include thermogravimetric analysis (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC), which are often used to study the existence, content and thermal weight loss temperature of organic groups on the surface of nanoenzymes. For the detection of nanoenzyme surface modifiers, appropriate characterization methods can be selected according to the unique properties of the modifiers themselves.
In addition to the methods mentioned above, other characterization techniques in the field of nanomaterials are also applicable to nanoenzymes. Each method has its advantages and disadvantages, and it often requires a combination of multiple techniques to have a more detailed understanding of the microstructure of nanoenzymes. With the progress of nanoscience and technology, new characterization methods will continue to emerge in the future, which will promote researchers to deeply understand the complex relationship between nanostructure and catalytic properties, and develop a new generation of more efficient and intelligent artificial nanoenzymes.
3.3 Synthesis of nanoenzyme
Nanozyme polymerizes the superior properties of natural and artificial enzymes, including high activity, good selectivity, high stability and reusability, and shows broad application prospects in the fields of medicine, agriculture, environment, food and pharmacy. How to realize the engineering preparation of nanoenzymes will become a key issue for the practical application of nanoenzymes. There are two ways to synthesize nanoenzymes: "top-down" and "bottom-up". The former is to decompose large-sized materials into smaller nanoparticles through various etching techniques, while the latter is to assemble smaller structural units (such as atoms, molecules, nanoparticles, etc.) Into nanosized complex structures through interaction[279,280][281]. Based on the above two preparation strategies, this section outlines the possible ways to achieve nanoenzyme engineering, including physical synthesis, chemical synthesis, and biosynthesis (Fig. 36).
3.3.1 Physical synthesis method
Physical synthesis is a typical "top-down" nanomaterial synthesis strategy, which uses optical and electrical technologies to evaporate materials in vacuum or inert gas, and then makes molecules or atoms form nanoparticles, as well as mechanical and spraying preparation technologies based on mechanical processes (Fig. 37). Although the purity of nanoparticles prepared by physical methods is high, the yield is low and the equipment cost is very high. In this section, several physical synthesis methods of nanoenzyme will be listed, including ball milling, ion sputtering, and plasma.
The ball milling method is to grind micron or submicron particles for a long time and then disperse them into the medium to obtain nanoparticles, such as micron-sized long-chain MoO3 nanobelts through ball milling.Then co-intercalated with Na+ and H2O to obtain Na+/H2O co-intercalated MoO3 nanobelts (NH-MoO3). Compared with the inert MoO3, the NH-MoO3 nanobelts exhibited good peroxidase-like activity[282]. Activated carbon and metal sulfides can be used to obtain well-dispersed peroxidase-like nanoenzyme by ball milling, which can be used in medical diagnosis fields such as glucose detection[283⇓~285]. In addition, the ball milling method is also an effective method for preparing monatomic nanoenzymes, which relies on mechanical force to break the chemical bonds between carrier molecules and form defects to capture metal atoms[286,287]. Gan et al. Used Ce (Ⅲ) acetate tetrahydrate and Au (Ⅲ) acetate as precursors to realize the kilogram-scale preparation of Au/CeO2 single-atom catalyst by ball milling[288]. High-loading Pt single-atom catalysts can also be prepared directly and rapidly by ball-milling Pt precursors and nitrogen-doped carbon supports[289]. Therefore, this environmentally friendly and low-cost ball milling method is expected to be an effective method for nanoenzyme engineering.
Ion sputtering is the main method to prepare metal nanoparticle clusters and nanostructured films. In that method, metal is Mel by a hot cathode, and the hot cathode is impacted by high-speed gas ion generated by high-voltage discharge, so that molten atoms or molecules are evaporate and deposited on the surface of a carrier to form nano particles, such as a Cu/CeO2-x nano catalyst prepared by a radio frequency plasma sputtering method, which can be used for the oxidation reaction of carbon monoxide[290]. Wang et al. Synthesized TiO2 encapsulated Au nanoparticles (Au@TiO2) by ion sputtering and atomic layer deposition technique with good peroxidase-like activity, which can be used for the detection of glucose and hydrogen peroxide[291]. Sputtering method has many advantages, including large evaporation area of target material, high particle recovery rate, uniform particles and narrow particle size distribution, which is suitable for the preparation of high melting point metallic nanoparticles.
Plasma preparation of nanoenzyme has the advantages of high efficiency, low cost and cleanness. Wang et al. Utilized plasma-assisted nitrogen doping of MoS2 nanosheets, resulting in efficient nanozymes[262]. The peroxidase-like activity of the nanosheets was significantly enhanced with the plasma treatment time. Using an efficient plasma treatment strategy, carbon-based green nanoenzyme (N-QG) can be obtained by doping nitrogen into graphene at room temperature, and its catalytic activity is nearly 5 times higher than that of undoped nanomaterials. Therefore, plasma-assisted nitrogen doping can improve the electrical conductivity of QG and create surface defects, thereby facilitating the enhanced transfer of electrons to the catalytic activity of N-QG[292].
3.3.2 Chemical synthesis
Chemical synthesis is the preparation of nanomaterials from molecules, atoms and ions through appropriate chemical reactions (Figure 38), including hydrothermal and solvothermal methods, chemical reduction methods, sol-gel methods and coprecipitation methods. Chemical synthesis is the main means of nanoenzyme preparation at present.
Hydrothermal method and solvothermal method are to heat water or solvent in a sealed pressure vessel to form a high temperature and high pressure environment in the reaction system, so as to realize the controllable synthesis of nanomaterials[294]. Fe-doped MoS2 nanosheets (Fe-MoS2) were prepared by a one-pot hydrothermal method, and the peroxidase-like activity could be controlled by adjusting the molar ratio of Mo/Fe, and the established Fe-MoS2 nanoenzyme colorimetric method has the feasibility of practical application[295]. Liu et al. First synthesized novel FeS2@ covalent organic framework (COF) nanozymes (FeS2@SNW-1) with peroxidase-like activity by hydrothermal and solvothermal methods inspired by natural enzymes[296]. Ji et al. Prepared ZIF-8 by a solvothermal method, using phosphorus atoms as the precursor of the synthesis system to anchor the metal active center and adjust the local coordination structure, and then polymerically coated Fe ions and poly (cyclotriphosphazene-4,4 ′ -aromatic copolyamide) (PZM) on ZIF-8 to prepare Fe/ZIF-8 @ PZM core-shell composite single-atom nanoenzyme (FeN3P-SAzymes), and the precise coordination structure of the designed FeN3P-SAzymes was beneficial to improve the catalytic performance[15]. In addition, hydrothermal and solvothermal methods are also widely used for the preparation of CuO nanoenzyme, CoO nanoenzyme and Fe3O4/Ag/Bi2MoO6 nanoenzyme, etc[297][298][299].
Chemical reduction has become the most commonly used method for the preparation of nanocatalysts because of its simplicity and rapidity. The morphology and particle size of nanoparticles can be effectively controlled by changing the molar concentration of reactants, the type of reducing agent and the reaction temperature[300]. The selection of appropriate reducing agent is the key factor for the efficient preparation of nanomaterials by chemical reduction. In the synthesis process, too fast reaction rate can lead to smaller particles, while too slow reaction rate may lead to particle aggregation[301][302]. Wang et al. Prepared hollow CuS nanocubes with an average particle size of 50 nm by chemical reduction method, which had a larger specific surface area than CuS of general solids, and showed better electrocatalytic and photothermal properties[303]. A peroxidase-like AuNPs with an average particle size of 20 – 30 nm was synthesized by chemical reduction, which can be used for rapid, accurate, and on-site detection of pathogens in water or biological fluids by binding to Pseudomonas aeruginosa specific nucleic acid aptamers[233]. A multifunctional artificial enzyme was synthesized by combining chemical reduction and electrodeposition to modify Pt NPs on a metalloporphyrin organic framework (PMOF). In the Pt @ PMOF (Fe) composite, PMOF (Fe) can prevent the aggregation of Pt NPs, resulting in high stability of Pt NPs[304]. Zhang et al. Successfully doped 6-aminocaproic acid-stabilized CeO2 with single-atom Pd by electrostatic adsorption of the precursor PdC followed by further reduction with NaBH4[305]. Compared with CeO2, single-atom Pd/CeO2 nanostructures have excellent peroxidase-like and catalase-like activities.
The sol-gel method is to disperse the raw materials in a solvent, generate active monomers through hydrolysis reaction, form a sol through polymerization, and then generate a gel with a certain spatial structure. By adjusting the concentration, type and temperature of chemical additives, the physical and chemical properties of nanoenzymes, such as crystallinity, morphology and magnetism, can be controlled[306]. The method has the advantages of simple operation, high nanoparticle purity, good uniformity, narrow particle size distribution and the like. Yu et al. Synthesized peroxidase-like Pt nanoparticle polyaniline hydrogel (PtNP/PAni) with a diameter of about 100 nm by sol-gel method using phytic acid as a complexing agent[307]. He et al. Proposed a mesoporous nanoenzyme, which not only has peroxidase-like activity, but also can cooperate with anticancer drugs to treat tumors[308]. Fe-doped mesoporous SiO2 nanoenzyme (FeMSN) was prepared by sol-gel method. The introduced Fe endows FeMSN with peroxidase-like activity, which can decompose H2O2 into ·OH under acidic conditions for chemokinetic treatment of tumors.
Coprecipitation method is to prepare nano-materials with uniform chemical composition from two or more cations in the solution through the action of precipitant. This method is easy to prepare nano-powder materials with small particle size and uniform distribution. The co-precipitation method is a good choice when higher purity and better stoichiometric control are required. Lan et al. Used the co-precipitation method to synthesize two kinds of nanomaterials, namely carbon spheres (Mn-MPSA-HCS) and hollow carbon cubes (Mn-MPSA-HCC) with a size of 100 – 200 nm, where a monolayer of Mnx(PO4)y was uniformly dispersed on the surface of the carbon structure, thus providing a large number of active sites for the reaction of analytes[309]. This method can be used to prepare transition metal phosphates of various shapes and sizes. The coprecipitation method has also been widely used to prepare layered double hydroxide nanoenzyme (LDH), such as Co/Fe LDH with oxidase activity, Ni/Co LDH with peroxidase activity and FeII/FeIIILDH[310][311][312].
In addition to the above chemical synthesis methods, polymerization and polycondensation methods, wet chemical methods, and electrochemical deposition methods are also widely used for the chemical synthesis of nanoenzymes[313⇓~315][316⇓~318][319,320].
3.3.3 Biosynthesis
Compared with traditional physical and chemical synthesis methods, biological synthesis of nanomaterials has the advantages of cost-effectiveness, eco-friendly and low energy consumption, and is expected to become an effective way for large-scale preparation of nanoenzymes. Various microorganisms such as bacteria, fungi, algae or plants can be used as "nanofactories" (Fig. 39)[321]. At present, nanomaterials synthesized by biological methods have been used in various applications, such as heavy metal removal, industrial wastewater treatment, biomedicine and biosensors[322].
Compared with other organisms, prokaryotic bacteria are relatively easy to manipulate and have become the main type of bacteria for the synthesis of metal-containing nanoparticles. Studies have shown that nanoparticles can be synthesized in the plasma membrane of lactic acid bacteria to produce a variety of highly structured Au and Ag nanocatalysts[324]. The synthesis of Au nanoparticles was realized by changing the pH of Shewanella. At pH = 7, the size of the synthesized nanoparticles was 10 ~ 20 nm. When pH = 1, the particle size is adjusted to 50 ~ 500 nm[325]. In addition to affecting the particle size, pH also has a significant effect on the morphology of nanoparticles. When Rhodopseudomonas cupulosa was used to synthesize Au nanoparticles, the synthesized nanoparticles were spherical at pH = 7, while the nanoparticles were flat at pH = 4[326]. In addition, Bacillus, Halobacterium, Brevibacterium, etc. Can be used for the biosynthesis of nanocatalytic materials[327,328][329][330].
Fungal synthesis of nanomaterials has many advantages, such as large surface area, high bioavailability, easy amplification and downstream processing[331]. At the same time, fungi are relatively easy to grow under laboratory and scale-up process conditions, and have better application prospects for large-scale preparation of nanomaterials. In addition, extracellular enzymes, peptides and secondary metabolites produced by fungi contribute to the synthesis of nanoparticles with different morphologies and sizes. Several fungal species such as Fusarium oxysporum, Aspergillus oryzae, Verticillium dahliae, Alternaria and Colletotrichum can be used for the synthesis of nanoparticles[332]. For example, Fusarium oxysporum was used to synthesize pure Ag nanoparticles with a particle size of 5 ~ 15 nm, and the nanostructure was stabilized by fungal protein[333]. Mukherjee et al. Reported the method of producing Au nanoparticles by Verticillium sp[334]. Riddin et al. Successfully realized the intracellular and extracellular synthesis of Pt nanoparticles using Fusarium oxysporum[335].
The preparation of nanoparticles from algae has the advantages of low toxicity and low temperature, and the speed of nanoparticle synthesis from algae is faster than that from other biological agents. A variety of algae have been used to prepare nanoparticles of various sizes and shapes. For example, Chlorella vulgaris can convert more than 90% of tetrachloroaurate into metallic state and deposit it inside and outside the cell, showing icosahedral, decahedral and tetrahedral structures[336]. Chlamydomonas reinhardtii can synthesize Ag nanoparticles in vitro and in vivo, but the synthesis rate in vitro is slow, and the synthesized nanoparticles are circular with the size of 5 ~ 15 nm, while the synthesis rate in vivo is fast, and the synthesized nanoparticles are rectangular with the size of 5 ~ 35 nm[337]. Spirochaeta can convert Au3+ to Au0+, so they were able to synthesize spherical, hexagonal, and triangular Au nanoparticles with particle sizes ranging from 2 to 50 nm[338]. In addition, Spirulina, Sargassum, Pinellia and so on can be used for the synthesis of metal nanoparticles[339,340][341][342].
There are relatively few studies on the preparation of nanoenzymes by biosynthesis, most of which use natural plant extracts as reducing agents to synthesize nanoenzyme materials in vitro, such as Nath et al., using cerium ammonium nitrate and zirconium oxychloride as raw materials and β-cyclodextrin as stabilizer.Cerium zirconate (CeZrO4) nanoenzyme was synthesized in vitro by using neem leaf extract as a reducing agent, which showed peroxidase and oxidase activities at the optimum pH of 4.0, and had potential anti-tumor applications[343]. Nguyen et al. Synthesized Ag NPs with a particle size of 14 nm by using saponin extract as a reducing agent, which showed peroxidase activity and had a good application prospect in glucose and ascorbic acid detection[344].
3.3.4 Brief summary
Nanoenzyme engineering is a technical system for large-scale preparation of nanoenzymes with stable physical and chemical properties to replace natural enzymes for industrial production. At present, the preparation methods of nanoenzyme mainly include physical synthesis, chemical synthesis and biological synthesis, among which the chemical method is widely used in scientific research and production because of its mild reaction, easy control, small equipment investment, and avoiding the introduction of other impurities in the physical method and the oxidation of particles in the air. In particular, the sol-gel method and the precipitation method in the chemical method can prepare the particles with uniform dispersion, good particle shape and high catalytic activity in the catalytic reaction. Although some achievements have been made in the preparation of nanoenzymes, there are still many problems to be solved in the actual production of nanoenzymes.It is mainly reflected in the following aspects: (1) The existing preparation technology is still in the stage of laboratory and small-scale production, and there are still many technical bottlenecks to be solved when it is applied to large-scale production, which will be the first problem to be solved in the realization of nanoenzyme engineering; (2) Due to the small particle size of nanoenzyme, it is easily oxidized, hygroscopic and agglomerated in the air, and its performance is very unstable, which brings many difficulties to industrial production and application, and reduces its performance; (3) The mechanism of the change of nanoenzyme activity and its kinetic and thermodynamic processes in the preparation still need to be further studied; (4) In today's implementation of green chemistry and advocacy of green synthetic chemistry, the research on biosynthesis and engineering application of nanoenzymes will become a difficult, hot and cutting-edge issue for nanoenzyme workers in the future.
4 Application of nanoenzyme
Catalysts have a wide range of important applications in chemical production, medicine and health, environmental protection and other fields, and are the cornerstone of modern social and economic development. According to statistics, the global catalyst market will be 35.5 billion US dollars in 2020 and is expected to reach 57.5 billion US dollars in 2030, involving more than 90% of the production process of chemical products[345]. However, due to the limited catalytic efficiency of traditional chemical catalysts, the catalytic process still requires high temperature, high pressure, strong acid, strong alkali and other conditions, as well as the support of pressure-resistant and corrosion-resistant materials, and the reaction process involves the use of a large number of organic solvents, which has the characteristics of high energy consumption and high pollution. In recent years, with the rapid growth of energy and material demand and the upgrading of industrial structure under the background of green development, academia and industry are committed to developing various environmentally friendly catalysts. Compared with chemical catalysts, enzymes are green and environmentally friendly biocatalysts with mild reaction conditions, unparalleled high catalytic activity and selectivity. However, the shortcomings of industrial enzymes in application are particularly obvious. First of all, most industrial enzymes need to be produced by microbial fermentation, and only high-yielding strains that have been strictly selected and studied for a long time can be used, and the fermentation process should strictly prevent contamination by miscellaneous bacteria. The enzyme produced by fermentation has to go through tedious screening, identification, separation, purification and preservation processes to obtain usable enzyme preparations. In addition, because the enzyme is very sensitive to the environment and is easily inactivated under non-mild conditions, the reaction conditions should be strictly controlled during catalysis. In addition, it is difficult to recover and separate the enzyme in the reaction mixture from the catalytic system, and thus it is difficult to realize continuous production. To sum up, although more than 3000 kinds of enzymes have been identified, only more than 60 kinds of enzymes can be produced on a large scale, which are mainly used in food processing. Although it is closely related to various life activities in organisms, the application of enzymes in the field of medicine and health is very limited because they are easily degraded and inactivated by proteases.
As a new generation of mimetic enzyme, nanoenzyme has a broader application prospect than traditional chemical catalysts and enzymes. Because of its enzyme-like catalytic activity, nanoenzyme has mild working environment, low energy consumption and low pollution, which meets the needs of green development. In addition, compared with enzymes, the structure of nanozymes is more stable and easier to produce on a large scale. In addition to efficient catalytic function, many nanoenzymes also have unique physical and chemical properties such as light, sound, electricity and magnetism. On the one hand, clever use of the bifunctional properties of nanozymes can lead to the design of more practical application strategies. For example, combined with the magnetic property and peroxidase-like catalytic activity of Fe3O4 nanoenzyme, a new enzyme-linked immunosorbent assay method integrating magnetic enrichment substrate and catalytic coloration can be created[148]. On the other hand, because the physical and chemical properties and catalytic characteristics of nanoenzymes are determined by their nanostructures, the use of magnetic, optical, electrical and other media to regulate the structure of nanoenzymes is expected to regulate their catalytic activity. In contrast to the high specificity of enzymes, nanozymes have a broad spectrum of substrates and often have multiple catalytic activities. This pan-catalytic property makes nanoenzyme have certain advantages in sewage treatment, antibacterial, tumor inhibition and other applications with broad-spectrum catalytic requirements. In addition, some catalytic activities of nanozymes can be catalyzed in cascade, which has been proved to have synergistic functions in antioxidant and other fields. However, the generally low selectivity makes it difficult for nanozymes to be applied in areas where there is a specific need for catalysis. Therefore, how to improve the selectivity is one of the key directions of future application research of nanoenzyme.
In recent years, with the continuous expansion of catalytic types and the continuous improvement of catalytic activity, the application research of nanoenzymes has become increasingly extensive and in-depth. At present, the application research of nanozymes mainly focuses on the biomedical field, involving disease diagnosis, biochemical detection and sensing, anti-tumor, anti-microbial, anti-inflammatory and antioxidant. In addition, some nanoenzymes have also been developed for applications such as sewage treatment, chemical synthesis, and chemical decontamination. It is particularly noteworthy that with the gradual clarification of the catalytic mechanism and structure-activity relationship of nanoenzymes, people are gradually able to rationally design and develop high-performance nanoenzymes for practical problems. Although no nanoenzyme has yet been used in practice, these studies have accumulated valuable experience for the development of real nanoenzyme products. It is believed that with the deepening of mechanism and application research, nanoenzymes will shine brilliantly in various fields related to catalysis in the future.
This section reviews the application research progress of nanoenzymes, mainly involving several aspects of nanoenzyme sensing, anti-tumor, anti-bacterial, anti-virus and environmental governance.This paper focuses on how to develop nanoenzymes with unique catalytic activity and physicochemical properties for practical problems, and looks forward to the feasible design strategies and potential application directions of nanoenzymes.
4.1 Nanoenzyme sensing
The emergence of nanozymes provides new ideas and methods for the sensing field[1]. Nanoenzyme not only has low cost, good stability and easy to prepare in large quantities, but also has the "dual characteristics" of enzyme and nanomaterials, which makes nanoenzyme can be used as a bifunctional or multifunctional material[346]. Based on these characteristics of nanoenzymes, scientists have developed a variety of new sensing technologies and methods, covering infectious disease prevention and control, in vitro diagnosis, environmental monitoring, food safety, forensic evidence and other fields[11,13,36,37]. This section will introduce the progress of nanoenzyme in sensing in recent years and look forward to the future development direction according to the methodological classification of nanoenzyme application.
4.1.1 Colorimetric analysis
Nanoenzyme colorimetric analysis is based on the color reaction of colorless substrate catalyzed by nanoenzyme to produce colored compounds, which has the advantages of simplicity, rapidity, low equipment requirements and visualization of results. Among them, the nanoenzyme with oxidoreductase-like activity realizes color change by catalyzing the oxidation of chromogenic substrates, for example, the corresponding chromogenic substrates of peroxide nanoenzyme include TMB, DAB, ABTS, etc. The nanoenzyme with hydrolase activity catalyzes the hydrolysis of a chromogenic substrate to initiate color change, in which the representative reaction is the hydrolysis of a phosphate ester bond, and the corresponding chromogenic substrate is pNPP (p-nitrophenylphosphate, whose phosphate ester bond is broken to produce 4-nitrophenol).
Following the report of enzyme-like catalytic properties of nanomaterials in 2007, Wei Hui and Wang Erkang constructed a cascade colorimetric analysis system based on GOx and Fe3O4 peroxide nanoenzyme in 2008.GOx catalyzes the oxidation of glucose to provide H2O2,Fe3O4 nanozymes catalyze ABTS coloration in the presence of H2O2 to monitor the concentration of glucose, and this study demonstrates the feasibility of coupling native and nanozymes for cascaded biosensing[8]. Since then, nanoenzyme colorimetric analysis has been combined with a variety of detection technologies, such as nanoenzyme-based colorimetric sensors, test strips, enzyme-linked immunosorbent assay, immunohistochemical analysis, etc., and has been widely used in clinical biomarker detection, environmental pollutant monitoring, food regulation, forensic identification, and other fields[347].
Xia et al. developed a Ni-Pt nanoenzyme, whose activity is 10,000 times higher than that of natural peroxidase. When it is applied to the ELISA immunodetection of carcinoembryonic antigen, it can achieve an ultra-sensitive detection of 1.1 pg/mL, which is hundreds of times lower than the traditional ELISA immunodetection based on natural enzyme (376 pg/mL), which reflects that improving the catalytic activity of nanoenzyme is essential to improve the detection sensitivity (Figure 40)[40].
Qiu et al. Established a colorimetric sensor based on peroxide nanoenzyme for the detection of arsenate by using the catalytic properties of arsenite and arsenate, which can inhibit some nanoenzymes[348][349]. CoOOH nanosheets with peroxidase activity can bind arsenate through As — O bonds and electrostatic attraction, resulting in the inhibition of its nanoenzymatic activity and the weakening of the ABTS colorimetric signal. Compared with other monitoring techniques, the system is highly sensitive to arsenate with a detection limit As low as 3.72 ppb (the maximum content in drinking water according to the World Health Organization guidelines is 10 ppb).
Stevens and Bhatia et al. Designed nanosensors that respond to the disease microenvironment using the peroxidase-mimicking activity of ultra-small gold nanoclusters (AuNCs) and the precise nanoscale filtration of the kidney[350]. Specific proteases highly expressed in the tumor environment cleave AuNCs from the vector, followed by renal excretion of ultra-small AuNCs into the urine. When TMB and hydrogen peroxide are added to the urine, under the catalysis of peroxidase-like AuNCs, the urine will undergo a significant color change, which can intuitively and quickly distinguish tumor-bearing individuals.
In 2012, Yan et al. Developed a new immunohistochemical technique based on ferritin nanozyme[178]. Among them, the heavy chain ferritin shell is responsible for targeting and recognizing the highly expressed transferrin receptor on tumor cells, while the internal iron core plays the role of peroxide nanoenzyme, which is responsible for catalyzing the substrate DAB to produce brown precipitates, thus achieving rapid and sensitive detection of tumor tissues. In addition, by coupling different antibodies, NANase can target and visualize a variety of biomarkers on tissue sections, such as the tumor marker vascular endothelial growth factor, the hepatoma cell-specific peptide SP94, and others[30][351]. These studies further demonstrate the feasibility and universality of using the nanozyme immunohistochemistry platform for pathological diagnosis.
4.1.2 Chemiluminescence method
Nano-enzyme chemiluminescence analysis is based on the chemiluminescence reaction catalyzed by nano-enzyme, which is a process in which the substrate absorbs chemical energy to produce electronic excited state and releases light radiation. Typical examples are peroxi-nanoenzyme/oxi-nanoenzyme and its substrate luminol, alkaline phosphate nanoenzyme and its substrate 1,2-dioxane derivative (AMPPD), etc.
In 2015, in response to the severe situation of the Ebola outbreak in West Africa, Yan et al. Used Fe3O4 magnetic nanoparticles as a peroxidase nanoenzyme probe to efficiently integrate the separation, identification and visual test strip detection of Ebola virus glycoprotein, which overcame the bottleneck problem of the traditional test strip method limited by its low sensitivity and long-term application[148]. At present, the nano-enzyme detection kit developed based on this technology has obtained a number of medical device registration certificates. After the outbreak of the new coronavirus epidemic, the team established a more sensitive chemiluminescent rapid detection test strip platform by using Co-Fe @ hemin peroxide nanoenzyme, which can achieve rapid and sensitive detection of SARS-CoV-2 virus within 16 minutes, and the detection limit of pseudovirus reached the same 360 TCID50/mL as ELISA method, which is conducive to early virus screening and epidemic prevention and control (Figure 41)[352].
Hu et al. Found that cerium oxide nanoparticles had catalytic activity similar to alkaline phosphatase, which could catalyze the dephosphorylation of CDP-star, a chemiluminescent substrate of alkaline phosphatase, thus generating a chemiluminescent signal, while the presence of Al3+ significantly inhibited the catalytic activity of cerium oxide nanoparticles[353]. Based on this phenomenon, they successfully achieved highly sensitive chemiluminescence detection of intracellular Al3+ levels. Subsequently, they and their collaborators further applied cerium oxide alkaline phosphate nanoenzyme to sandwich enzyme-linked immunoassay for prostate specific antigen, and established a nanoenzyme-based chemiluminescence detection platform with a detection sensitivity of 53 fg/mL[354].
4.1.3 Fluorescence detection
Fluorescent detection methods based on nanozymes can be achieved in a variety of ways, including direct catalysis of fluorescent substrates by nanozymes to generate fluorescent signals, and indirect enhancement or quenching of fluorescent signals by products of nanozymecatalyzed reactions.
Su et al. Constructed a ratiometric fluorescent sensing platform based on peroxidase nanoenzyme for glucose detection[355]. Firstly, glucose oxidase catalyzes the oxidation of glucose and provides H2O2 for the subsequent reaction, and then the fluorescent substrate o-phenylenediamine (OPD) is converted into the fluorescent substance 2,3-diaminophenylhydrazine (DAP) under the catalysis of the peroxidase activity of copper-doped carbon-based nanoenzyme (CuAA), which has a maximum emission at 558 nm. The fluorescence of Mg/N-doped carbon quantum dots (Mg-N-CQDs) at 444 nm was quenched by DAP due to the fluorescence inner filter effect, and the proportional fluorescence signal I558/I444 increased linearly with the glucose concentration in a certain range, which could quantify the glucose concentration. The method was validated in human serum samples with a detection limit of 1.56 μmol/L.
Ju and Wei et al. Developed a novel dual-mode microfluidic sensing platform based on CuO nanoenzyme as a Photoelectrochemical (PEC) -fluorescent multifunctional signal marker for the detection of neuron-specific enolase (NSE, one of the biomarkers for small cell lung cancer)[356]. The specific antibody immobilized on the PEC sensor captures NSE, which is subsequently recognized and bound by the primary antibody and the nanozyme-labeled secondary antibody in succession. CuO nanoenzyme, as an oxidase mimetic, efficiently catalyzes the oxidation of ascorbic acid with oxygen as an oxidant, resulting in the decrease of photocurrent signal. At the same time, the product of oxidative dehydrogenation of ascorbic acid reacts with OPD to release a fluorescence signal. This sensing strategy has been validated in human serum samples with good stability, reproducibility, and selectivity, achieving ultrasensitive detection of NSE in the range of 0.0001 – 150 ng/mL (PEC method) and 0.001 – 150 ng/mL (fluorescence method). Moreover, the introduction of microfluidic devices has enabled the miniaturization and automation of biosensors. Here, the nanozyme acts as a quencher of the PEC signal and a starter of the fluorescence signal at the same time, providing a new strategy for building an efficient dual-mode biosensing platform (Figure 42).
4.1.4 Electrochemical detection
Electrochemical biosensors can be divided into two categories: biocatalytic sensors and affinity sensors[357]. In the biocatalytic sensor, the nanoenzyme directly catalyzes the chemical reaction of the target analyte to produce an electroactive substance for signal transduction, thus realizing the quantitative detection of the substance. Some small molecules, such as H2O2, dopamine, uric acid, 3,4-dihydroxyphenylacetic acid, norepinephrine, and catechol, can be directly catalyzed by redox nanozymes to generate detectable electrical signals[358][359][360][361][362][363]. For example, Zhang et al. Synthesized Co and N Co-doped three-dimensional porous carbon composite with oxidase activity, and successfully detected dopamine in human serum and artificial cerebrospinal fluid samples electrochemically[359].
Affinity sensors rely on the interaction between the biological recognition element and the target analyte to cause changes in the electrochemical signal, in which the nanoenzyme plays a role in signal amplification. For example, Yang et al. Found that the esterolytic activity of Pt NPs could be activated in the presence of H3N-BH3 with redox function, based on which an electrochemical sensor for detecting thyroid-stimulating hormone (TSH) was developed[364]. The specific antibody immobilized on the electrode captured TSH, and then the antibody-labeled Pt NPs bound TSH and exerted hydrolase activity to catalyze the substrate 4-aminonaphthalene-1-acetate to 4-amino-1-naphthol, resulting in an electrochemical signal (Figure 43). Zhu et al. Synthesized mesoporous trimetallic AuPtPd nanospheres with efficient oxidase and peroxidase activities, which can synergistically catalyze the oxidation of 4-chloro-1-naphthol and produce precipitates on the surface of photoactive materials, resulting in the reduction of photocurrent[193]. Urease can catalyze urea to produce ammonia, and its proton consumption characteristic will lead to the decrease of nanoenzyme activity. Therefore, the photocurrent increases gradually with the increase of urease concentration, thus realizing the quantitative detection of urease.
The signal of Raman spectroscopy comes from Raman scattering produced by Raman reporter molecules on the surface of plasmonic nanomaterials (such as Au, Ag, Cu). Peroxidase nanozymes incorporated into plasmonic nanostructures can alter the SERS signal by catalyzing a reporter molecule. Trau et al. Designed a nanoenzyme-based SERS sensing platform to detect biomarker miRNAs[365]. When the target miRNA is present, the nucleic acid probe-labeled Au peroxidonanase is connected to the hollow nanoframe through base complementary pairing and assembled into a plasmonic nanostructure. The subsequently added TMB is oxidized by the peroxidase NANase catalysis, and its oxidation product, oxTMB, acts as a Raman reporter to generate a signal. The strategy was validated in clinical urine samples with a minimum detection limit of 0.7 fmol·L-1.
4.1.6 Other detection methods
According to the catalytic properties of nanozymes, some unique detection strategies have also been developed. Hydrogen peroxide nanoenzyme, for example, catalyzes the decomposition of H2O2 to produce oxygen and water. Using this property, Tang et al. Developed a pressure sensing system based on Pt NPs nanoenzyme[366]. Antibody-conjugated Pt NPs recognize carcinoembryonic antigen, and simultaneously exert catalase activity to catalyze the decomposition of H2O2 to produce a large amount of oxygen, which causes the change of air pressure in the closed device. The concentration of carcinoembryonic antigen can be quantitatively reflected by the highly sensitive detection of the pressure sensor.
In addition, the reaction catalyzed by nanozyme is also helpful for imaging. For example, Tremel et al. Demonstrated that manganese oxide nanoenzymes with SOD activity catalyze superoxide anions to enhance the contrast of magnetic resonance imaging[367]; The Prussian blue nanoenzyme can exert catalase activity to catalyze the decomposition of H2O2 to provide O2 at neutral pH, so it can be used as an ultrasound contrast agent to enhance ultrasound imaging[368].
4.1.7 Multi-modal detection
The icing on the cake is that the physicochemical properties of nanoenzymatic materials (such as light, heat and other effects) combined with enzymatic characteristics show good application potential in the design of multimodal detection platforms. Shi et al. Designed a multimodal nanoenzyme-linked immunosorbent assay with in situ generation of Prussian blue nanoparticles (PBNPs) to determine aflatoxin B1 (AFB1)[369]. First, AFB1 is captured by a specific antibody immobilized on the carrier, and then the aptamer-labeled magnetic nanoparticles (MNPs) recognize AFB1 and are anchored to the carrier. The number of anchored MNPs is proportional to the concentration of AFB1. In the presence of HCl and K4Fe(CN)6, MNP can act as a precursor to generate PBNPs in situ. PBNPs with excellent photothermal effect generate heat energy under the irradiation of near-infrared light, and portable quantitative detection is realized by a thermometer. On the other hand, PBNPs have peroxidase activity and can catalyze the oxidation of TMB, resulting in a colorimetric signal. In addition, MNP also acts as a fluorescence quencher, quenching the preincubated fluorescent probe Cy5. The in situ generation of PBNPs was accompanied by etching of MNPs, which restored the fluorescence of Cy5, thus enabling ultrasensitive fluorescence detection. Compared with the detection method providing a single signal, this multi-modal nano-enzyme immunosorbent assay strategy, which combines photothermal, colorimetric and fluorescent signals, has the characteristics of higher sensitivity, and is conducive to reducing false positive/negative results caused by accidental factors such as unstable operating conditions and biological environment.
4.1.8 Brief summary
The versatility, high stability, low cost and easy batch preparation of nanoenzymes endow them with unlimited potential and prospects in the field of sensing. Of course, the application of nanoenzymes in the field of sensing still faces many challenges and problems, which also attracts researchers in related fields to invest more efforts to solve and innovate.For example, the design and development of nanoenzymes with higher activity has been the goal of researchers for a long time, and improving the catalytic activity of nanoenzymes can improve the detection sensitivity and reduce the detection limit[370]. Generally speaking, the development of nanoenzymes with smaller particle size can improve the catalytic activity, because the smaller the size of nanoenzymes, the larger the specific surface area, the more the substrate binding, and the stronger the activity. For example, for iron oxide nanoenzyme particles, the peroxidase activity of 30 nm particles is higher than that of 150 and 300 nm particles under the same conditions[1]. Pt NPs with smaller particle size also had stronger ascorbate oxidase activity and peroxidase activity[371][372]. The morphology, structure and surface modification of nanoenzyme materials also have a considerable impact on their catalytic activity. For example, Zhu Junjie's research group reported that the peroxidase activity of iron oxide nanoenzymes with different shapes is: cluster sphere > triangular sheet > octahedron[236]. The activity of Co3O4 nanoenzyme was in the order of nanosheet > nanopolyhedron > nanorod > nanocube[124]. Yan et al. Found that modification with histidine could increase the kcat/KM of Fe3O4 nanoenzyme by 20 times[70]. In addition, different assemblies and doping of nanoenzymes have also been used to enhance enzyme activity. For example, Nagvenkar and Gedanken used Zn-doped CuO NPs to form nanocomposite (Zn-CuO) NPs to enhance their peroxidase activity[373]. Kim et al. Embedded the heme-like Fe—N4 active site into graphene (Fe-N-rGO) to mimic the heme cofactor in natural peroxidase to improve its catalytic activity, and the experiment showed that its catalytic activity was 700 times higher than that of pure graphene[374].
When nanozymes are used in sensing technology, another problem that needs to be paid attention to and solved is how to improve the specificity of nanozymes. Currently reported nanozymes often have multiple enzymatic activities (such as peroxidase and catalase activities), which often produce inevitable background signals in the detection system, thus reducing the sensitivity or accuracy of detection. Wei Hui and Gao Xingfa worked together to obtain a nanoenzyme with single peroxidase catalytic activity by incorporating nitrogen atoms (N) into reduced graphene oxide (rGO), which was applied to the detection of H2O2 and found to have a better linear range than rGO without nitrogen atoms[67]. In addition to improving the specificity of enzyme-like activity, improving substrate selectivity is also a challenge. At present, coating molecularly imprinted polymers (MIPs) on the surface of nanoenzymes is often used to improve the selectivity of substrates. Liu Juewen et al. Used this strategy to improve the specificity of peroxidase activity of Fe3O4 to two substrate molecules, TMB and ABTS, by about 100 times[101]. Specificity can also be improved by mimicking the structure of natural enzyme. For example, Dong et al. Developed catechol oxidase-like MOF-818 by mimicking the active center of natural catechol oxidase.It has high catechol oxidase-like activity and good specificity, and can specifically catalyze catechol to produce the corresponding o-quinone, but does not show peroxidase activity[138].
The in-depth study of the catalytic mechanism of nanoenzymes can provide theoretical support and guidance for improving the catalytic activity and specificity of nanoenzymes, which is also an important research direction in the field of nanoenzymes in recent years. At present, ultraviolet-visible spectrophotometer, electron spin resonance, X-ray photoelectron spectroscopy and density functional theory calculation are mainly used to explore the catalytic reaction mechanism of nanoenzyme. Gu et al. Found that the standard redox potential of Fe3+/Fe2+ was 0.771 V, while that of TMB was 1.13 V, so Fe3+/Fe2+ could not directly transfer electrons from TMB to H2O2.And ESR results confirmed the presence of ·OH in the reaction system under acidic conditions, therefore, the catalytic mechanism of iron oxide nanoenzyme should be that Fe3+/Fe2+ first reacts with H2O2 to produce ·OH, and then hydrogen donor substrate (such as TMB) is oxidized by ·OH to produce color reaction[9][375]. Qu et al. Found that the carbonyl group (C = O) on the surface of carbon-based nanoenzyme is its active site, while the carboxyl group (— COO —) is the substrate-binding site, which can bind to H2O2 molecules and convert them into ·OH, while the hydroxyl group (— C — OH) on the carbon atom can inhibit the activity of POD[376]. This study provides a new direction for the POD activity mechanism and the controllable design of the activity of carbon-based nanoenzyme materials.
The application of nanoenzyme in the sensing field can not only make full use of the versatility and easy preparation of nanoenzyme to construct convenient, inexpensive and rapid detection methods, but also break through the application limitations of traditional enzyme-based sensors caused by harsh application conditions by using its high stability. At present, a variety of nano-enzyme detection kits have obtained medical device registration certificates, and it is believed that commercial products will soon enter the market.
4.2 Nanoenzyme anti-tumor
4.2.1 Current application of nanoenzyme in tumor catalytic therapy
According to the Global Cancer Observatory (GCO), approximately 30 million patients will die of cancer each year by 2030[377]. There is currently no effective method to completely eradicate this life-threatening disease. Drug resistance, recurrence and metastasis are still the key factors of cancer treatment failure. Therefore, there is an urgent need to develop effective new strategies for cancer therapy.
As a cutting-edge science, nanotechnology has become a cutting-edge tool for solving various world-class scientific problems, including cancer. Nanoscale materials can improve permeability and biodistribution, while exhibiting unique physicochemical properties, such as high specific surface area, enhanced conductivity, superparamagnetic behavior, optical absorption spectrum shift, and fluorescence characteristics[378]. Among many nanomaterials, nanozymes with enzyme mimicry ability have shown great potential in cancer therapy. Since the introduction of nanozymes in 2007, reports on the use of nanozymes in anti-tumor therapy have been increasing year by year (Figure 44). Studies have shown that nanozymecatalytic therapy has unique advantages: on the one hand, nanozymes can be used as a therapeutic agent to directly kill tumor cells or inhibit tumor growth through its unique enzyme-mimicking ability. On the other hand, nanoenzyme can improve tumor microenvironment conditions through catalytic activity, which is beneficial to combining and enhancing the therapeutic effects of chemotherapy, radiotherapy, photothermal therapy, photodynamic therapy, sonodynamic therapy and immunotherapy. Therefore, the catalytic microenvironment is directly related to the function of nanoenzyme and the ultimate therapeutic effect.
The tumor microenvironment (TME) plays an important role in dynamically regulating cancer progression and influencing treatment outcomes. TME has the following characteristics: (1) Hypoxia: Due to the rapid proliferation of tumor cells, their oxygen consumption increases, and the oxygen supply of abnormal tumor vasculature is insufficient, the high oxygen consumption and hypoxia in TME will increase the osmotic pressure[379]. (2) Abnormal glucose metabolism leads to pH imbalance: Under hypoxic conditions, tumor cells mainly metabolize through the glycolytic pathway, so that the pH values of the cytoplasm and extracellular fluid in the TME are stabilized at 7.4 and 6.6, respectively[380]. (3) The increase of ROS level leads to the increase of H2O2 and glutathione (GSH) level: tumor cells respond to the relatively high level of ROS in cells by up-regulating the antioxidant system, and the level of H2O2 in TME increases to a certain extent. The abnormal oxidative stress makes TME show a higher redox potential level than normal tissues[381][382]. Therefore, TME offers unique advantages for nanozyme catalyzed therapy.
4.2.2 Anti-tumor mechanism of nanoenzyme
4.2.2.1 ROS production through POD activity
When nanoenzyme is used in anti-tumor therapy, the core mechanism is to produce ROS to kill tumor cells[383]. Among them, POD activity is the most commonly used type of activity for tumor therapy, and the reaction formula is as follows:
In this reaction, H2O2 acts as an electron acceptor to produce an intermediate dominated by ·OH under the catalysis of nanozyme, and ·OH further reacts with an electron donor (AH) to generate its oxidized form (A). Because of the strong reactivity of ·OH, various small organic molecules, nucleic acids, proteins, lipids and other macromolecules in cells can be oxidized as their reaction substrates, thus causing serious damage, which will lead to cell death when the damage accumulates to a certain extent.
4.2.2.2 Regulation of ROS levels by other enzyme activities
In addition to directly killing tumor cells through POD-like activity, nanozymes can also indirectly play an anti-tumor role by regulating intracellular ROS levels through other enzyme-like activities, which can be divided into the following types.
The CAT-like activity of nanoenzyme is mainly used to promote the generation of oxygen in the tumor. Although this type of activity consumes intracellular H2O2 and reduces the ROS level, it can improve the hypoxic microenvironment of the tumor, and use the generated O2 to promote the generation of singlet oxygen (1O2) after radiotherapy/photodynamic therapy/sonodynamic therapy, thereby killing the tumor (see 3.2.1 for details).
The SOD-like activity of nanozyme converts ROS in the form of superoxide anion () into H2O2, which itself has an antioxidant effect on cells, but if combined with POD nanozyme, it can also promote the generation of ROS and further convert H2O2 into ·OH to kill tumors[385].
The OXD-like activity of nanozyme can also produce ROS in the form of H2O2 and , but it is difficult to achieve the same tumor-killing ability as ·OH when acting independently, and it often needs to combine with other enzyme-like activities to kill tumors[386][387].
The GPx-like activity of nanozymes is mainly used to scavenge the high content of GSH in tumor cells, thereby weakening the antioxidant capacity of tumor cells, and can further improve the tumor-killing effect when combined with other means to promote the production of ROS.
The GOx-like activity of nanozyme is not only used to consume glucose for energy metabolism of tumor cells, but also the H2O2 generated by nanozyme can further generate ·OH to kill tumors by combining with POD-like activity[389].
In summary, there is a correlation between the reactions catalyzed by different activity types of nanozymes (fig. 45). Among them, POD-like activity, SOD-like activity, OXD-like activity and CAT-like activity can realize the mutual conversion between different types of ROS, and among the several forms of ROS existing in cells, the conversion of endogenous or H2O2 into ·OH is the main strategy to enhance the oxidative damage effect on tumor cells[390]. In addition, GPx-like activity and GOx-like activity play an important auxiliary role in regulating intracellular redox levels (fig. 45).
4.2.3 Application strategy of nanoenzyme in anti-tumor
4.2.3.1 Nanoenzyme for tumor catalytic therapy
Based on the above mechanism of action, there have been many studies on the application of nanoenzymes in cancer therapy. Specifically, a single type of nanoenzyme can play a catalytic therapeutic role, or the design can be combined with multiple nanoenzymes after integration. In addition, researchers are also expanding new directions on the basis of existing nanoenzymes to broaden the application prospects of nanoenzyme-catalyzed therapy.
It was found that NANase with POD-like activity could be used as an effective therapeutic agent for tumor catalytic therapy. On the one hand, the acidic microenvironment in tumor stroma is beneficial for nanozyme to exert POD-like activity to catalyze abnormally elevated H2O2 to produce ·OH and directly kill tumor cells[391]. On the other hand, Gu et al. Observed that Fe3O4NPs could exert POD-like activity more effectively in the acidic environment of lysosomes in tumor cells, resulting in a significant killing effect on human glioma U251 cells[9]. Since then, the antitumor potential of NANase itself has received increasing attention. For example, Weng et al. Designed and developed a biodegradable boron nitride (BON) nanostructure[392]. The material has both excellent biocompatibility and strong POD-like activity, and can induce significant apoptosis of tumor cells after intratumoral injection, showing therapeutic ability similar to anticancer drugs (Fig. 46).
Nanozyme can also further improve its biocompatibility and tumor therapeutic effect by incorporating protein scaffolds (such as ferritin) into biomimetic synthesis. On the basis of traditional ferritin nanoenzyme, Huang et al. Oligomerized multiple ferritin nanocages to prepare FTn-Ner-Pt-POD nanoenzyme, which enhanced the long circulation ability in vivo and the targeting ability to tumor cells, providing a new idea for nanoenzyme-based catalytic therapy strategy[393].
Although nanozyme has been used in tumor catalytic therapy, the ultimate efficacy is still limited by the insufficient catalytic activity of nanozyme itself. Monoatomic nanoenzyme is a new type of nanoenzyme based on the concept of monoatomic catalyst, which has atomically uniformly dispersed active centers and a clear coordination structure. Compared with traditional nanoenzymes, its catalytic activity has been greatly improved, so it has attracted much attention in the catalytic therapy of tumors[394]. For example, Ji et al. Developed a FeN3P single-atom nanoenzyme (FeN3P-SAzyme) with POD-like activity, and constructed an active center similar to natural horseradish peroxidase by precisely coordinating phosphorus and nitrogen elements to adjust the electronic structure of the single-atom iron active center (Fig. 47)[15]. The results showed that the kcat/KM and KM of FeN3P-SAzyme were comparable to those of natural horseradish peroxidase. In vivo experiments, the FeN3P-SAzyme showed significantly improved tumor therapeutic effect compared with the single traditional nanoenzyme, and there was no significant toxicity after intravenous injection. However, most of the single-atom nanozymes still have the problems of easy aggregation during preparation and difficult to accurately optimize the local coordination structure. Therefore, how to improve the preparation process is an important challenge for the application of single-atom nanoenzymes in tumor catalytic therapy. Liu et al. Designed and synthesized a five-coordinate FeN5 single-atom nanoenzyme with an active structure similar to that of the native enzyme[395]. By coating a SiO2 shell on the surface of the zeolite imidazolate framework precursor, the aggregation of the material in the high-temperature pyrolysis process can be avoided, and the obtained carbon nanoparticles have a better pore structure and a higher specific surface area, which is beneficial to the formation of Fe single atom sites. The kcat/KM of the FeN5 monoatomic nanoenzyme was 7.64 and 3.45×105 times higher than that of the traditional FeN4 monoatomic nanoenzyme and the commercialized Fe3O4 nanoenzyme, respectively, and showed significantly enhanced anti-tumor effect in both in vivo and in vitro experiments.
4.2.3.1. Catalytic therapy based on nanoenzyme cascade reaction
Compared with a single nanoenzyme catalytic therapy system, the strategy of combining multiple nanoenzymes can take advantage of cascade catalysis, using multiple substrates (such as H2O2, glucose, GSH) that are increased in TME compared with normal tissues to enhance the persistence of catalytic reactions. Qu et al. Reported MnO2@PtCo nanoflower materials based on nanoenzyme self-assembly[276]. Among them, PtCo exerts OXD-like activity and MnO2 exerts CAT-like activity. The oxygen generated by the decomposition of H2O2 catalyzed by MnO2-CAT nanoenzyme can be used as the reaction substrate of PtCo-oxidized nanoenzyme, which is further converted into and ·OH with killing ability, thus inducing cell apoptosis and playing a significant role in tumor inhibition in tumor-bearing mouse models. In addition to cascading CAT-like activity with OXD activity, another common strategy for nanoenzyme-catalyzed therapy is to cascade GOx-like activity with POD-like activity. Shi et al. Integrated Au NPs-GOx nanoenzyme and Fe3O4NPs-POD nanoenzyme into the channels of dendritic mesoporous silica nanoparticles (DMSN NPs) to prepare a combined nanoenzyme system capable of triggering a cascade reaction under TME[396]. Au NPs not only consume intracellular glucose to cause tumor starvation, but also promote Fe3O4NPs to catalyze the release of more highly toxic ·OH to kill tumor cells (Fig. 48). In vivo experiments show that the cascade reaction system will not be triggered in normal tissues with relatively lower glucose concentration and relatively higher pH, thus avoiding the generation of toxic and side effects.
4.2.3.2 Combined Strategies for Nanozyme Catalyzed Enhanced Cancer Therapy
At present, there are more and more clinical treatments for cancer, including surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, photodynamic therapy, hyperthermia, stem cell transplantation, hormone therapy and so on. However, the single traditional treatment usually faces the problems of hypoxia, immune escape, drug resistance, toxic side effects and so on, which seriously limits the therapeutic effect. Nanoenzyme can improve tumor microenvironment conditions through catalytic activity, and is widely used to assist a variety of commonly used cancer treatment methods, and enhance the efficacy through combination strategies.The enhancement effect can be reflected not only in the compensation of its catalytic activity for the shortcomings of various therapeutic methods, but also in the benefits brought by its non-catalytic activity, such as drug loading ability, photothermal conversion ability, magnetothermal conversion ability and other physical and chemical properties.
4.2.3.2. 1 nanoenzyme assisted aerobic therapy
Oxygen production catalyzed by CAT nanoenzyme can effectively enhance the efficacy of aerobic therapies such as radiotherapy (RT), photodynamic therapy (PDT) and sonodynamic therapy (SDT).
RT is one of the clinical first-line therapies for tumor treatment, which relies on various rays produced by radioisotopes to treat tumors[397]. Water and oxygen can be decomposed by radiation to form ROS, which can cause damage to tumor cells. However, the efficacy of RT has been limited by the hypoxic environment of the tumor site for many years. To this end, researchers have used the catalytic activity of CAT nanoenzyme to decompose the high concentration of H2O2 in tumor and produce oxygen, thus improving the problem of tumor hypoxia. For example, Choi et al. Reported that the porous Pt NPs-CAT nanoenzyme increased the oxygen concentration of the TME, while the platinum element itself, as a high-Z element, sensitized the radiation, thus improving the efficacy of conventional RT (Figure 49 a)[398].
The core principle of PDT and SDT, two frontier therapies in rapid development in clinical research, is that the photosensitizer/sonosensitizer is activated by laser/ultrasound, respectively, thereby converting oxygen into singlet oxygen (1O2)[399,400]. There have been many studies utilizing CAT nanozymes as sensitizers for PDT and SDT due to insufficient oxygen supply to the tumor site. For example, Hu et al. Used MnO2-BSA nanoparticles in combination with calcium peroxide nanoparticles (CaO2NPs)[401]. At the tumor site, CaO2NPs can react with water molecules to produce O2 and H2O2, which directly provide more reaction substrates for MnO2-BSA-CAT nanoenzyme, and the oxygen production rate of CaO2NPs can also be improved under the catalysis of nanoenzyme. After binding the photosensitizer chlorin E6 (Ce6) by BSA, the in vivo experiment achieved a significantly enhanced PDT therapeutic effect (fig. 49b). Zhang et al. Synthesized hollow-structured Fe3O4 nanoparticles (HIONs) for SDT to alleviate tumor hypoxia by using a hot flux method. After loading the sonosensitizer hematoporphyrin inside, modifying polydopamine outside and connecting polyethylene glycol, the obtained material (HP-HIONs @ PDA-PEG) also effectively increased the O2 content at the tumor site, thereby promoting the generation of 1O2[402]. HIONs-CAT nanoenzyme also has special magnetothermal conversion ability, and animal experiments show that it can mediate magnetothermal therapy after applying an alternating magnetic field, which has a synergistic effect with ultrasound-mediated SDT (Fig. 49c).
4.2.3.2. 2 Nano enzyme assisted immunotherapy
Because most tumors have acquired the ability to evade the body's immune surveillance mechanism, how to adjust and improve the TME to relieve immune suppression has become a key issue in immunotherapy[403]. NANase plays an auxiliary role in immunotherapy through three main mechanisms.
First, hypoxia can promote the formation of an immunosuppressive microenvironment at the tumor site through signaling pathways such as HIF-α, which can be regulated by the ability of nanozyme to catalyze the production of oxygen. For example, hypoxia causes tumor-associated macrophages (TAM), which are abundant within tumors, to induce differentiation into the M2 phenotype, which promotes tumor growth. To this end, Liu and Wei et al. Constructed polyethylene glycol-modified iron-manganese silicate nanoparticles (IMSN) loaded with TGF-β inhibitors. After IMSN entered the tumor site, it not only exerted CAT-like activity to alleviate the hypoxic environment to promote the differentiation of TAM to the M1 phenotype of anti-tumor growth, but also exerted POD-like activity to trigger iron death. Under the synergistic effect of the two activities, it produced a good tumor therapeutic effect[395].
Secondly, the acidic microenvironment of tumors can also promote the immune escape of tumor cells, which can be regulated by nanoenzymes with the ability to regulate pH. Chen and He et al. Reported tin selenide nanosheets (SnSe NSs) capable of mimicking lactate dehydrogenase and loaded with natural carbonic anhydrase IX inhibitor ABS[404]. ABS inhibits the hyperactive glycolytic process in tumor cells, and SnSe NSs convert the accumulated lactate into pyruvate. In combination with PD-L1 antibody, SnSe @ ABS triggered significant tumor ablation and effectively inhibited tumor metastasis.
Third, tumor cells themselves can also induce immunogenic death through ROS-producing nanozymes, release tumor-associated antigens and be ingested by antigen-presenting cells, thereby activating T-cell-mediated immune responses. He et al. Designed a bioadhesive injectable hydrogel loaded with palladium single-atom nanoenzyme (DA-CQD @ Pd SAN) and immunoadjuvant CpG oligodeoxynucleotide (CpGODN)[405]. After injection into the tumor site, the ·OH produced by the POD-like activity of DA-CQD @ Pd SAN not only killed tumor cells, but also decomposed the sulfide around it into sulfur radicals, which mediated the gelation of the hydrogel. CpG ODN enhanced the activation effect on antigen presenting cells with the assistance of tumor-associated antigen released by dead cells (fig. 50). After treatment, the proportion of CD8+T cells in tumor tissue increased, while the proportion of regulatory T cells, myeloid-derived suppressor cells and M2 macrophages decreased, indicating that the system significantly improved TME.
4.2.3.2. 3NM enzyme adjuvant chemotherapy
Chemotherapy is an important clinical treatment for cancer. There are widespread problems of drug resistance and toxic side effects.
Aiming at the problem of drug resistance, the pro-oxidant activity of nanozymes can be used synergistically. Yu and Zhan designed a carbon nanosphere @MnFe2O4 nanoenzyme with GPx-like activity and POD-like activity, and loaded it with gemcitabine, a chemotherapeutic drug[406]. This system improves the therapeutic effect of gemcitabine by consuming intracellular GSH, increasing the level of lipid peroxidation, and then inducing iron death of cells. In addition, intratumoral bacteria are also one of the important reasons for chemoresistance. Gao et al. Developed nitrogen-doped carbon nanosphere-POD nanoenzyme in a mouse model constructed from bacteria-containing tumors.The nanoenzyme kills intratumoral bacteria by producing ·OH, reverses gemcitabine resistance induced by bacterial cytidine deaminase, and further enhances the killing effect on tumor cells[407].
How to reduce the toxic and side effects of chemotherapeutic drugs is also the focus of current research. Therefore, the catalytic activity of nanoenzymes can be used to construct chemotherapeutic prodrug activation strategies, which is expected to improve the selectivity of chemotherapeutic drugs. Huang et al. Synthesized Pd NANase with POD-like activity and P450BM3 mutant-like activity in situ inside ferritin nanocages[408]. The Pd-NANase first exerts a POD-like activity in the acidic environment of the lysosome, resulting in permeabilization of the lysosomal membrane, and then converts the released hydroxycamptothecin prodrug molecule (pro-HCPT) into a toxic HCPT through a bioorthogonal reaction mediated by a P450BM3 mutant activity (fig. 51). The drug delivery system effectively avoids systemic toxicity caused by intravenous administration while inhibiting tumor growth in an in vivo model.
4.2.4 Brief summary
Although the field of nanozymes has made remarkable progress in recent years, research on nanozymes for tumor therapy is still in its infancy. In order to increase the potential for clinical translation of nanozymes, several key issues need to be addressed.
(1) Low catalytic efficiency
Compared with natural enzymes and organocatalysts, the catalytic activity and efficiency of nanozymes are relatively low, limiting their application in the treatment of diseases. The uneven atomic composition and complex crystal structure of nanozymes make it difficult to identify the actual catalytic active center and reveal the enzyme-like activity mechanism. Therefore, it is necessary to have a full understanding of the enzyme active center of natural enzymes, and according to the characteristics of nanoenzymes, the activity of nanoenzymes can be improved by adjusting the size, crystal face, composition, surface modification, mixing nanoenzymes and other methods. At present, single-atom nanozymes have highly dispersed catalytic active sites and atom utilization efficiency, which can effectively improve the catalytic performance of nanozymes, but the problem of limited loading efficiency of single-metal atoms remains to be solved. In addition, different nanozymes may have synergistic effects in the anti-tumor process. The rational design of cascaded nanozymes may be a promising strategy to improve catalytic efficiency.
(2) poor specificity
Intravenous injection is one of the main methods to administer antitumor nanoformulations, however, after nanoformulations enter the blood circulation, they may be captured and eliminated by the mononuclear macrophage system, which will affect their bioavailability and increase drug accumulation in healthy organs. The controllable design of nanozymes is of great significance to achieve their enzyme-mimicking activity and high specificity for diseases. One of the methods is to use the characteristics of the microenvironment to construct environmentally responsive nanoenzymes or modify the surface of nanoenzymes (functional groups, targeting peptides, etc.) To bind tumor-targeting molecules on the surface of nanoparticles, so as to improve the affinity and specificity of nanoenzymes and enhance the specific uptake of tumor cells. In addition, designing hybrid multifunctional nanomaterials with synergistic effects is an effective way to improve specificity. In recent years, many studies have shown that a variety of exogenous stimuli (such as light, magnetism, heat, ultrasound, etc.) can be used as triggers to control the activation of nanozymes. These modes may provide adjustable options to improve the specificity of nanozyme at tumor sites and reduce the harm to non-specific tissues.
(3) Poor biological safety
While nanozymes have great potential for disease therapy, however, ideal biocompatibility still faces challenges. Unlike traditional enzymes, nanozymes are mostly inorganic nanoparticles, which can enter the lysosome, mitochondria or nucleus of cells, and may cause damage to cells through irreversible interaction with intracellular components. Taking metal-based nanozymes as an example, although many studies have demonstrated the cytoprotective effect and biocompatibility of nanozymes, metal ion release is still considered to be a factor that causes side effects on normal tissues due to metal overload. For example, excess copper or iron in normal cells may trigger Fenton or Fenton-like reactions that severely damage biological macromolecules and nucleic acids. Studies have shown that coating the surface of nanoparticles with polymers such as polyethylene glycol and cell membrane can improve their biocompatibility. However, as a foreign substance, it may still cause an immune response in vivo. At the same time, the interaction mode between nanoenzyme and biological environment remains to be clarified, which requires in-depth study of its pharmacokinetics, absorption, distribution, metabolism, treatment sustainability, excretion and so on.
To sum up, although the antitumor application of nanozymes still faces challenges, nanozymes are of great significance in the field of tumor therapeutics due to their excellent enzymatic activity and unique physicochemical properties. Based on the cross-research of enzyme chemistry, nanomedicine, bio-nanotechnology and oncology, nanozymes have shown great potential and development prospects in cancer therapy.
4.3 Nano-enzyme antibacterial and antiviral
In recent years, more and more attention has been paid to personal and public health events related to microbial infections. Bacterial, fungal, and viral infections are important threats to human health and safety. Currently, the most common treatment for bacterial infections is the use of antibiotics[409]. However, the overuse of antibiotics has led to the emergence of multidrug-resistant superbugs, which pose a great threat to public health and the environment[410]. In addition, fungal infections are difficult to treat because there are few antifungal drugs available, and many of them have side effects, such as hepatotoxicity, nephrotoxicity, and neurotoxicity, or cause allergies and gastrointestinal discomfort[411]. More importantly, with the outbreak and development of the COVID-19 epidemic, people's daily life has been seriously affected due to the lack of effective antiviral drugs and vaccines, and has caused immeasurable losses to countries around the world[412]. Based on this situation, it is necessary to develop non-traditional therapeutic drugs with antibacterial, antifungal and antiviral properties, but without drug resistance, in order to effectively kill pathogenic microorganisms and prevent and control major infectious diseases.
Nanozyme has biomimetic activity to regulate the level of reactive oxygen radicals produced[36]. Excessive reactive oxygen radicals can damage cell membranes, proteins and nucleic acids, and kill pathogenic microorganisms, including bacteria, fungi and viruses[28,413⇓ ~415]. Therefore, in recent years, nanoenzyme has shown broad application prospects in antibacterial and antiviral therapy and adjuvant therapy[416]. For example, some nanozymes have horseradish peroxidase activity, lipid peroxidase activity, halogenated peroxidase activity and oxidase activity, which can directly catalyze substrate molecules to produce free radicals to kill pathogenic microorganisms[417⇓⇓~420]. Some nanoenzymes have glutathione peroxidase activity and catalase activity, which can neutralize excessive active oxygen free radicals produced by microbial infection, avoid excessive inflammatory reaction and improve body function[421,422]. Some nanozymes have nuclease activity or protease activity to degrade nucleic acids or proteins of pathogenic microorganisms[423,424]. In addition, nanoenzymes with special functional properties such as photothermal, sonodynamic and magnetic properties can also realize the construction of space-time controllable, deep, efficient and responsive nanotherapy platforms[99,425,426]. Therefore, nanozymes can be an alternative to antibiotics by combining their inherent enzyme-mimicking activity and physicochemical properties, and at the same time, nanozymes are less likely to cause resistance compared with conventional anti-microbial drugs.
This section will briefly summarize the latest research progress of nanoenzymes in the fields of antibacterial and antiviral, and look forward to the opportunities and challenges that need to be paid attention to in the future.
The skin, composed of dense epithelial cells, acts as a mechanical barrier against the invasion of foreign pathogens. When the skin is injured and the wound is formed, it is vulnerable to bacterial infection[410]. In particular, some chronic wounds, such as burns, scalds, eczema and skin lesions in diabetic patients, are not easy to heal when infected, requiring long-term treatment and leading to serious consequences when treatment fails[427⇓~429]. In addition, bacterial infections sometimes occur under the skin, causing subcutaneous abscesses. Subcutaneous abscess is a superficial abscess caused by acute bacterial infection and sometimes a bacterial capsule that is difficult to remove[423]. It can occur anywhere on the skin and may cause fever, lymphadenopathy, and bacteremia[416]. For bacterial infections of the epidermis, the usual approach is to use antibiotics at the local site of infection or systemically. However, overuse of antibiotics can lead to the emergence of drug-resistant bacteria, which has become an important challenge in the treatment of in vivo infections. In recent years, various functional and responsive nanoenzyme platforms have been successfully used for external wound healing and local treatment of subcutaneous abscesses.
Nanozyme usually has a broad spectrum of antibacterial properties and can treat infections caused by a variety of bacteria. Willner et al. Designed Au3+ functionalized UiO-67 metal-organic framework nanoparticles as a mimic of POD and OXD enzymes to catalyze hydrogen peroxide and oxygen to produce ·OH and to kill Escherichia coli and Staphylococcus aureus and promote wound healing (Figure 52 A)[419]. In addition, Gram-selective bacterial clearance can be achieved through the rational design of nanozymes. Qu et al. Designed an intelligent Gram-selective antibacterial system (Cit-MoS2) based on molybdenum disulfide (MoS2) and photoacid molecules to endow the nanoenzyme with the ability to selectively kill bacteria[430]. Depending on the cell wall composition and structure of different strains, the proportion of positive and negative charges on the surface of MoS2 and pH can be regulated by adjusting the illumination time, and then the selective recognition and killing of different bacteria can be achieved (Fig. 52b). In addition, the biomimetic design of the surface of the nanoenzyme can realize the capture and adhesion of the nanoenzyme to bacteria, reduce the distance between ROS and bacteria, and enhance the bactericidal effect. For example, Ren et al., inspired by the concept that surface roughness can enhance adhesion and defects can improve catalytic activity, constructed Cu@MoS2 nanoenzyme, which can bind bacteria in situ and efficiently kill bacteria through ROS generated by catalysis[43].
For most nanozymes, activity is optimal only in acidic solutions, whereas most physiological conditions have pH values exceeding 7.0 (and even > 8.0 in chronic wounds)[431,432]. At the same time, the concentration of hydrogen peroxide often has a great impact on the sterilization and healing of epidermal wounds, and excessive concentration of hydrogen peroxide will cause damage to normal cells[433]. Therefore, how to design a nanoenzyme antibacterial system to effectively exert its antibacterial effect on epidermal wounds without the limitation of pH and hydrogen peroxide concentration is an urgent problem to be solved. Based on this, Qu et al. Constructed a metal-organic framework-based self-activating hybrid nanoenzyme platform (Figure 52C)[433]. In this work, ultrathin two-dimensional sheet MOF was selected as the model peroxidase to adsorb glucose oxidase at the same time, and glucose-responsive pH reduction and hydrogen peroxide production were achieved at the wound site, thus obtaining efficient enzyme activity and minimal side effects, and the efficient antibacterial effect of hybrid nanoenzyme was verified in vitro and on mouse wound surface.
In recent years, the potential application of nanozymes in oral hygiene has also attracted the attention of researchers. Plaque on teeth provides excellent adhesion sites for colonization and growth of many bacteria[434]. Certain types of acid-producing bacteria, particularly Streptococcus mutans (S. mutans), are commensal microorganisms present in the human mouth that can form colonies on tooth surfaces and cause damage to tooth structure when sugars such as sucrose and fructose are present[435]. In order to solve this problem, Gao et al. Found that Fe3O4NPs with POD-like activity could promote the degradation of Streptococcus mutans membrane matrix and cause the death of bacteria in the membrane by catalyzing H2O2 to produce free radicals in situ[436]. In addition, Fang et al. Synthesized oxidized nanodiamond with POD-like enzyme activity, which can catalyze low concentration of H2O2 to produce ·OH, enhance the destruction of bacterial cell membrane and capsule, and accelerate the healing of wound around periodontal infection[437]. Recently, Hwang et al. Used nanoenzymatic robots (CARs, ferromagnetic nanoparticles) with catalytic antibacterial activity for human tooth capsule removal[438]. In this study, the most challenging capsular formation area inside the tooth, the isthmus area, and the common capsular formation area, the alveolus, were targeted to prevent oral caries by using a magnetic field to control the movement of CARs and special shapes to enter and move, as well as to target harmful bacterial capsules.
It is worth noting that the development of bacterial resistance is inextricably linked to bacterial biofilm[439]. For this reason, nanoenzymes can be used to degrade the membrane matrix and kill the pathogenic bacteria inside. Qu et al. Designed a MOF/Ce-based nanoenzyme with dual DNase-like and peroxidase-like enzymatic activities[423]. As shown in fig. 52d, on the one hand, the Ce complex on the MOF can enable the nanoenzyme to exhibit excellent DNA-like enzymatic activity, efficiently catalyze the degradation of eDNA, and destroy the established biofilm. On the other hand, MOF doped with Au showed enhanced peroxidase-like activity in the presence of hydrogen peroxide, which could efficiently kill the bacteria in it and had a good therapeutic effect on subcutaneous abscess.
4.3.2 Nanoenzyme-mediated deep bactericidal
Nanozyme not only has a good therapeutic effect on epidermal bacterial infections, but also shows broad application prospects for the treatment of some deep bacterial infections in vivo. For example, methicillin-resistant Staphylococcus aureus induced pneumonia, Helicobacter pylori infection induced gastritis, Salmonella induced enteritis, bacterial cholecystitis, peritonitis, sepsis, and bacterial contamination of implanted medical devices[440][441,442][443,444][445][446][29][447]. Tan et al. Developed an in vivo activatable pH-responsive nanoenzyme PtCo @ Graphene (PtCo @ G) to selectively treat Helicobacter pylori[441]. The nanoenzyme resists gastric acid corrosion, exhibits oxidase-like activity, stably produces reactive oxygen species only in an acidic gastric environment, and exhibits superior selective bactericidal properties. It exhibited highly effective selective bacteriostatic activity in a mouse model of Helicobacter pylori infection, with negligible side effects on normal tissues and commensal flora in vivo (fig. 53 a).
Gao et al. Designed and synthesized carbon nanospheres (CNSs) coated ultrasmall FeS2NPs, which has a good therapeutic effect on acute peritonitis caused by Escherichia coli in mice[446]. Due to the protection of sulfur, Fe2+ is continuously released, which triggers bacterial death by oxidizing the surface and internal molecules of bacteria (Figure 53b). Sun et al. Constructed an ultrasound-switchable (US) Pd @ Pt-T790 nanoplatform for the treatment of MRSA-induced bacterial myositis by combining Pd @ Pt nanoplates with CAT-like activity and organic sonosensitizer T790[99]. The modification of T790 blocked the CAT mimic enzyme activity of Pd @ Pt, but under the action of US, the enzyme activity was restored to catalyze the conversion of H2O2 to O2, which could be used to achieve the treatment of deep bacterial infection by ROS generated by sonodynamic induction (Figure 53C).
Bioorthogonal nanocatalysts based on transition metal catalysts have an enzyme-like substrate binding mechanism and can mimic the catalytic behavior of natural enzymes, which are called bioorthogonal nanocatalysts[448]. In recent years, Rotello et al. Designed a bio-orthogonal nanoenzyme system for the synthesis of proantibiotic drugs by adding a transition metal catalyst (TMC) into the nanoscaffold, and constructed an efficient and safe antibacterial platform with space-time controllable and reversible activation[449⇓~451]. As shown in Fig. 54a, Huang et al. encapsulated TMC in a quaternary ammonium salt polymer with hydrophobic pockets to construct a self-assembled polymer nanoparticle[450]. The designed bioorthogonal nanoenzyme can penetrate into the bacterial envelope and generate antibiotics with antibacterial activity in situ.
Qu et al. Synthesized a biomimetic urchin-like PdCu nanoenzyme that can capture bacteria. The nanoparticle has POD-like enzyme activity, and can bioorthogonally synthesize antibiotic molecules in situ, which can efficiently resist bacteria in the presence of hydrogen peroxide and precursor molecules (Fig. 54b)[452].
4.3.4 Nanoenzyme-mediated marine antifouling
Marine biofouling refers to the phenomenon that marine organisms adhere to the surface of marine artificial facilities and have a serious impact on the normal operation of artificial facilities. The formation of marine biofouling is based on the physical attachment of prokaryotic microorganisms such as bacteria to the basement membrane accumulated on the surface of the mechanism in advance, forming a microbial capsule, which facilitates the attachment of subsequent fouling organisms and damages marine artificial facilities[453]. In recent years, nanoenzymes with haloperoxidase activity have emerged in marine antifouling[420,454,455]. Seawater contains about 1 mmol/L of Br- and 500 mmol/L of Cl-, which can provide sufficient halide ions for halogenation reaction (Figure 55 A)[456].
Tremel et al. Found that cerium oxide nanorods (CeO2-x) had haloperoxidase-like activity and could produce HOBr and 1O2 under seawater conditions[454]. The cerium oxide nanorods were mixed in a commercially available coating and coated on the surface of stainless steel, which could significantly inhibit microbial adhesion in seawater, effectively prevent the formation of biofilm, and have strong antifouling performance (Fig. 55b). Although this kind of nanoenzyme has its advantages in marine antifouling, there are still some problems in practical application, such as the low catalytic activity of nanoenzyme and the low content of hydrogen peroxide in the ocean. Based on this, Wang et al. Explored visible-light-responsive photothermal HPO-like nanoenzymes to combat biological pollution[455]. The Mo atom enzyme can catalyze the oxidation of Br- to produce HOBr with high efficiency. At the same time, the photothermal effect can enhance its catalytic oxidation activity and antibacterial ability, so that it also has good antibacterial performance when used as a surface coating additive in the marine environment.
4.3.5 Nanoenzyme-mediated antifungal therapy
The delayed diagnosis of fungal infection, the lack and toxicity of antifungal drugs, and the low immunity of the host defense system make it difficult to be treated[416]. The uniqueness of the fungus also makes antifungal treatment more difficult, such as thicker cell walls and the formation of hyphae. In addition, fungi are eukaryotic cells and share similar cellular structures and metabolic pathways with host cells, which makes it challenging to develop specific antifungal drugs without cytotoxicity[457]. In addition, although there are few kinds of antifungal drugs available, drug resistance has also become an important issue in the clinical treatment of fungal infections. Therefore, there is a need to develop new antifungal agents with different mechanisms of action. Nanozyme may provide a new option for antifungal therapy through a catalytic killing mechanism.
Abdelhamid et al. Synthesized a cerium-based MOF (Ce-MOF) with various enzyme-like activities such as POD, CAT, and SOD, which has good antifungal activity against fungi such as Aspergillus flavus (A. flavus), Candida albicans (C. albicans), Rhodotorula (R. glutinis), and human opportunistic pathogens isolated from hospitals, but the study did not clarify the mechanism of its antifungal effect[458]. In addition, Li et al. Synthesized nitrogen and iodine doped carbon dots (I-CDs), which can be used as POD mimics to eliminate Candida albicans[459]. In the presence of hydrogen peroxide, I-CDs catalyzed the formation of ·OH, and its antibacterial activity against Candida albicans reached 90%. At the same time, visible light irradiation promoted the production of ·OH and successfully inhibited the growth of Candida albicans. However, at present, there are few studies on the treatment of fungal infections with nanoenzyme, which also indicates that nanoenzyme has a broader development space in this field in the future.
4.3.6 Nanoenzyme-mediated antiviral therapy
Currently, strategies to combat viral diseases are prevention (vaccines) and treatment (antiviral drugs and antibodies). Of the more than 220 viruses known to infect humans, there are only a dozen clinically approved antiviral drugs[460]. In addition, the efficacy of most antiviral drugs is limited to certain strains of virus and may be diminished if mutant strains evolve. In fact, drug resistance caused by highly variable mutations in the viral genome is currently a major challenge in the development of antiviral drugs[461]. To this end, nanozymes use their catalytic and physicochemical properties to directly or indirectly inactivate viruses, providing a new direction for combating viral infections[462]. The role of nanozymes in virotherapy is mainly to increase ROS levels, regulate the immune environment of the body, prevent viral replication, and oxidize viral lipid envelope and proteins.
As shown in Figure 56A, iron oxide nanoenzyme can cause lipid peroxidation of the lipid envelope of influenza A virus (IVAs), destroy the integrity of adjacent proteins (including matrix protein 1, hemagglutinin and neuraminidase), and inhibit the infection of IVAs to cells[415]. At the same time, the nanoenzyme has a broad spectrum of effects on various subtypes of IAVs, including H1 ~ H12 subtypes. The application of iron oxide nanoenzyme on masks can enhance the protective effect of masks on humans and reduce the threat of three important subtypes of H1N1, H5N1 and H7N9 viruses. In addition, chitosan-modified iron oxide nanoenzyme can also be used as a vaccine adjuvant to inactivate virus and improve the immune response to influenza virus (fig. 56B)[463].
The COVID-19 epidemic has affected the lives of people around the world, but there is still no effective treatment. Wang et al. Tried to use nanozyme to eliminate the virus[317]. They designed a titanium dioxide-supported single-atom nanoenzyme (Ag-TiO2SAN) containing atomically dispersed Ag atoms to eliminate the virus. Compared with traditional TiO2NPs, Ag-TiO2SAN has a high adsorption capacity for SARS-CoV2 pseudovirus, which is beneficial to the formation of SAN/virus complex. The complex is then phagocytosed by macrophages and enters lysosomes, whose strongly acidic microenvironment favors the production of ROS by Ag-TiO2SAN with POD activity to clear the virus (Figure 56 C). In addition, nanozyme has an antiviral effect on human immunodeficiency virus (HIV). Singh et al. Found that vanadium pentoxide (V2O5) nanosheets could mimic GPx activity, neutralize cellular ROS caused by HIV-1 infection, and inhibit HIV-1 reactivation and replication[421]. In addition, in addition to typical viruses, nanozymes can also prevent related diseases caused by prion infection, showing broad application prospects in antiviral therapy.
Nanozyme has the characteristic of eliminating pathogenic microorganisms because it can mimic a variety of natural enzymes, and its catalytic mechanism is not easy to produce drug resistance. In addition, as a nanomaterial, nanoenzyme has special physicochemical properties, which can enhance its ability to kill pathogenic microorganisms when combined with photothermal, magnetothermal and sonodynamic therapy. It has good therapeutic effect on surface bacterial infection, deep bacterial infection, marine antifouling, fungal and viral infection. However, there are still many problems and challenges to be solved in the future clinical application of nanozyme. (1) The catalytic activity of most nanoenzymes is far from that of natural enzymes, and it is still necessary to design highly active nanoenzymes according to the structure-activity relationship of natural enzymes; (2) Studies have shown that there are many kinds and quantities of human symbiotic flora, which play an important role in maintaining human health. How to further enhance the interaction between nanoenzymes and pathogenic microorganisms, enhance the specific killing of harmful bacteria by nanoenzymes, and avoid their damage to normal tissues and symbiotic flora needs further study; (3) When pathogenic microorganisms invade the human body, they tend to fall into immune cells and survive, and continue to infect normal cells and immune cells in the body. Such intracellular bacterial infections are often difficult to treat. The research of nanozyme in this area is still in its infancy. How to design a safe and efficient intracellular bactericidal nanoenzyme platform is full of challenges. To sum up, we believe that through ingenious design, nanoenzymes can become a new powerful weapon to effectively deal with the challenges of pathogenic microorganisms to the environment and human health, and promote the development of nanoenzyme production, learning and research.
4.4 Application of nanoenzyme in wastewater treatment
The development of efficient, stable and low-cost catalysts for the detection and degradation of pollutants in wastewater is of great significance for environmental protection and human health. Nanozymes have shown great potential in the detection and treatment of environmental pollutants due to their low cost, high stability, multiple catalytic activities and low environmental impact. By combining the unique physicochemical properties and enzyme-like catalytic activity of nanomaterials, researchers have developed a large number of nanoenzyme-based pollutant detection methods and treatment technologies. Through the development of new nano-enzyme sensors, the highly sensitive detection of metal ions and organic pollutants in wastewater has been achieved. Through the preparation of nanoenzyme materials with efficient enzyme catalytic activity, the efficient removal of phenol, aniline, dyes and other pollutants was achieved. This section reviews the recent advances in the use of nanozymes for the detection and treatment of pollutants in wastewater, and proposes possible future challenges for this technology.
4.4.1 Introduction
Environmental pollution has become a global problem, which not only affects our normal way of life, but also may threaten the future living environment. In the process of protecting ecology and maintaining environmental safety, the sensitive detection and efficient removal of environmental pollutants and toxic compounds accumulated over a long period of time are two basic problems. Enzymes are highly efficient natural catalysts present in living organisms, which provide effective ways to solve these environmental problems. However, the poor stability of the native enzyme and its easy deactivation under non-physiological conditions have seriously affected its application in wastewater environments.
Nanoenzyme is a nanomaterial with enzyme-like activity itself, which is different from the "nano-immobilized enzyme" that immobilizes natural enzymes to nanomaterials. Since the first report of intrinsic peroxidase-like activity of Fe3O4 NPs in 2007, more than thousands of nanozymes have been reported and have attracted increasing attention due to their high catalytic activity, low cost, high stability, and wide applicability from environmental detection to pollutant treatment[1][465]. With the emergence of a new concept of "nanoenzymology", nanoenzymes have become an emerging field linking nanotechnology and biology, opening up a wider range of applications for nanomaterials[466].
Compared with the natural enzyme, NANase has the following significant advantages: (1) high stability. Inorganic nanomaterials have much higher stability than natural biological enzymes, and can be used in a wider range of pH values (1-12) and temperatures (4-90 ℃). While the native enzyme is usually inactivated under the above-mentioned extreme conditions. (2) Low cost. The production process of natural enzymes is complex and costly, while the production of inorganic nanomaterials is efficient and relatively inexpensive. (3) Reusability. The nanoenzyme can be recovered and maintain high catalytic activity in subsequent cycles. (4) easy to be multifunctional. The nanoenzyme has a large enough specific surface area for various ligand conjugation to achieve surface functionalization of the nanoenzyme.
Researchers have developed new technologies and methods based on nanoenzymes for the detection and removal of pollutants in water environment by using the above characteristics of nanoenzymes.
4.4.2 Water Environmental Pollutant Detection
Since its discovery, nanozymes have been widely used in the detection of proteins, nucleic acids, metal ions, and small environmental molecules. In the initial work of Yan et al., an iron oxide nanoenzyme-based immunoassay was first developed for the detection of hepatitis B virus, followed by an iron oxide nanoenzyme-based test strip for the detection of Ebola virus[1][148]. Due to the high catalytic activity and unique physicochemical properties of iron oxide nanoenzyme, the sensitivity of the developed nanoenzyme test strip is 100 times higher than that of the traditional colloidal gold test strip. By combining the physicochemical properties and catalytic activities of nanozymes, many nanozyme-based platform technologies have been developed for the detection of aquatic environmental pollutants (Table 6).
4.4.2.1 Direct use of nanoenzyme activity
Peroxidase can catalyze H2O2 to oxidize the substrate into a colored product, and then can use the color change of the product to detect the target. Based on this principle, Wei and Wang et al. First developed a colorimetric method based on nanoenzyme to detect H2O2 using Fe3O4NPs[8]. The colorimetric detection of H2O2 was achieved by the Fe3O4 nanoenzyme, which catalyzed the oxidation of the substrate ABTS to a colored product by H2O2. The detection limit was as low as 3×10-6mol·L-1, and the linear detection range was 5×10-6~1×10-4mol·L-1. In addition, many nanoenzymes have been applied to hydrogen peroxide detection.Including Fe3O4, Co3O4, Cu2O,FeS、CeO2 、CoFe2O4 、BiFeO3 、MnFe2O4, CdS, FeSe, FeTe, ZnFe2O4NPs, graphene oxide, fullerene and carbon nanotube, etc[495].
Kim et al. Prepared a paper microfluidic device using DNA-copper hybrid nanoflowers with laccase activity, which can be used for colorimetric detection of various phenolic compounds such as dopamine, catechol, and hydroquinone[488]. In the presence of phenolic compounds, GNFs can catalyze their oxidation with 4-aminoantipyrine to form colored adducts. The detection limits of the three phenolic compounds were 4.5, 3.0 and 4.5 μg/mL, respectively.
4.4.2.2 Inhibition of nanoenzyme activity
The color rendering of the substrate can be reduced by inhibiting the activity of the nanoenzyme, and the detection of the substance to be detected can be realized according to the change of the color rendering. Liang et al. First developed a nanoenzyme and natural enzyme cascade reaction system for rapid detection of organophosphorus pesticides and nerve agents[467]. In the presence of acetylcholine, the native enzymes acetylcholinesterase (AChE) and choline oxidase (CHO) catalyze the generation of H2O2, activate the Fe3O4 NANase, and catalyze the oxidation of colorimetric substrates to produce a chromogenic reaction (Figure 57). After incubation with organophosphorus compounds (OPs), AChE enzyme activity was inhibited, and the production of H2O2 was reduced, resulting in a decrease in the catalytic oxidation of the contrast-colored substrate by the Fe3O4 nanoenzyme and a concomitant decrease in color absorbance. In this work, 1 nmol·L-1 sarin, 10 nM methyl paraoxon, and 5μmol·L-1 acephate could be detected. Also based on the enzyme cascade reaction, Liu et al. Constructed a ternary cascade enzyme reaction system of UsAuNPs/2D MOF, AChE and choline oxidase (ChOx), and realized the colorimetric detection of trichlorfon based on the inhibition of organophosphorus pesticides on acetylcholinesterase[468]. Huang et al. Prepared a bioactive paper for organophosphorus pesticide residue detection based on the cascade catalytic reaction of SACe-N-C monoatomic nanoenzyme with POD activity and AChE, and constructed a 3D printing platform to realize the field portable detection of omethoate, methamidophos, carbofuran and carbosulfan[469].
The detection of metal ions in the environment is very important. Taking advantage of the ability of metal ions to inhibit enzyme activity, researchers have developed a series of nanoenzymatic materials to achieve the detection of low concentrations of metal ions. For example, Fu et al. Realized the detection of Hg2+ in aqueous solution with a detection limit of 7.2 nmol·L-1 and a linear response range of 0~120 nmol·L-1 by using platinum-based perPOD nano-mimics synthesized by BSA-based nucleation template[472]; The detection of the Ag+ is realized by utilizing that the Ag+ can obviously inhibit the peroxidase catalytic activity of the Pd nanoenzyme, and the detection limit is 1.2 nmol·L-1[475]. Considering that copper ions are not only involved in the synthesis of a variety of enzymes, but also in metabolic processes, Liu et al. Used Cu2+ to inhibit the enzyme activity of gold nanoclusters and demonstrated a simple strategy to detect Cu2+[496][477]. Peng et al. Firstly realized the detection of Pb2+ based on colorimetric method by using the property that the catalytic activity of Au @ PuNP nanoenzyme was reduced by Pb2+-S2[478]. Cheng et Al. Developed a single-atom nanoenzyme with phosphatase activity (SACe-N-C), and found that Al3+ could specifically bind to O atoms to form Al — O bonds, thereby inhibiting its phosphatase activity, thus achieving rapid, portable and efficient detection of Al3+[518]. Wu et al. Established a porous Co3O4 nanosheet with peroxidase nanoenzyme activity for rapid and ultrasensitive detection of a variety of heavy metal ions[479].
It is also based on the ability of the substance to be detected to inhibit the activity of the nanoenzyme that the detection of a variety of organic substances has been achieved. For example, Singh et al. Developed a colorimetric sensor for malathion-sensitive detection using palladium-gold bimetallic nanoenzyme[482]. In the presence of hydrogen peroxide, the palladium-gold nanoenzyme showed good peroxidase-mimicking activity with o-phenylenediamine as the chromogenic substrate. Moreover, the developed palladium-gold nanoenzyme is stable in a wide temperature range (4 to 70 deg C) and shows high peroxidase activity in a pH range of 2 to 6, so that the detection of malathion under extreme conditions can be realized. The lowest detection limit of the method was 60 ng·mL-1. Taking advantage of the ability of malathion to inhibit both Ir NPs POD and OXD activities, Han et al. Developed a highly selective colorimetric assay for malathion, in which the linear range of detection was 0.1~5.0μmol·L-1 and the detection limit was 6 nmol·L-1[483]. Wei et al. Developed a colorimetric nanoenzyme sensor array based on heteroatom-doped graphene, which realized the detection of aromatic pesticides (Fig. 58)[497]. Graphene materials have adsorbability, that is, when different pesticides are adsorbed on graphene, the active sites of nanoenzymes may be covered to different degrees, resulting in the decrease of their POD activity. Five pesticides (lactofen, fluroxypyr, bensulfuron-methyl, fomesafen and diafenthiuron) in the range of 5~500μmol·L-1 were successfully identified based on this method. In addition, methods based on nanoenzymatic materials for the detection of cyanide, pesticides atrazine, simazine, and phenolic compounds have been reported[481][486][487][170].
4.4.2.3 Enhanced nanoenzyme activity
It is also an important method to realize the detection of the analyte through the enhancement of the activity of the nanoenzyme by the analyte. Mercury ion (Hg2+) is recognized as one of the most harmful pollutants in the environment, and its accumulation in the human body can cause damage to the central nervous system[498]. Yang et al. Developed an efficient nanoenzyme strategy based on Au/Fe3O4/ graphene oxide hybrid nanomaterials for highly sensitive detection and rapid removal of Hg2+ in aqueous solution, based on the fact that Hg2+ can be reduced by sodium citrate to form Hg0, which can be dispersed on the surface of Au NPs to change their surface properties and improve their POD activity[471]. The formation of Au-Hg amalgam (alloy) can significantly enhance the enzyme activity. Based on this, Kwon et al. Also developed a gold nanozyme-based paper chip for colorimetric detection of Hg2+, with a detection limit of 0.06 ng and a linear response range of 0.02 – 2000 ng (Fig. 59)[473]. Similarly, the oligoethylene glycol modified AuNPs (OEG-AuNPs) prepared by Cao et al. Also achieved the specific detection of Hg2+[474].
The enzymatic activity of the material can be restored by binding of the analyte to the aptamer. Based on this, Bansal et al. combined the inherent peroxidase-like activity of gold nanoparticles (GNPs) with the high affinity and specificity of ssDNA aptamer to develop a surface plasmon resonance sensor for kanamycin detection (Fig. 60), in which the aptamer molecule has high affinity and specificity with kanamycin attached to GNPs[480]. When the target molecule is present, the aptamer is released from the GNPs and adheres to kanamycin, while the free GNPs exert catalytic activity. The method provided visual detection of kanamycin within 3 – 8 min with a detection limit of 1.49 nmol·L-1.
In recent years, nanomaterials have shown great potential in the detection of pollutants in water environment. Detection methods based on nanomaterials mainly include fluorescence, chemiluminescence, Raman and electrochemical sensing, but their detection methods often have difficulties such as low sensitivity, poor repeatability and complex operation. Based on the color change caused by the interaction of the nanoenzyme with the substrate, a colorimetric nanoenzyme-based sensor can be easily constructed. It has the advantages of simple operation, rapid response, simple instrument and equipment, visualization and semi-quantification, and can be used for sensitive and rapid visualization detection of metal ions, pesticides, nerve agents, toxins and other organic substances in water environmental pollutants.
4.4.3 Water Environment Pollutant Treatment
The development of efficient, robust, and low-cost catalysts for the removal of environmental pollutants from wastewater is of great significance to the environment and health. The enzyme-like catalytic activity of NANase has been widely studied for the catalytic degradation of environmental pollutants such as phenol, aniline, rhodamine B, methylene blue, and xylenol orange (Table 7). Using nanoenzyme as catalyst to treat water environmental pollutants has the advantages of low cost, easy preparation, high stability, small environmental impact, recyclability and so on.
4.4.3.1 Peroxidase
Peroxide nanoenzyme can act on peroxide to produce transient free radical active intermediates, and then can use the strong oxidizing property of free radicals to degrade pollutants.
In 2008, Yan et al. First used Fe3O4 nanoparticles as a mimic of peroxidase-like enzyme to remove phenol from wastewater[499]. Under the condition of 16 ℃ and pH = 3, the Fe3O4 nanoenzyme can catalyze hydrogen peroxide to produce hydroxyl radical, which can effectively remove 85% of phenol, and the nanoenzyme can be recycled for many times. Zuo et al. Designed a carbon nanotube/magnetic nanoparticle nanocomposite for efficient catalytic oxidation of phenol and its removal from industrial wastewater[500]. The synthesized nanocomposite retains the magnetic properties of individual magnetic nanoparticles and thus can be effectively separated under an external magnetic field. In addition, the formation of nanocomposite enhanced the inherent POD-like activity of magnetic nanoparticles, achieving about 99% phenol removal efficiency after 10 H of treatment time. Moreover, the resulting insoluble polyaromatic products can be easily separated from the wastewater. In 2012, Fang et al. Studied the removal of bisphenol A by using the peroxidase-like catalytic activity of ferromagnetic nanoenzyme, and achieved a clearance rate of up to 95%[505]. In addition, people have optimized the synthesis method of iron oxide nanoenzyme and the preparation of composite materials to improve its peroxidase activity, and achieved the degradation of a variety of phenolic compounds, aniline, dyes, humic acid and so on[516,537][499][502,504,512⇓ ~514,521,534,538][533]. Tang et al. Found that Fe3O4 magnetic nanoparticles could activate sulfate anions to produce free radicals, and then degrade sulfadiazine, and further studied the catalytic mechanism[503].
In addition to iron oxide nanoenzymes, non-ferryl peroxide nanoenzymes for pollutant degradation have also been reported. In 2015, Feng et al. Developed a copper oxide nanoparticle with POD activity to remove phenol pollutants[525]. Under the optimal conditions, phenol could be completely removed within 35 min. In 2014, Jiang et al. Developed a bismuth bromide nanoenzyme for the removal of methylene orange organic dye pollutants[511]. In 2016, Yang et al. First reported the peroxidase-like catalytic activity of cubic boron nitride to decompose H2O2 and produce hydroxyl radicals, and achieved the removal of rhodamine B[524]. In the work of Tang et al., porous Co3O4 nanorods/graphene oxide hybrid nanomaterials were used for methylene blue removal using the peroxidase-like activity of hybrid nanomaterials[526]. The hybrid nanomaterials exhibited significantly enhanced pollutant adsorption capacity and enhanced peroxidase activity compared to Co3O4NPs or graphene oxide alone. Xu et al. Also reported the peroxidase activity of deficient vanadium oxide nanomembranes, which can be used to degrade rhodamine pollutants[527]. Moreover, the authors propose that different oxygen vacancies in nanozymes can mimic the complexity and functionality of native enzymes.
4.4.3.2 Oxidase
The oxidase can oxidize a substrate in the presence of a O2. Oxidase materials are often combined with activated persulfate in pollutant treatment processes for the purpose of degrading organic matter. For example, Huang et al. Reported the novel Fe-Bi bimetallic MOF-derived carbon-supported Fe-N4 and Bi-N4 two-site FeBi-NC SAzyme single-atom nanoenzyme with high OXD activity, which can be used for cascade catalysis and persulfate (PMS) activation to degrade dye pollutants (Figure 61)[522]. Both Fe-N4 and Bi-N4 in FeBi-NC show strong binding energy and electron donating ability, which can promote PMS activation to generate highly reactive intermediates for rhodamine B degradation, which can achieve 100% degradation within 5 min at 25 ° C environment. In 2020, Huang et al. Fabricated Co-based bimetallic hollow nanocages (HNCs) (C-CoM-HNC, M = Ni, Mn, Cu, and Zn) by ion-assisted reaction and low temperature reaction[528]. The copper-doped C-CoM-HNCs nanostructure has stronger oxidase activity and bisulfate (PMS) activation ability than other C-CoM-HNCs, which can be applied to the degradation of rhodamine B, and has good reusability in eight cycles.
In addition, nanomaterials with both oxidase and peroxidase activities have also been used for pollutant treatment. In 2021, Zuo et al. Reported a 3D layered BSA-NiCo2O4@MnO2/C multifunctional micromotor with both oxidase and peroxidase activities, which can rapidly move forward in aqueous solution through the bubble recoil mechanism, not only to enhance the colorimetric sensing performance, but also to improve the removal efficiency of Cu2+ through enhanced mass transfer in the detection and catalytic degradation system[529]. In addition, the micromotor showed good catalytic activity for the degradation of tetracycline hydrochloride, and the degradation efficiency was > 95% within 180 min at room temperature.
4.4.3.3 Other
Laccase has the function of oxidizing polyphenols, which can be used to remove phenols in water environment. For example, Zhang et al. Prepared iron single-atom-anchored N-doped carbon materials (Fe1@CN-20) that can be used as laccase mimics in order to solve the inherent defects of laccase sensitivity, poor denaturation stability, and high cost of purification process[523]. Which exhibited kinetic constants comparable to those of laccase. The material has excellent stability, that is, it can remain stable under the conditions of extreme pH (2 ~ 9), high temperature (100 ℃), strong ionic strength (500 mmol·L-1NaCl), high ethanol concentration (40% by volume) and long storage time (2 months). The material can be applied to the detection and degradation of phenolic compounds, and the degradation of 2,4-dichlorophenol and 2,6-dimethoxyphenol was achieved in 12 and 4 H, respectively, with an efficiency of 65% and 100%, respectively. Huang et al. Prepared a novel NANase (CH-Cu) with laccase-like activity by coordination of Cu+/Cu2+ with cysteine (Cys) -histidine (His) dipeptide[530]. Compared with laccase, CH-Cu nanoenzyme has a higher efficiency in the degradation of chlorophenols and bisphenols, and the degradation efficiency can reach 90% in 12 H. Huang et al. Prepared a novel MOF-based amorphous nanoenzyme (CA-Cu) through a facile solvothermal reaction of cyanuric acid and Cu2+ to mimic the N-Cu coordination environment between its active site imidazole and Cu2+, which has both laccase and catecholase activities and can achieve degradation of phenolic compounds in a neutral (pH = 6.8) environment[532]. Rao et al. Synthesized CuNi/CoMoO4 nanoenzyme with laccase-like activity by hydrothermal method, calcination and impregnation techniques, which can be used as persulfate (PMS) activator (Fig. 62)[531]. CuNi/CoMoO4 can activate PMS to produce ·OH and S for the degradation of organic pollutant acid fuchsin. The degradation efficiency can reach 99.45% within 40 min.
Vanadium haloperoxidase can catalyze the oxidation of halides (Cl- and Br-) with hydrogen peroxide to form hypohalous acids (HOCl and HOBr). Based on this, Tremel et al. Embedded CeO2-x nanorods with vanadium haloperoxidase activity into a polymer mat to prevent biological contamination in aqueous environments[541]. Under the action of H2O2, CeO2-x nanorods catalyze the oxidative bromination of Br- to HBrO, which will destroy the quorum sensing of microorganisms and prevent the biological pollution of algae.
In addition to this, NANase with phosphatase activity has also been used to degrade organic matter. In 2015, Jano Janoš et al. Showed that CeO2 nanomaterials had phosphatase activity and could be used for the rapid degradation of organophosphorus pesticide methyl parathion and some nerve agents[517]. The composite maintains good magnetic properties and shows efficient degradation performance, with a reaction half-life of about 10 min and a conversion degree of nearly 100%. In 2016, Mugesh et al. Found that the vacancies of cerium oxide nanoparticles could mimic the catalytic activity of phosphotriesterase to rapidly degrade neurotoxic substances such as paraoxon, parathion and malathion, so as to reduce or even eliminate their harm to human body[542]. They demonstrated that the hydrolytic action of the nanozyme is due to a synergistic effect between Ce3+ and Ce4+ ions located in the active site.
To sum up, based on the unique enzyme-like catalytic activity of nanoenzyme itself, it can be directly used for the removal of a variety of organic matter in wastewater. Compared with other nano-catalytic technologies using photocatalysis and electrocatalysis, the direct use of nano-enzyme itself can produce strong oxidative free radicals, which can effectively achieve the degradation of organic matter. The process can reduce the consumption of high energy and the use of a large amount of oxidant, reduce the cost and avoid secondary pollution.
4.4.4 Brief summary
As an emerging field, nanozymes also have many challenges in the detection and removal of environmental pollutants. In the detection of environmental pollutants, even though the detection limits of organic pollutants and metal ion pollutants have been reduced to the nanomolar concentration range, nanoenzyme-based sensors are vulnerable to the interference of complex pollutants in wastewater when detecting various wastewater samples. Therefore, it is necessary to further improve the stability and anti-interference ability of the detection, and enhance the specificity of the detection. In recent years, there are also many strategies to improve the detection activity of nanozymes, such as molecular blotting, which makes nanozymes have artificial selective binding sites. Another solution is to establish a cascade system of nanoenzymes and biological enzymes, such as acetylcholinesterase and Fe3O4 nanoenzymes mentioned in this paper to complete the rapid detection of organophosphorus pesticides and nerve agents. For the application of nanoenzymes in the degradation of pollutants in wastewater, although the catalytic activity of some nanoenzymes is higher than that of natural enzymes, there is still an urgent need to improve their catalytic activity to deal with a large number of pollutants in the actual environment. In the past decade, researchers have used various modification methods to improve the catalytic activity of nanoenzymes, so that they can be better applied to the removal of pollutants in the actual environment, such as compounding carbon nanomaterials with magnetic nanoparticles, multi-metal doping, etc[500,512,513,522]. On the basis of the above materials, it is urgent to continue to enhance the catalytic activity of enzymes, improve the degradation efficiency and reduce the cost. Secondly, although a variety of enzymatically active nanoenzyme materials have been developed for pollutant degradation in recent years, nanoenzymes with peroxidase-like activity are still the main catalysts for pollutant removal.And the peroxide-like nanoenzyme showed high catalytic activity only at low pH, indicating that the nanoenzyme-based treatment technology can only be used for acidic wastewater. The application of nanoenzyme degradation technology to the degradation of pollutants in a wide range of pH environments is an urgent problem to be solved. Thirdly, in practical application, microbial degradation is a common method used for sewage treatment. The method refers to the use of microbial metabolism to degrade organic matter in wastewater. However, microorganisms are vulnerable to interference from high toxicity or biological toxicity of high concentration organic matter, which reduces their degradation function. A new idea is to combine nanoenzyme with microorganisms, and use the activity of nanoenzyme to improve the activity of microorganisms to degrade harmful substances. Qu et al. Found that surface-protonated nanopolymers enhanced the bioactivity of algae to remove harmful substances[539]. Metabolomic analysis has shown that nanomaterials can induce moderate oxidative stress in algae and upregulate the biosynthesis of extracellular polymers to resist physiological damage caused by toxic substances in water. In addition, temperature is also an important factor affecting the ability of microbial degradation. Too low ambient temperature not only interferes with the formation of microorganisms, but also leads to the reduction of degradation activity. Therefore, it is the key to explore a method with the advantages of high efficiency, low cost and not easy to be disturbed by low temperature environment to degrade organic matter in wastewater. Nano-enzyme has stronger environmental adaptability, that is, it can maintain strong enzyme activity in low temperature environment. Therefore, the development and utilization of nano-enzyme materials for sewage treatment can overcome the interference of low temperature and solve the problem of wastewater treatment in low temperature and cold areas. Although the field of nanoenzymes has been substantially developed for environmental applications, future breakthroughs in nanoenzyme technology are still needed before nanoenzyme technology can be applied to the detection and degradation of actual wastewater pollutants on a large scale.
Carbon sequestration, nitrogen fixation and clean energy development are also important issues in the environmental field. Carbon dioxide is the main component of greenhouse gases, which directly induces global temperature rise. Active capture of carbon dioxide from the atmosphere is a solution, in addition to reducing carbon dioxide emissions. Ammonia plays an important role in the field of agriculture and is an important agricultural fertilizer. Most of the production of ammonia today comes from the Hubble process. The process uses nitrogen and hydrogen, which is an energy-intensive process that also produces large amounts of carbon dioxide. The use of fossil fuels has greatly promoted the development of industry, but at the same time, it has brought bad effects on human living environment. The development and utilization of clean energy and the promotion of green industry are of great significance to environmental protection. At present, a series of corresponding technologies have been developed in the above fields, such as mineralizing carbon dioxide into building materials such as limestone, changing soil microorganisms and plants to improve the carbon sequestration capacity of soil.Converting biomass energy into electric energy/heat energy or hydrogen energy, fixing nitrogen by catalyzing nitrogen to produce ammonia, improving nitrogen fixation efficiency by changing catalytic metal or using photoelectrocatalysis, and producing hydrogen by biomass fermentation or photolysis and electrolysis of water. Although there are various methods, each has its own limitations, such as land use, inefficiency, the use of a large number of precious metals, and the lack of long-term sustainable development models. Of course, different enzymes that exist in nature will also play a role in environmental protection. Photosynthetic plants or microorganisms can convert carbon dioxide and water in the air into glucose, nitrogen-fixing bacteria can convert nitrogen in the air into ammonia, and hydrogenase can reduce protons into hydrogen at normal temperature and pressure. However, the environmental tolerance of the above natural enzymes is weak, or the corresponding enzymes only exist in a small number of bacteria. Therefore, the development of nano-enzyme materials with enzyme-like activity, high environmental tolerance and large-scale preparation to achieve the purpose of improving catalytic efficiency will have a significant impact on the development of carbon fixation, nitrogen fixation and clean energy industries. To sum up, nanoenzymes also show strong application prospects in carbon fixation, nitrogen fixation and clean energy industries.
By combining unique physicochemical properties and catalytic characteristics, nanozymes have shown great promise for environmental pollutant detection and degradation treatment[534,535]. So far, nanozymes have successfully achieved highly sensitive detection of a variety of water environmental pollutants, such as heavy metal ions, pesticides, nerve agents, toxins and other organic pollutants. The enzymatic activity of nanoenzymes has been used to remove a variety of water environmental pollutants, including wastewater containing phenols, amines and dyes. The application of nanoenzymes has shown great advantages in the removal of pollutants from water environment, which usually combines the high oxidation efficiency and magnetic separation characteristics of magnetic nanoparticles, and also shows the characteristics of high catalytic activity, mild application conditions and easy recovery. Therefore, the introduction of nanoenzymes into more environmental research and the continuous improvement of the catalytic efficiency and specificity of nanoenzyme materials can make them play an important role in the process of environmental protection.
5 Prospects for the field of nanoenzymes
5.1 Key Scientific Issues
(1) Analysis of the key catalytic sites of nanoenzymes: Except for a few single-atom nanoenzymes, we still know little about the key structural information of the catalytic sites of most nanoenzymes. The surface structure of nanomaterials is complex, and it is not clear whether the catalytic activity of nanoenzymes is dominated by some or a specific nanostructure. Uncovering these key structural elements will not only help to understand the catalytic mechanism of nanozymes, but also provide evidence for the design of high-performance nanozymes.
(2) Analyze the relationship between the confined microenvironment and the catalytic selectivity of nanoenzyme: The path selection of catalytic reaction is often determined by the catalytic microenvironment around the active site. An in-depth understanding of the interrelationship between the confined microenvironment of nanozymes and their catalytic selectivity will be of great significance for the development of nanozymes with high substrate specificity and single catalytic activity.
(3) Analysis of the catalytic mechanism of nanozyme: Although nanozyme has similar catalytic ability to natural enzyme, its structure is quite different from that of natural enzyme. Therefore, the nanozyme has a unique catalytic mechanism that is different from that of the native enzyme. However, most of the current nanoenzyme research focuses on the optimization of catalytic activity, and less on the analysis of catalytic mechanism. Although some studies have predicted the catalytic pathway of nanozymes by theoretical calculation and other methods,However, few studies have been able to clarify the key information such as the key sites where nanoenzymes play a major catalytic role, the dynamic changes of nanoenzyme structure during catalysis, the structural forms of nanoenzyme-substrate complexes and reaction intermediates from the level of experimental verification. It is very important for the rational design and synthesis of high-performance nanoenzymes in the future to deeply analyze the catalytic mechanism of nanoenzymes by using advanced nanocharacterization methods, understand the whole process of nanoenzyme-catalyzed enzyme-like reactions at the atomic/molecular level, and establish the catalytic kinetics suitable for nanoenzymes.
(4) Expanding the catalytic type: The catalytic reaction of the existing nanoenzyme is a relatively simple redox reaction. The catalytic type of nanoenzyme can be expanded from two aspects. On the one hand, redox reactions with important social and economic value, such as methane monooxygenation, can be studied; On the other hand, we should expand the application of nanoenzymes in non-redox reactions.
(5) Expanding the application field: At present, the application of nanoenzymes is concentrated in the field of medicine and health, and many important advances have been made. However, the difficulty of clinical transformation of nanomedicine and the low market recognition make it difficult for nanoenzyme to create significant social benefits in a short time. Under the background of green development, nanoenzyme itself, as a green and environmentally friendly catalyst, has great application potential in chemical synthesis, new energy development, environmental protection and other fields. Based on the advantage of short development cycle, once the appropriate nanoenzyme is developed, it is expected to achieve high market recognition, and is expected to have a greater impact in the short term, expanding and activating the application research field of nanoenzyme.
(6) Establishment of nanoenzyme Database: At present, the number of nanoenzyme literature and data has reached a considerable scale, and there are many types and catalytic types of nanoenzymes. Through the establishment of nanoenzyme database, it will be conducive to the integration of large data analysis methods to analyze the mechanism and structure-activity relationship of nanoenzymes, and lay the foundation for the establishment of theoretical system of nanoenzyme catalysis.
And (7) establish a nanoenzyme Library (Library): establishing a corresponding nanoenzyme library accord to that type of the reaction catalyzed by the nanoenzyme or the type of the material based on the standardization of nanoenzyme research. This will facilitate the large-scale production application of nanozyme research.
(8) Large-scale production and quality control: In order to meet the transformation application, it is necessary to establish the large-scale production process and quality control standards of nanoenzyme. At present, most nanoenzymes are synthesized in small systems in the laboratory for material characterization and performance testing. However, the mass production and pilot scale-up of materials are less developed, and there is a lack of quality control methods and standards for materials and enzymes, including nanoscale, morphology, dispersion, enzyme activity and stability. Therefore, it is necessary to establish a large-scale production and quality control system to lay a material foundation for transformation and application.
5.2 Conclusion
As a new scientific discovery, nanoenzyme has played a subversive role in the development of enzymology, biocatalysis and nanotechnology. On the one hand, nanozymes establish a new paradigm for nanobiological research. Since the discovery of nanoenzymes, people have begun to study nanoeffects and design nanomaterials from the perspective of biocatalysis by using enzymatic research theories and methods. This new research paradigm makes it easier for us to understand the catalytic behavior of nanomaterials in biological systems, which is conducive to promoting the biomedical applications of nanomaterials.
On the other hand, nanozymes make us realize that although the concept of "enzyme is protein or RNA" is deeply rooted in people's minds, enzymes may still be an iceberg that has not yet shown its full picture. People's objective understanding of things is always improving with the progress of science and technology. Throughout the history of enzyme research, we can find that the core connotation of enzyme is "efficient biocatalysis", which is not limited to its material properties (cells, proteins, RNA or DNA, etc.), but refers to any substance that can efficiently catalyze biochemical reactions under physiological conditions. The progress of protein characterization technology has led to the discovery of protein enzymes, the progress of nucleic acid characterization technology has led to the discovery of nucleic acid enzymes, and nanoenzymes have been discovered with the progress of nanocharacterization technology. With the progress of science and technology, more forms of enzymes may be revealed in the future.
As a kind of "new material", the core connotation and scientific significance of nanoenzyme are still further clarified with the deepening of research. It is undeniable that nanozymes have efficient biocatalysis, which can efficiently catalyze known biochemical reactions in physiological environments, and can intervene in life activities in vivo through catalytic behavior. In addition, the catalytic activity of nanozyme is determined by its own intrinsic structure and does not depend on any known biological enzyme molecules. Therefore, we believe that with the deepening of nanoenzyme research and the gradual improvement of the theoretical system, people will have more and more accurate positioning and evaluation of nanoenzymes, and it is expected to have a far-reaching impact on biomedicine, environmental protection, green synthesis and other fields.
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