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Application of Fluorescence Nanomaterials in Pathogenic Bacteria Detection

Dan Zhao , Changtao Wang , Lei Su , Xueji Zhang

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (9) : 1482 -1495.

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Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (9) : 1482-1495 DOI: 10.7536/PC201104
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Application of Fluorescence Nanomaterials in Pathogenic Bacteria Detection
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Abstract

Pathogenic bacteria contamination brings severe safety problems to human health. Fast, accurate and sensitive detection of pathogenic bacteria is an important way to reduce pathogenic bacteria pollution. Traditional methods for detecting pathogenic bacteria have the disadvantages of being time-consuming and laborious. Fluorescent nanomaterials have the advantages of high fluorescence intensity, good stability and excellent biocompatibility, which provide a new research approach for the application of fluorescent nanomaterials as biosensors for pathogenic bacteria detection. This review summarizes the applications of various fluorescent nanomaterials in pathogenic bacteria detection in recent years, including semiconductor quantum dots, fluorescent metal nanoclusters, carbon nanomaterials, upconversion nanoparticles and fluorescent silicon nanoparticles. It mainly focuses on the analysis and comparison of the optical properties and detection mechanisms of different types of fluorescent nanomaterials. The bioconjugation of nanomaterials plays an important role in the whole process of pathogenic bacteria detection, which is closely related to the detection specificity. This article introduces the characteristics of different recognition elements, including antibodies, aptamers, phages and antibiotics. The conjunction methods between different recognition elements and nanomaterials are also discussed. Finally, the advantages and limitations of different nanomaterials for detecting pathogenic bacteria are reviewed and the development prospects in practical application and research priorities in the future are addressed.

Contents

1 Introduction

2 Application of fluorescent nanomaterials in detection of pathogenic bacteria

2.1 Quantum dots

2.2 Metal nanoclusters

2.3 Fluorescent carbon nanomaterials

2.4 Up-conversion nanomaterials

2.5 Fluorescent silica nanomaterials

3 Recognition methods

3.1 Specific recognition

3.2 Non-specific recognition

4 Conclusion and outlook

Graphical abstract

Keywords

fluorescence / nanomaterial / pathogenic bacteria / detection / recognition

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Dan Zhao, Changtao Wang, Lei Su, Xueji Zhang. Application of Fluorescence Nanomaterials in Pathogenic Bacteria Detection. Progress in Chemistry, 2021, 33(9): 1482-1495 DOI:10.7536/PC201104

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1 Introduction

Pathogen detection is closely related to human health. There are more than 250 diseases caused by different foodborne pathogens in the world. These diseases are mainly caused by Streptococcal, Salmonella typhimurium, Escherichia coli O157: H7, Listeria monocytogenes and Mycobacterium tuberculosis[1]. Early and rapid detection of pathogenic microorganisms is the key to the prevention of infection. The traditional methods for quantifying and detecting pathogens include plate counting and Polymerase Chain Reaction (PCR). Plate counting can estimate the number of viable bacteria in a sample, and PCR can detect specific DNA and RNA in target bacteria. These methods have high specificity and sensitivity, but often require complex sample preparation and long detection time[2].
Shortening the time required for the detection of pathogens and reducing the detection limit are long-standing challenges in the field of food detection. Biosensor-based detection strategies are currently a very promising approach to overcome these challenges. Biosensor is a kind of sensor surface modified with biological materials such as antibodies and aptamers as recognition elements, which converts recognition signals into measurable physical or chemical signals through biochemical reactions. Due to the high selectivity and sensitivity of biological components to some special molecules, the detection accuracy of biosensors is higher[3]. Currently available sensors for detecting samples are limited by factors such as response time, detection range, detection limit, signal to noise ratio, and specificity[4]. The unique physical and chemical properties of nanomaterials can improve the performance of biosensors, especially the functionalized nanomaterials with recognition sites have more efficient ability to capture analytes in the whole sensing and detection process[5,6].
Fluorescence analysis refers to an analysis method that some substances produce fluorescence under the irradiation of light with specific wavelength, and the qualitative or quantitative analysis of substances is carried out by using its intensity. This method has many advantages, such as good selectivity, high fluorescence intensity, high sensitivity, and a variety of detection techniques and methods[7]. Because of the advantages of fluorescence analysis technology, the most appropriate detection technology and scheme can be selected according to the actual detection conditions. Fluorescent probes are necessary for fluorescence analysis. In the early stage, researchers generally used traditional fluorescent dyes as fluorescent agents, but fluorescent dyes have the defects of high background, narrow excitation spectrum, wide and asymmetric fluorescence emission spectrum, and easy to be photobleached, which greatly limited their application in the biomedical field[8]. With the rapid development of nanomaterials and nanotechnology, new fluorescent nanomaterials are expected to gradually replace traditional luminescent materials due to their high fluorescence quantum yield and superior light stability. The advantages of fluorescent nanomaterials for bacterial detection lie in their small size, strong adsorption capacity and high surface reactivity, and one pathogen can be combined with multiple nanoparticles at the same time, so that the labeled target bacteria have higher fluorescence intensity[9]. With its high fluorescence stability, it can track the target in real time within minutes to hours. In addition, different strains can be detected simultaneously by using multicolor fluorescent nanoparticles, and the toxicity of the fluorescent nanoparticles is greatly reduced after the surface of the fluorescent nanoparticles is wrapped with an inert substance shell, which is far lower than that of organic dyes[10].
At present, fluorescent nanomaterials widely used in detection include inorganic fluorescent nanomaterials, such as carbon-based materials such as quantum dots, metal nanoclusters, carbon dots and graphene quantum dots. Rare earth up-conversion nanomaterials, fluorescent silicon nanoparticles, organic fluorescent nanomaterials, etc[11]. In order for these materials to recognize the target, they are often functionalized to recognize and attach to the bacterial surface. The immobilization of recognition elements on the surface of nanomaterials is an important step to realize the function of biosensors. In this paper, the application of different fluorescent nanomaterials in the detection of pathogenic bacteria was summarized, and the identification methods of fluorescent nanomaterials and pathogenic bacteria were summarized, which provided a reference for the application of fluorescence nanomaterials in this area.

2 Application of Fluorescent Nanomaterials in Detection of Pathogenic Bacteria

2.1 Semiconductor quantum dot

Quantum dots, also known as semiconductor nanoparticles or semiconductor nanocrystals, are a kind of atomic clusters composed of II ~ VI or III ~ V group elements with three-dimensional size in the nanometer scale. According to the quantum confinement effect, the band gap of quantum dots can be adjusted by controlling the size and material composition of quantum dots. CdX (X = S, Se, Te) quantum dots are the most widely studied quantum dots. Compared with organic fluorescent materials, quantum dots (QDs) have better photostability, higher quantum yield and wider absorption band. Different QDs can emit different fluorescence when excited by a single excitation source. Quantum dots (QDs) have been used as fluorescent probes and donors in fluorescence-based sensors due to their good fluorescence emission properties[12].
Magnetic nanoparticles have good operability and strong separation ability. Immunomagnetic separation technology based on antibody specific recognition can ensure the specific recognition of target bacteria, which makes the separation and enrichment accurate and effective, and the processing method simple and fast. Dogan et al separated and detected Escherichia coli by combining chitosan-modified CdTe quantum dots with Fe3O4 magnetic nanoparticles[13]. The method comprises the following steps of: respectively modifying specific antibodies of Escherichia coli on the surfaces of the two types of nanoparticles, wherein the magnetic nanoparticles are used for adsorbing the Escherichia coli and separating the Escherichia coli from other impurities; and then, labeling target bacteria with fluorescent quantum dots, and quantifying the concentration of the target bacteria by detecting the fluorescence intensity of the quantum dots. The detection limit of this assay for E. coli was 30 cfu/mL (Figure 1).
Xue et al. Used High Gradient Magnetic Separation Device to separate and detect Escherichia coli[14]. In the presence of a magnetic field, the magnetic nanoparticles modified with the monoclonal antibody of Escherichia coli O157: H7 are fixed in the channel and can specifically capture target bacteria, and other substances in the liquid to be detected flow out of the channel,Injecting the quantum dots modified with the Escherichia coli antibody into the channel to be combined with the Escherichia coli, removing the magnetic field, allowing a magnetic nanoparticle-target bacterium-quantum dot complex to flow out along with the channel, and performing fluorescence detection on the quantum dots by using a fluorescence detection device to quantify the content of the target bacteria. This high-gradient magnetic separation device effectively separated 86% of E. coli O157: H7 with a detection limit as low as 14 cfu/mL, and was able to complete the pathogen detection within 2 H (Figure 2). It is worth noting that in the process of immunomagnetic separation, there will always be a small part of the analyte loss, which will affect the accuracy of the detection results[15].
In addition, bacteria can also be detected by directly observing the change in fluorescence intensity of the reaction system without separating the pathogenic bacteria from the fluorescent marker. Because the surface of concanavalin contains lipopolysaccharide binding sites, Ebrahim et al. Used concanavalin A-modified CdTe quantum dots to specifically recognize Serratia, whose bacterial cell wall contains lipopolysaccharide[16]. The fluorescence intensity of CdTe decreased with the increase of Serratia concentration, and the mechanism of fluorescence quenching may be related to the internal filtration effect, electron transfer and non-radiative recombination[16].
It is verified that compared with the quantum dots with only a core, the core-shell quantum dots have better fluorescence properties, and the single quantum dot particles are easily affected by external impurities and lattice defects, so the fluorescence quantum yield is low. When used as a core/core and wrapped by another semiconductor material to form a core-shell structure, it can effectively eliminate the non-radiative relaxation path, prevent photochemical fading, and promote the fluorescence emission intensity to be improved[4].
Yu et al. linked the antibody of Escherichia coli O157: H7 with CdTe/CdS with a core-shell structure as a fluorescent marker,An Escherichia coli polyclonal antibody and a goat anti-rabbit antibody are respectively used as a detection line and a control line to be modified on a glass cellulose membrane; when the mixed solution of target bacteria and the antibody quantum dot complex moves to a detection area through capillary action on a sample addition plate,The Escherichia coli polyclonal antibody on the detection line captures the target bacteria, and simultaneously, the antibody quantum dot complex is specifically combined with the target bacteria on the detection line, and in addition, the antibody quantum dot complex is combined with the control line antibody, so that fluorescent bands are respectively presented on the detection and control lines, and thus the Escherichia coli O157: H7 in the sample is detected[17]. This method has been applied in daily life, such as early pregnancy screening test paper and Helicobacter pylori detection test paper, which has the advantages of simplicity, rapidity and convenience.
As that luminescent property of the quantum dot is closely related to the size of the quantum dot, the quantum dots with different sizes can generate different emission lights unde the excitation of the same light source, and based on the property, the method can be used for simultaneously detecting several different pathogenic bacteria. Wang et al. used three kinds of quantum dots with different emission wavelengths of 504 nm, 557 nm and 604 nm to connect with the antibodies of Salmonella, Escherichia coli and Staphylococcus aureus, respectively, and detected the target bacteria in nine kinds of foods through the immune recognition with the target bacteria. This method is simple and feasible, and has good anti-interference ability[18].
Quantum dots (QDs) have good optical and electrical properties in the detection of pathogens, but the high cost of synthesis and the high toxicity of the metal elements used have become the main problems limiting their application[19]. In addition, the stability and fluorescence intensity of QDs decreased with the further surface modification or bioattachment[20].

2.2 Metal nanocluster

Fluorescent Metal Nano Clusters (FM NCs) have broad prospects in environmental monitoring, biological imaging, disease monitoring, catalysis, optoelectronics and other fields due to their simple synthesis method, strong fluorescence intensity, good biocompatibility and good stability[21]. At present, a variety of fluorescent metal nanoclusters have been found, such as platinum nanoclusters, gold nanoclusters, silver nanoclusters, copper nanoclusters and various alloy nanoclusters. Fluorescent metal nanoclusters are fluorescent nanomaterials with a core size of less than 2 nm, which are composed of a protective agent and several, dozens or hundreds of metal atoms (such as Cu, Ag, Au, Pt). When the size of FM NCs is smaller than the Fermi wavelength of the metal, the continuous energy level of the electron will be split into various discrete energy levels, which makes FM NCs have unique strong fluorescence properties. The application of metal nanoclusters in the field of biosensing and detection has been continuously expanded due to their unique optical properties and low toxic side effects[22].
Due to the simple synthesis method and good luminescence properties of metal nanoclusters, more and more people use them to detect pathogenic bacteria. Cheng et al. Wrapped gold nanoclusters with chitosan, and modified the surface of gold nanoclusters with antibodies to capture Escherichia coli O157: H7, and modified the surface of magnetic nanoparticles with antibodies to capture target strains.When a target strain is combined with the two nanoparticles, the Escherichia coli O157: H7 is separated by a magnetic separation technology, and the fluorescence of a detection system is measured to quantitatively measure the target strain[23]. Because a large number of fluorescent gold nanoclusters are encapsulated by chitosan, the fluorescence of the detection system is greatly increased, and the detection limit is 1 cfu/mL.The detection sensitivity of the gold nanocluster microcapsule system is improved by 67. 6 times compared with the traditional method of directly using gold nanoclusters to quantify pathogens.
In addition to the direct use of metal nanoclusters for labeled detection signals, fluorescent gold nanoclusters can also be synthesized in situ on the surface of target bacteria after capture, thus realizing the quantification of pathogens. Hossein-Nejad-Ariani et al. Detected Listeria monocytogenes by in situ synthesis of gold nanoclusters[24]. As a specific peptide for capturing Listeria monocytogenes, Leu-a was immobilized on the glass surface.After capturing the target strain, Leu-a reacted with 3-mercaptopropionic acid and chloroauric acid to synthesize fluorescent gold nanoclusters.The concentration of bacteria was quantified by detecting the fluorescence intensity. This method does not require the isolation of bacteria and is simple to operate. The detection can be completed within 50 min using 10 μL of sample, and the detection limit is 2000 cfu/mL (Figure 3). The method for synthesizing the gold nanoclusters in situ provides a new direction for the detection of pathogenic bacteria.
By combining the unique luminescent properties of the metal nanoclusters with the characteristics of the pathogenic bacteria to be detected, the modification of an identification element is omitted, and the detection process is simplified. It is well known that Cu2+ can quench the fluorescence of gold nanoclusters, while the reducing enzyme NDH-2 of Escherichia coli can reduce divalent copper ions to monovalent copper ions, resulting in that it no longer has the property of quenching the fluorescence of gold nanoclusters. Based On this principle, Yan et al. Developed an On-Off-On mode for the detection of Escherichia coli[25]. The fluorescence of gold nanoclusters was quenched in the presence of Cu2 +, and the fluorescence of gold nanoclusters was restored after the addition of Escherichia coli. There was a good linear relationship between the degree of fluorescence recovery and the concentration of E. coli in the detection system, and the detection of E. coli could be completed within 0.5 H by using this method, with a minimum detection limit of 89 cfu/mL (Figure 4). However, other bacteria, such as Salmonella typhimurium, have the same effect on copper ions, so the specificity of this method needs to be verified[26].
Based On the principle that Sulfate-reducing bacteria (SRB) can reduce SO 4 2 - to S2-, Zheng et al added SRB to the GSH-Au (I) aggregation-induced luminescence system induced by Pb2+.The produced S2- reacted with the Pb2+ to form PbS precipitate, which led to the decrease of fluorescence in the aggregation-induced luminescence system.The SRB could be quantified by detecting the decrease of fluorescence signal.The detection limit of SRB was 10 cfu/mL, and the detection time was 4 d[27]. The gold nanocluster aggregation luminescence system induced by Pb2+ has a good linearity in the detection of pathogenic bacteria in the 1×10~107cfu/mL concentration, but the detection time is long. In addition, the improper treatment of heavy metal lead ions will cause environmental pollution.
Silver nanoclusters also have some applications in the detection of pathogenic bacteria. Zheng et al. Used silver nanoclusters with DNA as a template to detect Escherichia coli[28]. They first prepared a complex system containing magnetic nanoparticles, DNAzyme and acetylcholinesterase (MNP-DNAzyme-AChE), in which the target molecules in E. coli lysates bind to DNAzyme and cut it at the same time, so that the DNA chain connected with acetylcholinesterase (AChE) is released into solution.After the magnetic nanoparticles were separated in an electric field, DNA-AgNCs and acetylthiocholine (ATCh) were added to the system, and AChE could catalyze the hydrolysis of ATCh to thiocholine (TCh), which could significantly enhance the fluorescence intensity of DNA-AgNCs. E. coli was quantified by detecting the change in fluorescence intensity of the reaction system with a detection limit of 60 cfu/mL (fig. 5). The fluorescent silver nanoclusters with DNA as a template can be used as a potential universal detection system to detect different pathogenic bacteria and small molecular substances by changing the corresponding DNase recognition probes.

2.3 Fluorescent carbon nanomaterial

Fluorescent Carbon nanomaterials include CDs (Carbon Dots), GQDs (graphene Quantum Dots), GO (Graphene oxide) and CNT (Carbon Nanotube). Although the luminescence mechanism of fluorescent carbon nanomaterials has not yet been thoroughly studied, they have been widely used in biological detection due to their low toxicity and good biocompatibility[29,30]. The photoluminescence of CDs and GQDs is comparable to that of quantum dots, with high stability and tunability, depending on the size, surface functional groups, chemical composition, doping and excitation wavelength. The luminescence mechanism of CDs and GQDs may be caused by the radiative recombination and quantum confinement of excitons on the surface. Under infrared excitation, CDs and GQDs exhibit upconversion photoluminescence[31,32]. Because carbon-based materials generally have low toxicity, CDs and GQDs are more environmentally friendly than metal quantum dots, and renewable resources such as carbohydrates, fruit peels and bagasse can be used to synthesize carbon dots. Furthermore, carbon-based nanomaterials are generally biodegradable. Based on these properties, CDs and GQDs can be used as fluorescent probes or fluorescent donors in biosensors, providing a new means for the imaging and detection of pathogens[33].

2.3.1 Carbon Point

The carbon dots are amorphous or crystalline quasi-spherical carbon nanoparticles with a diameter of less than 10 nm. There are two modes of pathogen detection using carbon dots, direct and indirect. Direct detection requires certain groups or specific charges on the surface of carbon dots, which can be used for the labeling and detection of pathogens. For example, the surface of the amphiphilic carbon dot has high affinity with the bacterial cell membrane and can be used as a fluorescent marker for detecting pathogenic bacteria[34]. Indirect detection requires the modification of small molecules or groups such as antibodies and aptamers on the surface of carbon dots to achieve the recognition and labeling of target bacteria.
Yang et al. obtained CDs @ BONs by encapsulating carbon dots with organic silicon nanomaterials, which can specifically capture target bacteria after being modified with Staphylococcus aureus antibodies, and at the same time, bacteria can be separated by magnetic nanoparticles modified with antibodies[35]. The carbon dots in the silicone nanocapsules were released by the destruction of NaBH4, and the detection limit of this method for Staphylococcus aureus was as low as 1 cfu/mL. Because hundreds of carbon dots are wrapped in the nanocapsules, the fluorescence signal of this system is greatly increased compared with the detection method using only carbon dots, and the sensitivity is 108 times higher than that of the traditional detection method. The above signal amplification strategy provides a new way to detect pathogens with high sensitivity (Figure 6).
Bhaisare et al. Synthesized magnetic nanoparticles by FeCl2·4H2O and FeCl3·6H2O, and then reacted with chitosan to synthesize magnetic carbon dots (Mag-CDs). Because chitosan contains a large number of amino groups, the synthesized magnetic carbon dots have positive charges, which are collected by magnetic separation technology after combining with negatively charged pathogens through electrostatic interaction. The fluorescence intensity detected is linear with the concentration of pathogens[36]. The detection limits of this method for Staphylococcus aureus and Escherichia coli were 3×102cfu/mL and 3.5×102cfu/mL, respectively. They used MALDI-MS technology to analyze the composition of the isolated strains and identify the strains. Although the above methods based on the surface charge properties of carbon dots to identify strains are simple, they have poor specificity and can only identify a certain type of bacteria, but can not identify a certain target.
In addition, in order to avoid the complex surface modification of carbon dots, carbon dots can also be synthesized according to the special properties of the surface of the target bacteria. Zheng et al used boric acid, polymyxin and vancomycin, three molecules that can recognize different bacterial surfaces, to synthesize carbon dots with diamine citrate respectively[37]. According to the principle that the binding of the carbon dot surface receptor to the target bacteria results in the decrease of the carbon dot fluorescence, the liner discriminant analysis can be used to classify and distinguish the strains through the fluorescence changes of the target bacteria bound to three carbon dots. E. coli, Desulfovibrio desulfuricans, Staphylococcus sciuri, Listeria monocytogenes, Staphylococcus aureus and Pseudomonas aeruginosa with different surface adsorption properties were identified by this method, and the accuracy rate was 91. 6 percent. The specific recognition of bacteria is enhanced by the superposition of multiple recognition signals.
The above methods are all methods that directly use carbon dots as fluorescent markers to identify and detect target bacteria. In addition, by using its own optical properties, it can be used in combination with other nano-materials such as quantum dots, and the recognition of target bacteria can be achieved by modifying the surface of quantum dots, thus avoiding the problem that the surface of carbon dots is not easy to modify. Duan et al. synthesized CdTe quantum dots capable of emitting two different colors of fluorescence: green fluorescent quantum dots (gQDs) and red fluorescent quantum dots (rQDs), which were modified with aptamers of Vibrio Parahemolyticus and Salmonella typhimurium, respectively[38]. In the absence of the target strain, the π-π stacking interaction between the aptamer nucleic acid and the surface of the carbon dots brings the gQDs, rQDs and the carbon dots closer, resulting in fluorescence resonance energy transfer and fluorescence quenching of the quantum dots. When the target bacterial strain is mixed into the system, the aptamer is dissociated from the carbon point due to the conformational change after being combined with the bacteria, and the fluorescence of the corresponding quantum dot is restored. The fluorescence intensity is proportional to the concentration of the target bacteria, and since the luminescence peaks of the two quantum dots do not overlap, simultaneous detection of the two targets can be achieved (fig. 8).

2.3.2 Graphene quantum dot

Due to their unique properties, GQDs have become the ideal carbon nanomaterials. GQDs have a graphene-like lattice structure with a small number of atomic layers and a transverse size in the range of 3 ~ 20 nm[12]. According to their luminescent properties, the pathogens can be detected by fluorescence or electrochemiluminescence.
Safardoust-Hojaghan et al. Synthesized a graphene quantum dot emitting blue light at about 500 nm by hydrothermal method using citric acid and ethylenediamine[39]. When Escherichia coli or Staphylococcus aureus was mixed with it, the fluorescence was quenched, and the degree of fluorescence quenching was proportional to the concentration of target bacteria in the reaction system. This is because the luminescence properties of graphene quantum dots are closely related to their size, surface defects, functional groups and oxygen-containing groups[40]. The mixture of Escherichia coli and Staphylococcus aureus with graphene quantum dots caused the change of surface functional groups, which led to fluorescence quenching. The method can complete the detection of pathogenic bacteria within 2 hours, and the detection process is fast and simple.However, due to the lack of specificity for the detected strains, it is impossible to determine the concentration of a certain bacterium in a system mixed with multiple bacteria.
Chen et al. Used electrochemiluminescence to detect Escherichia coli through nitrogen-doped graphene (N-GQDs)[41]. Firstly, Escherichia coli O157: H7 and dopamine were immobilized on the substrate by electropolymerization based on Surface Imprinted Polymer (SIP) technology.After the Escherichia coli was removed by citric acid and sodium dodecyl sulfate, the bacterial vacancies left in the polydopamine were used to capture Escherichia coli. N-GQDs modified with E. coli polyclonal antibody were used to identify and quantify the captured E. coli.After potassium persulfate was added to the system, the whole SIP-E. coli O157: H7/PAb-N-GQDs could produce stronger electrochemiluminescence. Under the optimal conditions, the ECL intensity showed a good linear relationship with the concentration of the analyte, with a detection range of 10~107cfu/mL and a minimum detection limit of 8 cfu/mL (Figure 9). The method for detecting the Escherichia coli through the electrochemiluminescence method has good specificity and sensitivity, and can be used for detecting other pathogenic bacteria by changing corresponding antibodies. The detection of pathogenic bacteria requires high selectivity and sensitivity, and GQDs are promising detection materials due to their good photostability and rapid photoluminescence response[42].

2.4 Upconversion nanoparticles

Upconversion Nanoparticles (UCNPs), as a new type of fluorescent nanomaterials, are materials with diameters less than 100 nm formed by doping lanthanide ions with suitable inorganic Nanoparticles. Unlike quantum dots and traditional organic fluorescent materials, upconversion nanoparticles can emit visible light when excited by near-infrared light[43]. The emission spectrum of the upconversion material can be changed by adjusting the parameters of the dopant and the host material, such as doping concentration, particle size, and morphology. Due to their excellent photostability, narrow emission wavelength and low fluorescence background, upconversion particles have been widely used in biosensing as fluorescent probes and donors[44,45].
Because the up-conversion nanoparticles can be excited by infrared light, and the infrared light has stronger permeability and weaker light damage, the damage to the body can be reduced,Wu et al. Connected an aptamer capable of specifically recognizing pathogenic bacteria to the surface of the up-conversion nanomaterial, connected a magnetic nanoparticle to one end of an RNA sequence complementary to the aptamer, and mixed the two to prepare a detection probe[46]. When the probe meets the target bacteria, the RNA sequence with the magnetic nanoparticles is dissociated from the up-conversion nanomaterial, and the magnetic nanoparticles and their conjugates are separated under a magnetic field for fluorescence intensity detection. There was a negative linear relationship between the fluorescence intensity and the concentration of the pathogen. They achieved simultaneous detection of Staphylococcus aureus, Vibrio parahemolyticus and Salmonella typhimurium by attaching aptamers of different bacteria to three upconversion nanoparticles with different emission wavelengths (Figure 10). Due to the specificity of the aptamer, the method can realize the identification and quantification of specific bacteria, and has the advantages of convenience, sensitivity and strong specificity.
Kurt et al. Used upconversion particles and quantum dots to detect Salmonella and Staphylococcus aureus simultaneously[47]. Due to the different luminescent properties of the two materials, the corresponding spectra should be used for detection, which can effectively avoid signal interference. Firstly, CdTe quantum dots with different emission wavelengths and up-conversion material NaYF4:Yb,Tm are respectively connected with aptamers of two target bacteria to be detected, and then nucleic acid DNA chains complementary to the two aptamers are connected with magnetic nanoparticles. When the target bacteria are mixed and incubated with the nano material containing the corresponding aptamer for a period of time, the surplus aptamer which is not combined with the target bacteria is combined with the magnetic nano particle, and the magnetic separation technology is used for separating the aptamer for fluorescence detection. The concentration of the target bacteria is inversely proportional to the measured fluorescence intensity. The detection limits for Staphylococcus aureus and Salmonella were 16 cfu/mL and 28 cfu/mL, respectively. The detection system simultaneously uses CdTe quantum dots as a down-conversion material and NaYF4:Yb,Tm as an up-conversion material, so two fluorescence tests are needed to avoid signal interference. By changing the aptamer, the system can be used for the detection of more substances.
Jin et al. Detected Escherichia coli by fluorescence resonance energy transfer between upconversion nanoparticles NaYF4: Yb/Er and gold nanoparticles[48]. The gold nanoparticles are modified with an aptamer of Escherichia coli ATCC8739, the UCNPs are modified with a cDNA nucleotide chain complementary to the Escherichia coli aptamer, and the cDNA nucleotide chain is complementarily paired with the Escherichia coli aptamer in the absence of a target strain in the system,The distance between UCNPs and GNPs is shortened, and the fluorescence emitted by the UCNPs is quenched by the GNPs; when a target strain appears in the system, the aptamer is dissociated from the cDNA and combined with Escherichia coli, so that the UCNPs are dissociated from the GNPs, and the fluorescence is restored. Using this method to detect Escherichia coli in water and food, the detection limit is as low as 3 cfu/mL, which can be completed within 20 minutes (Figure 11). Upconversion nanoparticles have the advantages of good stability, low biological toxicity and long fluorescence lifetime, and have important application prospects in the fields of fluorescence biological detection and imaging.

2.5 Fluorescent silicon nanoparticles

Among many nanomaterials, silicon nanostructures have been widely used in many fields, such as biosensing, bioimaging, tumor diagnosis and therapy, due to their excellent optical/electrical properties, excellent biocompatibility, easy surface modification and compatibility with traditional silicon planar technology[49,50]. The silicon nano material has biodegradability, the degradation product in the organism is silicate, and the silicate is removed out of the body through kidney metabolism, so that the silicon nano material does not generate inflammation and toxic and side effects on the human body[51].
The optical properties of large-sized silicon are not good, but when its average particle size is less than or equal to 5 nm, it will convert from indirect band gap to direct band gap, resulting in quantum confinement effect and fluorescence[52]. There are many kinds of silicon nanomaterials, including silicon nanoparticles, mesoporous silicon nanoparticles, silicon nanocrystals and silicon nanoneedles, etc. Fluorescent silicon nanoparticles have a wide range of applications in biosensing and imaging due to their good biocompatibility and easy surface modification[53]. Due to the fact that a large number of biological recognition elements and dye molecules can be integrated into the surface and the interior of the silicon nanoparticles, the fluorescent dye doped silicon nanoparticles show higher detection sensitivity, and the encapsulation of silicon makes the fluorescent molecules more stable and less prone to quenching[54]. The current fluorescent silicon nanoparticles are mainly divided into organic fluorescent dye doped and inorganic fluorescent material doped[55]. The luminescent efficiency of the organic fluorescent dye is high, so that the fluorescent silicon nanoparticles doped with the organic fluorescent dye have more applications. For example, tetramethylrhodamine-doped silicon nanoparticles synthesized by reverse microemulsion method have efficient and stable luminescence properties and excellent signal amplification function, which can be used for trace detection of substances[56].
Borsa et al. Used mesoporous silica loaded with fluorescent dyes to detect Staphylococcus aureus[57]. The magnetic nanoparticles connected with the aptamer of the bacteria to be detected are used for separating the bacterial strains in the blood, micrococcal nuclease in the magnetic nanoparticles can be released after the magnetic nanoparticles are heated, and then mesoporous silicon particles blocked by nucleotide chains are added into the system,The specific binding of nuclease and the nucleotide chain loses the blocking effect, so that the fluorescent dye in the mesoporous silicon is released. The target bacteria are quantified by detecting the intensity of fluorescence, and the detection limit for Staphylococcus aureus in blood is 682 cfu/mL (Figure 12). The detection method of fluorescent silicon nanoparticles blocked by nucleic acid aptamers can achieve specific detection of target pathogens, but it has the problem that the blocking is not strict and the organic dye leaks out to produce background fluorescence, which leads to high detection limit.
Wang et al. Used ruthenium bipyridine (RuBpy) doped fluorescent silicon nanoparticles to detect Salmonella typhimurium[58]. The method comprises the following steps of: firstly, capturing Salmonella typhimurium by using a biotin-modified aptamer, and binding the captured Salmonella typhimurium with an avidin-modified fluorescent silicon nanoparticle after centrifugation, wherein the fluorescence intensity in a detection system is in direct proportion to the concentration of the bacteria to be detected. Compared with the traditional fluorescent dyes, the dye-doped silicon fluorescent nanoparticles have good photostability and biocompatibility, are indispensable excellent materials in the current detection field, and have good application prospects.
In addition, Chen et al. synthesized FITC-doped silica NPs (FSiNPs) by W/O microemulsion method[59]. The antibody of Escherichia coli O157: H7 is modified on the surface of the strain so as to realize the capture of target bacteria, and the generated fluorescence signal is detected by using a fluorescence microscope or flow cytometry so as to obtain the quantification of the strain.
Na et al. detected dipicolinic acid (DPA) in Bacillus anthracis spores by using europium-doped fluorescent silicon nanoparticles (Eu @ SiNPs)[60]. The fluorescent silicon nanoparticles can emit two kinds of fluorescence at 470 nm and 617 nm. The addition of DPA results in the antenna effect of Eu @ SiNPs, which leads to the enhancement of fluorescence at 617 nm, while the fluorescence at 470 nm is almost unchanged. DPA can be detected by changing the fluorescence ratio of the two wavelengths, so as to further provide a reference for the content of Bacillus anthracis. This is the first time to use the proportional fluorescence method to detect Bacillus anthracis spores, which avoids the interference of background fluorescence and is simple and rapid. It provides a new idea for the research of fluorescent silicon nanoparticles in the future.

3 Introduction of Identification Methods of Nanomaterials and Pathogenic Bacteria

A biosensor is an integrated device containing a biological recognition element that contains a physicochemical detector element or transducer capable of converting a recognition signal into quantitative or semi-quantitative analytical information[61]. In order to achieve good detection results, the specificity and sensitivity of recognition elements are very important, so they play a key role in biosensors.

3.1 Pecific recognition

3.1.1 Antibody linkage

Antibody (IgG) is a large Y-shaped protein, which is the most commonly used element in pathogen recognition because of its wide range of uses, high sensitivity and selectivity[62]. There are three types of antibodies used in antibody-based immunoassays: polyclonal antibodies, monoclonal antibodies, and genetically engineered antibodies[1]. Active sites on small molecules are usually used to react with amino, carboxyl, sulfhydryl or hydroxyl markers on proteins. The common coupling methods include carbodiimide condensation, glutaraldehyde, periodate oxidation and N-hydroxysuccinimide activation[63,64]. In addition to the direct chemical connection, the fluorescent nanomaterial can be coupled with biotin/avidin first, and then combined with the antibody. Antibody recognition has good specificity.How to connect the antibody to the nanomaterial while maintaining its activity has become a difficult point in the detection.In addition, the poor stability and high price of antibodies, especially monoclonal antibodies, have become one of the reasons for limiting their application[65].

3.1.2 Aptamer

Aptamer is a single-stranded DNA or RNA nucleotide sequence screened from a random single-stranded nucleic acid sequence library by using System Evolution of Ligands by Exponential Enrichment (SELEX), which has high affinity with the target substance[66]. The nucleic acid substance biological recognition element can be combined with a series of ligand substances, including small molecule substances such as ions, drugs, organic dyes or mycotoxins, and even whole cells such as bacteria have high specific recognition function[67]. Aptamer technology has the advantages of high affinity, high stability, low toxicity, low sensitivity to denaturation environment, easy synthesis, easy modification and low cost, so aptamer-based sensors have attracted more and more attention[68]. At present, aptamers have been used to detect many pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus and Salmonella[69,70].
Due to the narrow and tunable emission spectrum of the fluorescent nano material, the fluorescent nano particles with different emission wavelengths can be used for simultaneously detecting a plurality of bacteria after being modified with a plurality of recognition elements. Researchers have achieved the simultaneous detection of multiple bacteria using a combination of multiple aptamer nanoprobes in one detection system. Duan et al. reported the simultaneous detection of Salmonella and Staphylococcus aureus using aptamer-linked fluorescent nanoprobes of two colors[71]. When the target bacteria appeared, the fluorescent probe could adsorb the target bacteria and produce fluorescence under 980 nm excitation light. The detection limit of this method was 5 cfu/mL for Salmonella and 8 cfu/mL for Staphylococcus aureus. The detection range was 10~105cfu/mL. In addition, Duan et al. Used aptamer-linked dual-color quantum dots to detect Vibrio parahaemolyticus and Salmonella typhimurium simultaneously[72]. The application of aptamer-containing fluorescently labeled nanoprobes has a good prospect in the multiple and sensitive detection of bacteria, but it may lead to fluorescence quenching in complex matrix applications[73].

3.1.3 Bacteriophage

Bacteriophages are natural enemies of bacteria and can be isolated from soil, natural wastewater and other places. Phages lack their own metabolic mechanisms and need to absorb nutrients from host cells to reproduce. In nature, bacteriophages have strong specificity for host bacteria[74]. The stability of bacteriophage in different pH and temperature makes it a very advantageous probe in the field of microbial detection[75]. Because they can only interact specifically with some living host bacteria, phages can be used to detect viable but unculturable bacteria, but antibodies and nucleotides cannot be used for this purpose[55]. Because phages have special host recognition systems, careful screening of phage libraries is required[76].
Engineered bacteriophage uses genetic engineering technology to introduce the gene of interest into the bacteriophage, and through the expression of gene products, the target pathogen can be detected. Edgar et al. prepared a T7 bacteriophage capable of expressing biotinylated peptide, which propagated and produced the biotinylated peptide T7 bacteriophage after infecting the host bacterium Escherichia coli,The amount of E. coli is proportional to the phage produced by proliferation, at this time, avidin-modified quantum dots are combined with biotinylated peptides, and the quantification of E. coli is achieved by detecting the fluorescence intensity, with a detection limit of 10 cfu/mL[77]. Chen et al. Used T7 bacteriophage expressing biotin to combine with avidin-modified FeCo magnetic nanoparticles for the separation of pathogens, and the separation efficiency was comparable to that of antibody-labeled pathogens[78]. However, bacteriophages may cause lysis of bacteria during infection of E. coli, which may affect isolation. In addition, the bacteriophage is modified by means of genetic engineering to express reporter molecules such as green fluorescent protein and luciferase, the bacteriophage replicates after infecting target bacteria, and the quantification of bacteria is realized by detecting the signal of the reporter molecules[79][80]. Bacteriophage can be used to detect the live bacteria of pathogenic bacteria, and the detection limit is low, which provides a new platform for biological detection. However, this technique only detects bacteria that can be used as phage hosts, which limits its wide application.

3.2 Nonspecific recognition

3.2.1 Char property of pathogenic bacteria

And when that surface charge of the nano material is different from the surface charge of the bacteria, the nano material is combine in an electrostatic adsorption mode so as to identify and detect the pathogenic bacteria[81]. As a non-specific identification method, the electrostatic interaction detection method can be used to detect a certain type of bacteria, and can also be used to identify a certain genus of bacteria according to their special properties, depending on the charged properties of the nanomaterials used and the target bacteria.
Usually, the surface of bacteria is negatively charged, so it can be combined with positively charged nanomaterials for detection, which can be divided into indirect and direct methods[82]. The indirect method means that the luminescent material is not directly reacted with the pathogenic bacteria. Gold nanoparticles, carbon nanotubes, graphene oxide and other materials can interact with some enzymes and inhibit the activity of enzymes. Based on the reversible interaction, the restoration of enzyme activity can be used as an indicator of bacterial concentration. Li et al. Synthesized graphene oxide with positive charges, which can inhibit the activity of β-galactosidase[83]. After Escherichia coli was mixed into the detection system, the adsorption of β-galactosidase was reduced due to the electrostatic binding between bacteria and graphene oxide, resulting in the recovery of β-galactosidase activity, and the quantitative determination of Escherichia coli was indirectly realized through the determination of enzyme activity. In addition, Rotello et al. Reported the detection of bacteria using positively charged gold nanoparticles[84]. The activity of β-galactosidase (β-gal) was inhibited by gold nanoparticles due to electrostatic interaction. When the bacteria to be tested appeared, the gold nanoparticles were released from the AuNPs/β-gal complex and bound to the bacteria, thus the enzyme activity was restored. Different concentrations of bacteria lead to different recovery of enzyme activity, thus showing different color changes.The detection limit of this method for Escherichia coli and Staphylococcus aureus is 100 cfu/mL, and the detection can be completed within 10 min.
The direct method is to modify the surface of nano-materials with positively charged groups, such as chitosan or compounds with amino groups, and detect the concentration of microorganisms by detecting the changes of fluorescence or absorption light after combining with negatively charged microorganisms. Raj et al. modified the surface of gold nanoparticles with cysteine at the amino terminal to make them positively charged, and after combining with Escherichia coli O157: H7 through electrostatic interaction, the stability of colloidal gold was destroyed and aggregation occurred. With the increase of the number of Escherichia coli O157: H7,Coli O157: H7. The color of the solution gradually changed from red to blue, and the UV absorption spectrum showed a red shift.As a visual detection method, the detection limit of E. coli O157: H7 was 100 cfu/mL[85]. Bhaisare et al. Used magnetic carbon dots modified by chitosan to adsorb, separate and detect Enterobacter by fluorescence[36]. This method of detecting pathogenic bacteria by electrostatic interaction is not specific, but it has the advantage of convenience and rapidity in applications that do not distinguish between bacterial species but only quantify the population.

3.2.2 Structural characteristics of pathogenic bacteria

Pathogenic bacteria are divided into Gram-positive bacteria and Gram-negative bacteria according to their cell wall components. Detection strategies can be developed according to the differences in the composition of the bacterial outer wall.Antibacterial compounds, boric acid and carbohydrates are widely used in the identification of pathogens.
Due to the specific binding of many antibacterial agents to bacterial cell walls, more and more antibacterial agents are currently used for the identification and detection of certain types of bacteria[86,87]. Vancomycin is an antibiotic belonging to the glycopeptide family, which binds to the (D) -Ala- (D) -Ala peptide chain on the peptidoglycan of Gram-positive bacteria. Based on this property, gold nanoparticles or carbon dots modified with vancomycin can be used to identify Gram-positive bacteria[88][89]. Lysozyme is a hydrolytic enzyme that specifically acts on the cell wall of bacteria, also known as cell wall lysis enzyme. The enzyme has strong decomposition effect on Gram-positive bacteria, Bacillus subtilis and Deinococcus radiodurans; It also has a certain killing effect on Gram-negative bacteria such as Escherichia coli, Proteus vulgaris and Vibrio parahaemolyticus[87]. Liu et al. Used lysozyme to synthesize gold nanoclusters, which could identify Escherichia coli and detect its concentration, with a detection limit of 2×104cfu/mL[90]. Colistin sulfate can interact with lipopolysaccharide and phospholipid in the outer membrane of Gram-negative bacteria, resulting in leakage of cytoplasm and death of bacteria, but has no effect on Gram-positive bacteria (except Staphylococcus aureus and Streptococcus hemolyticus) and fungi. The bacteria can not only be used as antibacterial drugs, but also be used for the detection of bacteria. Chandra et al. Used colistin sulfate to synthesize carbon dots, which could detect Escherichia coli with fluorescence, and the detection limit was 460 cfu/mL[91]. Chandra et al. Also synthesized carbon dots using the antibiotic amikacin, and Cooper et al. Linked lectins to magnetic nanoparticles for the separation of microorganisms from blood samples[92][93].
Boric acid can bind to bacteria whose cell walls contain cis-diol molecules. Due to the distribution of lipopolysaccharide (containing cis-diol molecules) in Gram-negative bacteria, Zheng et al. Used boric acid to synthesize carbon dots for the identification of bacteria containing cis-diol molecules on their cell walls[37]. Carbohydrates can also be used for the recognition of pathogenic bacteria, because glycoproteins and glycolipids formed after carbohydrates are linked to proteins and lipids can be recognized and linked to bacteria. El-Boubbou et al. Used mannose- and galactose-modified magnetic nanoparticles for the differentiation of E. coli series[94]. The method of identifying bacteria based on the difference of cell wall components is simple and efficient, but it can only distinguish one type of bacteria, and the specificity is low.

4 Conclusion and prospect

In order to protect food and human health, the rapid detection of pathogenic bacteria is very important, and the application of fluorescent nanomaterial biosensors provides more possibilities for the detection of pathogenic bacteria. In this paper, the binding modes and detection methods of fluorescent nanomaterials such as quantum dots, metal nanoclusters and fluorescent carbon nanomaterials with pathogens are reviewed, and the advantages and disadvantages of the detection methods are compared.
Quantum dots and fluorescent silicon nanoparticles have strong fluorescence emission efficiency and have been widely used in the detection of small molecules. The core-shell structure of quantum dots is easy to be modified by biomolecules such as antibodies and aptamers, which greatly improves the sensitivity of detection. Because of that toxicity of the quantum dot, the commercial application of the quantum dots need to be improved. Upconversion materials doped with lanthanide metals have the advantages of low toxicity and unique luminescence properties, and have advantages in multiple detection of pathogens. In order to improve the application of upconversion nanomaterials in biosensors, it is urgent to improve the low quantum yield of hydrophilic upconversion nanomaterials[95]. Fluorescent metal nanoclusters have the advantages of small size, good biocompatibility, strong photoluminescence resistance to photobleaching, easy synthesis and energy donor, which have been increasingly used in recent years. Because of their unique luminescence properties such as FRET and AIE, they provide more modes for detection. Fluorescent carbon nanomaterials are easy to prepare, high quantum yield and good biocompatibility, which provide more possibilities for their application in detection. However, the difficulty of surface modification has become the main factor limiting its application[96].
With the continuous development of nanotechnology, the unique structure and performance advantages of fluorescent nanoprobes can break through the limitations of traditional methods in the detection of pathogens, and have obvious advantages and potential in shortening the detection time, improving the sensitivity and accuracy of bacterial detection, and reducing the consumption of samples and reagents. At present, the detection of pathogenic bacteria based on fluorescent nanoparticles is still in the laboratory research stage.How to transform it into a portable detector like colloidal gold test strip and blood glucose detector has become the focus of more and more researchers, and it is also the hot spot of future research on fluorescent nanoparticles.

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Funding

National Natural Science Foundation of China(31971382)

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