1 Introduction
According to the technical standard definition of the International Union of Pure and Applied chemistry (IUPAC), "solid state electroanalytical chemistry" (solid state electroanalytical chemistry;SSEAC) is a means to analyze the information of solid substances by electrochemical methods, which is particularly suitable for analyzing the elemental composition, phase composition and redox state of solid particles due to the differences in electrochemical behavior of electrochemically active substances under different conditions
[1]. SSEAC technology relies on techniques that can be traced back to a series of original works by Scholz and others in the late 1980s and early 1990s
[2⇓⇓~5]. The voltammetry of immobilized microparticles (VIMP) proposed by Scholz et al. enables rapid and efficient electrochemical analysis of solid particles. SSEAC has been successfully applied to obtain electrochemical information of natural pigments, plants, minerals and cultural relics, and to carry out qualitative and quantitative analysis, due to its small sample requirements, portability, rapid acquisition of fingerprints and recording of the behavior of target samples in electrochemical reactions.
Among them, the application of SSEAC technology in the analysis of plant samples began in 2015, which is the latest application field of this technology
[6]. Plant tissues are rich in electrochemically active substances, and different species of plants have different electrochemical behaviors due to the great differences in the types and proportions of electrochemically active substances, which makes SSEAC show potential application value in plant identification, plant phylogeny and plant physiological monitoring. In this paper, the development of SSEAC and its relationship with molecular electrochemistry and solid-state electrochemistry are briefly reviewed. Then, the applications of SSEAC in plant identification, plant phylogeny and plant physiological monitoring in recent years were reviewed in detail, and the application prospects and future research directions were prospected.
2 Background to SSEAC
2.1 Definition and Research Scope of SSEAC
SSEAC is a method for obtaining information about solid substances (quantitative elemental composition, phase composition, structural information, and reactivity) by electrochemical methods. The definition of this technical term was adopted and confirmed by the IUPAC report in 2013
[1]. In particular, the report points out that electrochemical characterization of solids is not only of great value for electrochemical applications of materials (such as batteries, fuel cells, corrosion prevention and electrochemical processing), but also helps to provide information on the physical structure and chemical composition of solid substances.
SSEAC is a branch of solid-state electrochemistry. In a narrow sense, solid-state electrochemistry refers to an electrochemical system in which at least one solid ionic conductor participates. However, Scholz, founder and editor-in-chief of the Journal of Solid State Electrochemistry, proposed in the first issue of 1997 that in a broader sense, Solid-State Electrochemistry includes all electrochemistry in which at least one solid phase plays a decisive role.Its research areas include solid-state electrochemical reaction mechanisms, semiconductor electrochemistry, electrochemical cells, batteries, and fuel cells, electrochemical mineral leaching, electrochemical metal plating, solid-state electrochemical sensors, and ion and electron transport in solid materials and polymers
[7]. SSEAC is a more subdivided field, which refers to the use of electrochemical methods to analyze the information of solid substances, mainly referring to the analysis of the properties of particles immobilized on the surface of electrodes.
The solid substance immobilized or embedded on the surface of the electrode in SSEAC technology is different from the so-called "surface modified electrode". Bond and Scholz proposed that "surface-modified electrode (SME)" should be strictly applied to an electrode modified by coating a thin film of a specific material on the surface of the electrode to cause a specific reaction or response
[8]. In general, SMEs are prepared to improve the analytical performance of the electrode for analytes in solution (increasing sensitivity, selectivity, or both), thereby allowing the SME to act as a potentiometric, conductometric (impedance), amperometric, or voltammetric sensor. However, in SSEAC technology, the solid carrier on the surface of the electrode is used to obtain the information of the solid substance, rather than using the properties of the solid to obtain the information of the analyte in the electrolyte solution. Moreover, these solid materials do not necessarily form a dense film. In fact, in the application of SSEAC technology, it is often faced with heterogeneous and random size solid samples. It should be recognized that there is a transition from "molecular electrochemistry" to "solid-state electrochemistry" in electroanalytical chemistry, depending on the size of the entities involved in the interfacial charge transfer process (and the degree of attachment to the substrate electrode) (fig. 1). In molecular electrochemistry, electrodes are seen as a means of transferring electrons to induce or activate a substance dispersed in a 3D solution space. In solid-state electrochemistry, electrodes are still regarded as a means of transferring electrons, but research substances can gradually be immobilized or embedded on the surface of electrodes in the form of particles or films by means of adsorption, deposition, assembly and modification.
2.2 Electrochemical methods for the study of solid substances
Electrography, proposed by Fritz and extended by Weisz, was used sporadically in the early days to analyze solid substances
[9][10]. Until the 1960s, with the use of carbon paste electrodes by Adams and Kuwana in electrochemical research, the analysis of solid substances by electrochemical methods began to receive some attention, especially in the field of mineral analysis
[11][12,13]. By the end of the 1980s, Scholz et al. Proposed the "immobilized particle voltammetry", which overcame the limitations of contact materials in the previous technology and extended the scope of application of SSEAC to many fields
[2⇓~4,14,15].
The principle of electrography is to press a solid sample against a paper soaked in an electrolyte solution (Fig. 2a). By anodizing the surface of the solid sample, the reaction product reacts with the reagent in the paper to obtain a colored reaction product, which shows the distribution of the reaction elements on the surface of the sample. However, this method is limited to the testing of some mineral samples, and requires a large amount of samples, which has been rarely used in SSEAC research.
Today, there are two main types of SSEAC analysis methods: the first is to press the solid particles to be measured, carbon paste and binder together to form the working electrode (Figure 2b)
[16]. This has always been the mainstream method to study the electrochemical behavior of solid materials. The binder is often an organic liquid that is not compatible with the electrolyte solution, or the electrolyte is directly used as the binder. Both methods have their advantages and disadvantages. The organic liquid binder can prevent the electrolyte from entering the slurry and ensure the stability of the pressed electrode in the electrolyte solution. However, such binders, such as paraffin oil, may diffuse to the surface of the graphite powder and solid particles. On the other hand, the use of an electrolyte as a binder sometimes dissolves the solid particles.
The second method is to immobilize the solid particles to be measured on the surface of the working electrode. When the particle size of the sample is in the micrometer range, the simplest way to immobilize the particles on a paraffin-impregnated electrode is to place a few milligrams of the sample on a glass plate or glazed tile, followed by gentle rubbing with an electrode rod (Fig. 2 C). Normally this will cause the solid particles to become partially embedded in the graphite surface. In some specific research work, due to the limitation of samples, some special alternatives have been developed
[17,18]. Even in some cases, the solid particles to be measured are uniformly dispersed in the electrolyte
[19,20]. The limitation of this method is that it is impossible to know exactly how many particles are immobilized on the electrode surface, which makes it difficult to quantitatively analyze the results of the study.
To obtain an electrochemical response of the solid particles, they need to be in direct contact with a conductive electrode or dispersed in a conductive matrix. So far, the most frequently used techniques in SSEAC are cyclic voltammetry and linear potential sweep voltammetry, followed by differential pulse voltammetry and square wave voltammetry
[4,21,22]. Chronocoulometry, chronoamperometry, chronopotentiometry, electrochemical impedance spectroscopy (EIS), electrochemical noise (EN) analysis, and electrochemical quartz crystal microbalance (EQCM) measurements have also been used as complementary means for the analysis of solid materials
[23][24][25][26][27⇓⇓~30].
2.3 Types of information obtained by SSEAC technology
The SSEAC can provide three different types of information about solid materials, including: (1) Elemental composition (qualitative and quantitative), such as Bi, Cu, and Pb, which can be identified in the Bi
1.8Pb
0.39Sr
1.99Ca
2.06Cu
3.15O
10.5 and the relative amounts of these three electroactive elements can be quantified
[4]. (2) Information on redox States of electroactive materials, identification and relative quantification of Fe (Ⅲ) and Fe (Ⅱ) redox States in clay and ceramic materials
[31]. (3) phase composition (also relate to qualitative phase identification and phase quantification), such as phase identification and quantitative analysis of mixed crystal of CuS
xSe
1-x and CuBi
xSb
1-xS
2[32]. Figure 3 shows the main types of information analyzed by the SSEAC technique.
According to the type of information obtained by SSEAC, the relevant research can be divided into two categories: qualitative analysis and quantitative analysis. Qualitative analysis refers to the identification of elements, redox centers, compounds and phases in particles by SSEAC technology. The sample may consist of a single compound (or phase) or a mixture of compounds (phases). In the analysis, the different components of the analyte are identified by means of voltammetric responses obtained in a suitable electrolyte. For example, the analysis of a sample from the fresco "La Trasfigurazione" painted by Sebastiano del Piombo in St. Peter's Basilica of Montorio Church in Rome from 1521 to 1524 shows that the voltammetric characteristics of the pigment sample correspond well to the electrochemical behavior of azurite and white lead, indicating that the pigment of the fresco contains these two mineral pigments
[33]. In addition, SSEAC in combination with VIMP is also able to distinguish and identify different crystal forms of minerals or compounds, or individual substances located in the matrix, such as pigment ions in glass materials or guest molecules and ions in microporous solids
[2,3,15][34].
In quantitative analysis, SSEAC can quantitatively analyze the components in solid substances by internal standard method. The internal standard substance is often a solid substance with a well-defined electrochemical response, and a known proportion of the substance is added to the sample to produce a measurement signal. The added internal standard may be the same signal as the analyte or it may be an auxiliary internal standard that produces a separate signal. Absolute quantification can then be obtained by VIMP
[35].
In addition, SSEAC technology can also be used to identify and trace the oxidation state and changes of electroactive substances. Uch as the study of the Cu
3+-Cu
2+-Cu
+ transition in the orthorhombic YBa
2Cu
3O
7-x phase, the study of manganese complexes entrapped in zeolites, the identification and quantification of different lead and tin species in glazed materials, and the speciation of manganese in marine sediment carbonates
[36][37][38][39,40].
3 Plant Research Based on SSEAC
3.1 Plant Identification
The identification and recognition of plants and plant products can provide theoretical guidance for the fields of plant resources and traditional Chinese medicine resources, and has a very intuitive practical application value in the identification of medicinal materials, food safety and quality control of economic crops. Because SSEAC is faster, cheaper, and easier to operate in the field than the commonly used chromatographic and spectral techniques, it has become a new recognition technology besides visual image recognition. SSEAC has now been applied to the study of electrochemically active substances in various tissues and organs of plants. In the plant kingdom, today's research has covered a large number of herbs, vines and woody plants.
In 2015, Dom Domínguez et al. First used SSEAC technology to study different types of tea, and identified different varieties of black tea, green tea and oolong tea. The required amount of samples can be as low as 0.01 mg
[41]. In the same year, Martini et al. Proposed the screening, identification and evaluation of herbal formulas using SSEAC technology
[42]. The method does not need the assistance of microextraction and uses organic solvents to simply extract polyphenol compounds with electrochemical activity. Studies have shown that some bioactive phenolic components, such as anthocyanins and proanthocyanidins, have electrical activity in various components of plant tissues. This method does not require the identification and quantification of individual compounds present in the sample as other techniques do, but rather uses the overall voltammetric data for detection and evaluation. Fig. 4 shows the identification of three varieties of Indian cactus (Caralluma adscendens) using ratios of volt-ampere characteristic data.
In 2016, Dom Dom Énech-Carb Carbé and others began to study the differences in electrochemical behavior of different parts of plants. They selected the leaves, bark, roots and fruits of Zanthoxylum chiloperone as the research objects, and observed the huge differences in electrochemical information of different organs of plants. They believed that this difference had great application potential in the rapid identification of plants
[44]. In 2017, they further applied SSEAC technology to the identification of raw materials of wooden cultural relics
[45]. A series of European wood products from 375 to 350 BC and American wood products from the 17th to 19th centuries were identified and identified according to the differences of electrochemically active substances remaining in wood fibers.
At present, plant recognition based on SSEAC mainly relies on the difference degree of the characteristic peaks in the fingerprint spectrum to judge the recognition method, which is based on dimensionality reduction thinking and requires human subjective judgment. This method can also be applied when there are fewer plant types, but it is difficult to summarize obvious rules in the identification of a large number of plant types, and it is difficult to meet the needs of real application scenarios, such as the identification of the authenticity of medicinal materials. Our research group started the research of SSEAC in 2017. In terms of plant recognition and identification, we no longer advocate the use of characteristic peaks to identify different plants, but directly use SSEAC maps under different conditions for pattern and image recognition.
In the first work, we selected the leaves of Lycoris radiata, Lycoris anhuiensis, Lycoris aurea, Lycori radiata and Lycori longituba as the research objects
[46]. The electrochemical fingerprints obtained in phosphate buffer solution, Tris buffer solution and acetate buffer solution were manually processed to form a 3D image (fig. 5A). In this work, we preliminarily verified the positive effect of introducing two-dimensional material graphene on the collection of electrochemical fingerprints of plant tissues, and realized the presentation of 3D mode by multi-dimensional construction of fingerprint data. Since this presentation is not suitable for effective plant species identification, we finally chose the more visually intuitive 2.5D map (Fig. 5B), 2D density map (Fig. 5C) and RGB color bands (Fig. 5d)
[47][48][49]. By using this method, the plant species can be easily identified with the naked eye, which lays the foundation for the subsequent intelligent identification.
We not only identified different species of plants within the same genus, but also further discussed the identification of different sexes of the same species of plants. We specifically selected dioecious plants with different commercial values, including Taxus × media, Dioscorea zingiberensis, and Dioscorea bulbifera
[50]. In this work, simple comparison of electrochemical voltammograms can not clearly distinguish the sex of plants, but must be based on the construction of multi-dimensional graphics after extraction with different solvents, and can be identified by pattern recognition. In addition, we have also applied SSEAC technology to the analysis and identification of plant products, such as tea drinks, seaweed fertilizer, vinegar and liquor
[51][52][53][54].
3.2 Phylogenetic study of plant
Around the 1960s, a new discipline, phytochemotaxonomy, emerged between plant taxonomy and phytochemistry, which uses chemical characteristics to study the relationship between plant groups and explore the evolution of the plant kingdom. The genetic variation of various chemical components formed by plants is basically consistent with the evolution of plant family and genus system. Certain types of chemical components are often distributed in certain plant families and genera, which can be used as a basis to solve the relationship between species and genera
[55]. In the past 20 years, phytochemotaxonomy has developed rapidly, and researchers have focused on the secondary components of plants, that is, the small molecular compounds of secondary metabolism (such as sugars, glycosides, flavonoids, alkaloids, terpenoids, essential oils and tannins). Because the distribution of these small molecular compounds in the plant kingdom has obvious discontinuity, this limited distribution is a valuable taxonomic character in the study of plant classification and phylogenetic relationship. However, chemical composition analysis based on chromatographic and spectroscopic techniques requires complex plant sample processing steps and large analytical instruments, and often only a limited number of compounds are used as classification criteria.As a result, the taxonomic conclusions drawn by this method often do not have a very strong degree of interpretation, so it is still not used as a conventional technique for plant classification research. SSEAC analysis technology can present the information of all electrochemically active substances in plant tissues in voltammetric scanning, which is an extremely simple and efficient way to obtain the common signal of multiple chemical components. In recent years, this method has been explored to study the phylogeny of different plants.
In 2015, Dom Dom Énech-Carb Carbé et al. modified the plant leaves on the surface of the glassy carbon working electrode, tried to identify and analyze 37 species of plants belonging to 24 families of 11 orders, and finally realized the identification of 16 species belonging to 7 families of Rosales by using the different peak positions (Fig. 6a), and then clustered the 7 families by using the normalized voltammetric data (Fig. 6B)
[56].
In 2016, Ortiz-Miranda et al. Of the Polytechnic University of Valencia further studied the application of this method in plant identification and taxonomy (Fig. 6B)
[57]. In this study, the electrochemical fingerprints of pine needles from six species in the genus Pinus were used to study the differences between the subgenera monovascular and bivascular Pinus, and the phylogenetic trees of the six species were compared with previous results from chloroplast genomes using a clustering method
[58]. Cebri Cebrián-Torrej TorrejÓn et al. 2017 reported a SSEAC study on Asparagus seeds
[59]. The samples studied include 20 species of 3 subgenera, and the results are basically consistent with the results of APG Ⅲ classification
[60]. These two works further demonstrate the connection between the chemical composition information presented by electrochemical fingerprints and plant phylogeny.
At present, the main application of SSEAC is based on the direct immobilization of plant tissues on the electrode surface and then analysis. Although the commonly used glassy carbon electrode is a stable and reusable electrode, the polishing process before each use and the detection method requiring an electrolytic cell to insert the three-electrode system not only require a stable laboratory operating environment,It is also necessary to add a film-forming agent to the plant tissue to prevent the plant tissue from falling off the electrode surface (fig. 7A). Moreover, plant tissues contain a large number of components with poor conductivity, such as lignin, cellulose and protein, so it is difficult to obtain the signal of electrochemically active components in plant tissues directly immobilized on the surface of electrodes with high sensitivity. Therefore, after the plant tissue is coated with the two-dimensional material with high specific surface, high conductivity and good immobilization capacity, the two-dimensional material is used for efficiently adsorbing the small molecular compounds of the plant tissue in the extraction process,The coated plant tissue was then directly immobilized on the surface of a printed electrode integrated with a three-electrode system for highly sensitive SSEAC acquisition (Fig. 7B). Imultaneously, that method can quantitatively control the immobilize plant tissue, overcomes the limitation that the immobilization of the particle on the surface of the traditional electrode cannot be quantitatively studied, and simultaneously can ensure that the tissue is kept stable in the electrochemical reaction of a liquid phase environment.
In our study, we specially selected the plants of Lycoris spp. As the research objects. Lycoris is a kind of perennial herbs with bulbs belonging to Amaryllidaceae. There are about 20 species in the world, mainly distributed in China, Korea and Japan in East Asia. Through close scientific cooperation with two botanical gardens, we have introduced and cultivated all the plants of this genus. An important characteristic of Lycoris plant is that interspecific hybridization is frequent, and natural hybridization phenomena exist in both nature and artificial cultivation environment, among which that taxonomic status of protospecies is clear,However, the interspecific relationship between hybrids and their parents is controversial in academic circles, so the species of this genus are ideal materials for the study of plant reticulate evolution. We used SSEAC to study the phylogeny of different organs of Lycoris radiata, including leaves, bulbs, flowers and pollen (Fig. 8)
[46][47][61][49]. The results confirmed the relationship between the information presented by the electrochemical fingerprint and the phylogenetic position of plants, and discussed the genetic relationship among species in Lycoris by using the results. For example, Lycoris chinensis (L. chinensis) and Lycoris aurea (L. aurea) are very similar in morphological analysis, but their leafing stages are different. The relationship between the two species has been reported in the literature, and our results suggest that the relationship between the two species is distant, which is consistent with the results of random amplified polymorphic DNA (RAPD) markers. For another example, Lycoris radiata (L. straminea) was previously considered to have originated from natural hybridization, and its parents may be Lycoris dwarf (L. radiata var. pumila) and a species with 2n = 6m + 10T karyotype. According to our results, another parent of Lycoris radiata is probably Lycoris chinensis, and the karyotype of Lycoris chinensis is 2n = 6m + 10T.
Recently, we extended the mature and stable SSEAC to study the interspecific relationships between Chimonanthus spp. And Pueraria spp
[62,63][64]. The application of SSEAC to phylogenetic studies at the genus level is a key to testing the technique as a taxonomic methodology, and we have obtained some positive results in the study of intergeneric relationships within Amaryllidaceae
[48].
3.3 Plant Physiological Monitoring
Plant organs contain different components at different stages of growth. The content of some compounds in the fruit will have a big difference before and after ripening. Because of the good electrochemical response to some compounds, such as rutin, chlorogenic acid, naringin and ascorbic acid, SSEAC technology can be used to detect the maturity of plant fruits and evaluate the quality of fruits planted under different agronomic conditions
[6].
Many plants respond to external stresses, for example by developing defenses against herbivorous insects. In this case, plants tend to release chemical volatile organic compounds to repel egg-laying insects or attract their parasites and natural enemies. Interestingly, plants seem to be able to distinguish between mechanical injuries and attacks by herbivorous insects
[65]. From a chemical point of view, the substances contained in the chemical defense of plants against external stresses are often alkaloids, terpenoids, cyanogenic glycosides and phenolic compounds that are toxic to insects
[66]. Moreover, volatile organic compounds released by damaged plants can induce defense responses in neighboring plants, and this chemical communication between plants and between plants and other organisms is a proven ecological phenomenon
[67]. The SSEAC technique monitored the production of chemicals associated with external stress and communication processes in neighboring plants of Peperomia obtusifolia after being eaten by beetles
[68]. In the monitoring, it was found that under the external stress of phytophagous beetles, the electrochemical behavior of the round leaf pepper grass would change accordingly, and the electrochemical oxidation behavior of gossypol was recorded (Fig. 9).
In 2019, SSEAC technology was developed and applied to detect the response of seedlings to salt stress
[69]. In situ electrochemical generation of reactive oxygen species (ROS) can react with plant polyphenols and produce electrochemical active signals. Applying a simple kinetic model based on the second-order reaction between ROS and polyphenol components to the electrochemical data, the apparent rate constants can be derived. This rate constant is related to the level of salt stress, thus revealing significant differences between the tested species. These results were compared with the response of seed germination to salt concentration, which verified the potential of SSEAC as an analytical tool for assessing plant tolerance to salt stress.
4 Conclusion and prospect
Plant analysis based on SSEAC is an interdisciplinary analytical technique between electroanalytical chemistry and phytochemistry in recent years, which can provide new academic understanding of interspecific relationships, variation, differentiation and adaptation of species.It provides theoretical guidance in the fields of biological resources, traditional Chinese medicine resources, natural medicinal chemistry and so on, and has a very intuitive practical application value in the identification of medicinal materials, food safety and quality control of cash crops. SSEAC can show the information of all electrochemically active substances in plant tissues in voltammetric scanning, which is an extremely simple and efficient way to obtain the common signal of multiple chemical components. In recent years, under the research led by European scholars, this method has been explored for the identification of different plants, phylogenetic studies and plant physiological monitoring.
Although SSEAC has been initially applied to some practical applications, the iteration and optimization of its underlying technology are slow. The signal recognition of SSEAC mainly depends on the judgment of the difference degree of the characteristic peaks in the voltammogram, and this recognition method based on dimensionality reduction still needs subjective judgment. Especially in the study of phylogeny, it is difficult to provide convincing evidence and guidance in some plant genera with complex and controversial interspecific relationships because of the introduction of large errors based on a single voltammetric signal. Future research in this field can integrate SSEAC signals of different plant organs, determine reasonable signal acquisition standards, eliminate duplicate expression, improve the expression abundance of fingerprints, and express the complete information of electrochemical active substances in plants as much as possible, so as to accurately reflect the differences between different plants. In plant recognition and identification, SSEAC signals under different conditions can be standardized, and the standardized data can be used to construct pattern signals and color signals as channel values of different color patterns. The patterns and color bands of the identified species of plants were used as standard maps to construct the database. The SSEAC signals of unknown species of plants are used to construct the same pattern or color band, and the computer is used to study the automatic processing and interpretation of the pattern and color by mathematical calculation, so as to match the most similar standard map and realize the intelligent identification of plants.
National Natural Science Foundation of China(22004026)
China Postdoctoral Science Foundation(2018M640523)
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