Electrochemical Profiling of Plants

Abstract

The profiling, or fingerprinting, of distinct varieties of the Plantae kingdom is based on the bioactive ingredients, which are systematically segregated to perform their detailed analysis. The secondary products portray a pivotal role in defining the ecophysiology of distinct plant species. There is a crucial role of the profiling domain in understanding the various features, characteristics, and conditions related to plants. Advancements in variable technologies have contributed to the development of highly specific sensors for the non-invasive detection of molecules. Furthermore, many hyphenated techniques have led to the development of highly specific integrated systems that allow multiplexed detection, such as high-performance liquid chromatography, gas chromatography, etc., which are quite cumbersome and un-economical. In contrast, electrochemical sensors are a promising alternative which are capable of performing the precise recognition of compounds due to efficient signal transduction. However, due to a few bottlenecks in understanding the principles and non-redox features of minimal metabolites, the area has not been explored. This review article provides an insight to the electrochemical basis of plants in comparison with other traditional approaches and with necessary positive and negative outlooks. Studies consisting of the idea of merging the fields are limited; hence, relevant non-phytochemical reports are included for a better comparison of reports to broaden the scope of this work.

1. Introduction and Background

The traditional approach to classifying living organisms is ‘uni’ or ‘multicellular’, wherein plants are defined to be majorly eukaryotic multicellular species. They are essential for nature and to maintain distinct domains of living species, with the ultimate ability of food and metabolite resource hubs. Along with representing a food supply to different strata of society, their products are equally vital for the sustenance of shelter, fuel, and innumerable counter-products. The use of sensor technology in agriculture is mainly focused on the soil [1], water [2], weed control [3,4,5], and nutrient analysis [6,7,8]. The platforms are designed to understand the external factors surrounding plants rather than the plant itself. The term “Internet of things” has evolved over time; in a recent report, the authors emphasized a nascent concept concerning plants [9]. Plants have signal communications similar to neurons, and these signals are the basis of sensors and cloud technology. The idea herein, is to understand or decode the signals and employ this knowledge for both agriculture and plant domain. These environmental factors are known to be proxies for estimating the well-being of plants. This further affects the food supply chain and the different stages of plant growth need monitoring to ensure proper yields. This paper focuses on plant profiling rather than surroundings to understand the basics of plants, their features, and characteristics. Plant-based sensor technologies provide all kinds of crucial understanding for the optimization of food production.

When plants are subjected to external stressors, various defensive compounds are released by their systems. These compounds are basically a mixture of secondary metabolites (terpenoids, alkaloids, phenolics, etc.), volatile compounds (ethylene, methylene chloride, salicylic acid, etc.), and chemical defenses (phenyl propanoids, glucosilonates, etc.), which exert interesting functions, as shown in Figure 1. Automated pattern recognition is implemented to categorize the phytonutrients. Unambiguous identification and quantification are based on the bio-activity assay, modes of action in the body systems, target sites, and their source of cultivation. Categorizing wild varieties/species can cause potential problems in variety identification, as well as quality aspects, with catastrophic consequences. Taxonomy is one such study that offers a clear understanding of plant origins, relationships, and evolutionary patterns. The concept of the ‘origin of species’ is such an understanding wherein a phylogenetic relationship is derived for clear infragenic identification. Many other fields contribute to the achievements of botany, involving molecular biology, ecology, genomic understanding, cytology, bioinformatics, and biochemistry. These together are penetrated or interlinked with each other, providing directions for new explorations and modelling/untangling the core of life’s existence.

Plants consist of a mix of complexes or a rather soup of bio-synthesized nutrients as a part of their metabolic pathway. These metabolites are not easy to segregate for qualitative and quantitative estimations. The electrochemical approach involves detecting only a fraction called phyto-actives with response to the current–voltage plot, while the rest remain silent. Fortunately, the combination of these secondary metabolites is often taxonomically distinct and causes oxidative stress to plants; consequently, these are detectable using electrochemical approaches. An interesting example of the Herbivory group is the aggression-induced profile of undamaged pepper from chemical reactions due to beetles. The divergence in signals points towards the alterations in chemicals involved in defense mechanisms upon oxidative stress (polyphenols and terpenes). These changes were regarded to be the chemical communications of plants using volatile organic compounds. The first report for a potentiometric sensor involving the tissue of the yellow squash plant variety was postulated by Kuriyama et al. for glutamate sensing [10]. Following this example, another report for dopamine using ‘Banantrode’ (a banana pulp modified clark oxygen electrode) was demonstrated by Sidwell et al. in 1985 [11]. In continuation, new electrochemical platforms were developed based on corn kernels for the sensing of pyruvate [12], whereas a cucumber-plant-leaf-based system was used for cysteine detection [13]. Transgenic plants used as platforms themselves were reported in 2002 [14]. This includes the genetic alteration of genes with green fluorescent protein (GFP) via the use of an alcohol dehydrogenase promoter under variable conditions.

In the plant kingdom, many different ubiquitous secondary metabolites have an immensely positive impact on human individuals. These metabolites are responsible for herbivore defenses that exert cytotoxicity towards pathogens; and these features can be exploited in the pharmaceutical industry as antimicrobial supplements. Defensive products against neurotoxin activity can be potent muscle-relaxants, anti-depressants, etc. In continuation, many secondary products evolve and target molecular cells and tissues in competing trophic levels. Meanwhile, species of plants can contribute as defenses against insects and can be utilized as insect repellents. Ginko, an ornamental plant, improves peripheral and cerebrovascular circulation and the treatment of medical conditions associated with tinnitus and vertigo, because it is particularly effective in the scavenging of free radicals [15,16,17]. Kava, an indigenous species of ‘Oceania’ islands, is an intoxicating beverage used for treating tension, insomnia, anxiety, and agitation issues, because their profile includes benzodiazapines [18]. The property of radical scavenging is attributed to the ‘antioxidant power’ of a plant variety. With its widespread empirical use, the varieties necessitate accurate and reliable information on their phytochemical and antioxidant properties. However, there is no universal method for accurately and quantitatively measuring power. In electroanalysis, the total electrochemical antioxidant power (TEAP) of various plant material is dependent on the peak maxima [19]. The other established methods include 2,2-diphenyl 1-picrylhydrazyl (DPPH) assays, Folin–Ciocalteu assays, oxygen radical absorbance capacity (ORAC), lipid peroxide inhibition, dichlorofluorescein-diacetate (DCHF-DA), and the ferric reducing ability of plasma (FRAP) for assessing their total antioxidant activity. The electrochemical approach makes the total estimation relatively rapid, simple, and low cost.

Intelligent systems/studies are now being used to simplify and interpret large sets of plant profiles developed as a collaborative initiative to systematically classify the folk taxonomies based on plant characteristics. Stobiecki et al. reported the profiling of Arabidopsis thaliana of phenolic glycosidic conjugates [20]. Umoh et al. compiled a phylogeny based on subjective knowledge and through a multidisciplinary approach using metabolite investigations [21]. Berkov et al. recently elaborated the basic chemodiversity, chemotaxonomy, and chemoecology of Amaryllidaceae alkaloids, which were subdivided into 59 genera [21]. Herein, we present a basic method for ‘taxa’ characteristics in order to understand the phylogenetic relationships and profiling of plants using an electrochemical approach. This review accounts for various approaches accounted to date, including non-electrochemical techniques. A brief comparative study of techniques is presented in Figure 2.

Electrochemistry has been used in the building of sensor platforms in order to detect biomolecules in a laboratory setting, which has been extended to miniaturized point-of-care kits [22]. Globally, it has changed the perspective of medical and clinical diagnostics [23]. The sample manipulation of biosensors enables a wide spectrum of bio-compound measurements [24]. The minute quantities of the sample sizes involve just a few microliters to several nanoliters and simple pre-treatment steps before the initiation of experiments. The investigation requires tens of seconds to few minutes, making it the best choice in establishing on-site or in-field biosensing and detection systems [25]. These sensors represent a perfect interface with the incorporation of methodologies establishing a bio-recognition system consisting of electrode coatings with distinct transducers [26], reporter molecules [27], probes [28], nanomaterials [29], polymers [30], dendrimers [31], bio-derived assemblies [32], etc., with the scope of improved biocompatibility [33], enhanced signal intensities [34], additional binding attributes [35], etc. The aim is to combine the two separate categories of nanomaterials and nanofabrication in a recent trend of designs including contributions of MEMS and portable platforms for the diagnostics of fluidic, gaseous, and solid interfaces. Thus, commercialized diagnosis devices are the end goal of these sensor developments, incorporating confined sample pretreatment, delivery, and analysis sections [36,37,38]. The signal readouts are an amalgamation of responses from the electrode, capture and/or reporter probe, molecular linkers, enzyme molecule, etc., which are integrated to enhance the performance [39]. The presence of a nanomaterial matrix on the electrode system is quite favorable in establishing a high surface-to-volume output, strengthening attachment to the target substrate, enhancing biological compatibility, uplifting signal intensity, and establishing a better recognition base for adjoining molecules. Thus, the extensive use of nano-molecules has become a trend in the approach for developing electrochemical sensors. Many nano-molecules, such as carbon nano-materials (such as MWCNT, SWCNT, DWCNT, and graphene), metal nanoparticles (such as TiO₂, Ag, and Ni), and bulk carbon (such as carbon black, mesoporous carbon, activated charcoal) have been developed. A number of extensive studies have been performed on these nano-molecules to establish well-defined relationships [40,41]. Carbon nanomaterials assure immobilization characteristics for enzymes, and at the same time, act as amperometric transducers [42,43,44]. There are a few key attributes of a potential sensor, including: the detection speed (complete recognition within a few minutes; easy signal readout), sensitivity (from several fM to aM, without any issues of sample amplification), specificity (stringency for recognition; single-base mismatch detection), convenience (MEMS + nanotechnology; portable in-field assays; point of care biosensing), and multiplexing (combination of different biomarkers/bio-molecules correlates better accuracy; simultaneous detection) [45]. These sensors enable device miniaturization with a choice of batch-fabrication and well-defined precision capabilities.

DNA oxidation via electrochemistry opens the door to powerful techniques assembling the kinetics and thermodynamic information on a same scale. This approach can be utilized for analyzing DNA damage [46]. Liang et al. reported ruthenium tris(bipyridine) immobilized on a tin oxide nanoparticle electrode to identify DNA oxidation based on each nucleotide [47]. The quest for DNA-specific binding agents is fueled by the desire to modulate gene expression, to search for new antitumor drugs, and to develop molecular probes for DNA. This feature review article is not intended to be an exhaustive review, but rather, a compilation of classical and new avenues for the profiling of various plants in recent studies. Additionally, a critical outlook towards the state of the art and important advancements for the electroanalysis field are the focus. The aim of this study was to provide a new outlook of merging the electrochemical setup with the Plantae kingdom and to exploit the various domains to extract inherent information.

2. Traditional Approaches for Profiling of Plants

The enormous potential of plant systems can only be prioritized based on completed analyses of bioactive compounds using different traditional protocols such as those detailed subsequently.

High-resolution proton and carbon nuclear magnetic resonance spectroscopy (NMR) measure for high concentrations of metabolites in simplified tissue extracts, biofilm, and intact tissue. It is quite insensitive to a wide range of genetic modifications, toxicology insults, and physiological stimuli using conventional ¹H NMR. This approach often suffers due to small chemical shift range, producing significant overlap in resonance, whereas ¹³C NMR is intrinsically less sensitive due to the lower gyromagnetic ratio of the ¹³C nucleus. Hence, an alternative is using hyphenated techniques such as GC–MS or LC–MS, thereby improving the sensitivity.

Gas chromatography–mass spectroscopy or liquid chromatography–mass spectroscopy represent better routes for lower concentrations of metabolic fingerprints [48]. Although the theoretical basis involves major detection advantages, no reports are available in the literature. LC in conjugation with coulometric arrays represents a much better technique for the identification of discrete serotypes. Furthermore, LC–MS or GC–MS help in directly importing the data into pattern recognition. Coupling HPLC with MS offers the analysis and quantification of folates present in their natural state, with the added benefit of analyte identification. It ensures the correct analyte identity when compared with a reference library. The confirmation of identities can be achieved by negative ion ESI–MS using authentic reported samples [49]. However, a robust technique to overcome issues of the non-selective extraction and determination of the comprehensive profiling of contents, i.e., folates, polyglutamated status, flavones, etc., has not yet been developed.

Thus, a combination of NMR, LC–MS, GC–MS, FT–MS, and HPLC electrochemical arrays for profiling can be used as a global analytical approach. These include non-directed and class-specific analyses.

Capillary electrophoresis is another alternative with different choices of detectors; analyses of the majority of compounds can be carried out for those of extremely high molecular weight with varying polarity and a thermo-labile nature [50].

Electron spray, ionization spray or atmospheric pressure chemical ionization can be used in conjugation with LC/MS for efficient metabolic profiling. This involves a step for the creation of ions (protonated or de-protonated), exhibiting low internal energy. Thus, the fragmentation of molecules is solely dependent on the potential difference applied between the capillary and entrance in the ion source.

Fluorescence-labeled oligonucleotides labeled with a fluorescent moiety in conjugation with surface modification techniques serve for high-density DNA sequence and gene expression analyses. The detection level can be up to sub-nanomolar.

Size exclusion chromatography–high-performance liquid chromatography coupled electrochemical detectors are a well-defined substitute for ensuring proper differentiation for small and larger molecular systems (especially secondary metabolites).

In contrast, cyclic voltammetry is a unique and simplified tool for characterization, providing specific oxidation–reduction potential based on the chemico-physical properties of molecules [51,52]. It is a relatively rapid technique with nano-concentrations of samples required for analysis. Most widely used is the technique with considerable output referring to thermodynamics with respect to redox properties and kinetics for chemical reactions or adsorbed processes. Various electrochemical techniques have been extended for sensing of plant based systems in

Table 1. Tabulation of distinct types of electrochemical sensors and the scope of their applications reported by researchers in since 2000.

Chemically Modified Electrode	pH	Profile Constituent	Technique	Real Sample	Comment	Ref No.
GCE (acetone/chloroform extracts)	7, PBS	Quercetin, rutin, morin	SWV; −0.25 V to +1.25 V	Rosid varieties	Taxonomical studies; repeatability with 3 different harvests	[53]
Au (electrochemical chip fabrication)	5.8 and 7.1, PBS	β-glucuronidase monitoring	CV, −1 V to 1 V; chronoamperometry at 0.7 V and −0.4 V	Tobacco and tomato varieties	Gene expression monitoring	[9]
Boron-doped diamond electrode (BDDE)	7, PBS	Flavone peaks	SEC–HPLC–ELC detector; 0.8 V to 0.4 V	Profiling of Betula verrucosa, Equisetum arvense, Polygonum aviculare, etc.	Relationships between Cu and Fe species and with flavonoids	[54]
GCE	7, PBS	Antioxidant power determination	CV; −0.4 V to +0.8 V	Buxus hyrcana, Rumex crispus, Archillea millefolium, Zateria Multiflora, Ginkobiloba, Lippa citriodora, etc.	The lower the potential, better the antioxidant power	[51]
Hanging Hg drop electrode	3.5, B-R	Indole butyric acid	Stripping voltammetry; N2 atm	Direct bio-chemical	Plant hormone monitoring	[55]
GCE/CNT/AO-HRP	7.6, PBS	Methyl salicylate	Amperometry; 0.45 V	Wintergreen oil	Volatile organic compounds	[56]
GCE/GO-Fe2O3-CS	Methanolic 0.1 M NaClO4	Gallic acid; ascorbic acid	CV, DPV, −0.5 V to +1.5 V	Inflorescences variety	Antioxidant activity	[19]
SPE	7, PBS	-	DPV; −0.3 V to 1.0 V	14 Lycoris flowers	Fingerprinting petal tissues	[57]
Ag/AgCl	pH 2–7	H2SO4, HNO3	Potential difference	Soyabean seedling	Effects of uncoupler (FCCP) and acid rain are studied	[58]
SP/TiO2 or SiO2	4, KPH	p-ethylene guaiacol	CV; −0.1 V to +0.4 V	Phytophthora catorum	Plant disease biomarkers	[59]
CF-UME	Bz + EtOH: H2SO4	-	SWV; 0.05 V	Edible oils	Antioxidant activity	[60]
GCE	9, Tris buffer	Scatter Pattern study	DPV; −0.2 V to 1.4 V	Chimonanthus praecox	Evolutionary studies	[61]
Ag/AgCl	In situ study	DNP	Potential Difference	Glycine Maxx Merrill	Interfacial ion transport	[62]
Hg drop electrode	5, acetate buffer	Cd(ll) and Pb	DPV; −0.2 V to 1.2 V	Maize and sunflower seedling	Trace element contamination determination	[63]
SPE/Tyr/GA	4.5; acetate buffer	catechol	DPV; 0.2 V to 0.6 V	Mushroom tryosinase	Antioxidant capacity	[64]
-	-	PAH’s	Electrochemical detectors	Pisum Satvium	Oxidative stress agent determination	[65]
Ag/AgCl	-	CCCP	Action potentials	Soyabean	Environmental biosensors	[66]
GCE/guanine, GCE/adenine,	4.8; PBS	AA, GA, coumaric acid, resveratrol	SWV;	Mayonnaise and margarine	Antioxidant capacity	[67]
CPE/stearic acid/DNA	7.4; PBS	herbicide resistance	SWV	Phosphinothricin Resistance	Barcode gene	[68]
Au wire/TGA	7; PBS	Binding site of plant protein	CV;0.0 to 0.8 V	Planthacyanin (Blue Cu-protein)	Binding site of proteins	[69]
SPE/MWCNT-NH2/AG/PNPG-PVA	7; PBS	Inhibition of AG enzyme represents the potential for the plant to inhibit glucose production	CV; Amp i-t	Ehretis laevis; Micromelum pubescens; Spondias dulcis	Anti-diabetic potential of medicinal plants	[70]
Au	13; NaOH	Mg, Zn, and Mn	Electrochemical detector	Folium betulae; Folium menthae; Folium salviae; radix Valerianae; radix Taraxaci	Speciation of Mg, Zn, and Mn in plants	[71]
GCE	HClO4	Trans-resveratrol	Adsorptive stripping SWV	21 Rioja red wines	Phytoalexin determination	[72]
GCE/MWCNT	7; PBS	Sesamol	DPV	Sesame seeds and oils	Phytonutrient content analysis	[44]
GCE/CPE/PVP	3; B-R buffer	Kaempferol	CV; SWV	Spinach, cabbage, broccoli, and chicory	Total phenolic compounds	[73]
GCE/GNR/guanine	7.4; PBS	OH scavenging by AA	SWV	Fruit juices	Total antioxidant capacity	[74]
GCE/carbohydrate	0.1 M KCl	Quercetin	CV; SWV	Chemical compounds	Flavonoid determination	[75]
Ir, Rh, Pt, Au, Ag, Cu, Co, Ni	4.8; K3PO4	Fruit juice characterization	Pulse voltammetry	Electronic tongue	Passiflora mollisima, Myrciaria dubia	[76]
GCE/Poly-CDDA	4; PBS	AA, dopamine, and UA	DPV;	Chemical compounds	Antioxidant capacity	[77]
GCE/MWCNT	7; PBS	Guaiacol	DPV	Whisky and brandy	Nutritive content	[78]
GCE	NaClO4	Standard reduction potential of OH.	CV	Melissa officinalis L, Fragaria L, Origanum majorana L, Salvia officinalis L, Equistum arvensis L, Calendula L, Alcea rosea L, Melilotus officinalis L	Total antioxidant potential	[79]
CPE	5; PBS	Electrochemical index	DPV	Red, white, and sparkling wine, and grape juice	Correlating DDPH with the electrochemical index	[80]
6B pencil graphite electrode	7; PBS	2,3;2,4;2,3,5 hydroxy benzoic acid	DPV	Commercial tea available	Tea quality testing	[43]
CPE	5; PBS	Electrochemical index; antioxidant power	CV; SWV; DPV	Coffee extracts	Total phenolic content	[81]
GCE	3.6; acetate buffer	delphinidin glucoside; cyanidin glucoside	DPV	Cabernet Sauvignon wine, raspberry	Antioxidant capacity	[82]
GCE	4.7;7; potassium phosphate	Normalized current plots	SWV	Potentilla Argentea; Sarcopoterium spinosum; Agrimonia eupatoria; Salvia valentina; Lavandula multifida	Fingerprinting of seeds	[83]

AO, alcohol oxidase; HRP, horseradish peroxidase; SP screen-printed; CF-UME, carbon fiber ultra-microelectrode; FCCP, carbonyl cyanide p-trifluromethoxypenylhydrazone; DNP, 2,4 dinitrophenol; Tyr, enzyme tyrosinase; GA, glutaraldehyde; Pah, polycyclic aromatic hydrocarbon; CCCP, carbonyl cyanide 3-chlorophenyl hydrazone; AG, α-glycosidase; PNPG, p-nitropenyl-α-D-glucopyranosidase; PVA, polyvinyl alcohol; CPE/PVP, carbon paste electrode with poly(vinylpyrrolidone); GNR, graphene nanoribbon; CDDA, 3-(5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic acid.

.

Flow injection analysis is a simplified chemical procedure performed with a flowing carrier stream and used in combination with the electrochemical system, and shows similar results to HPLC-coupled ECD.

3. Profiling of Biochemicals

Profiling details a whole plant cellular network constituting of transcription (DNA→mRNA; the first step), followed by translation (mRNA→nucleotide), and finally, post-translational modifications of the gene products. Each step of the analysis is extremely difficult due to the presence of a huge number of primary and secondary metabolites. Countless metabolites are present in cells based on the cell type, differentiation, and specialized type acquired. These essential bio-elements mix with organic moieties, forming natural complexes which are potentially important for survival. It is typical to identify the nucleotide sequences of nuclear and chloroplast regions. Hence, chemotaxonomy is one such classification that can further the understanding of chemical compositions [21,53,84,85].

The chemical analysis concept includes vast domains of estimating nutritional, pharmacological, and chemological properties. Alternatives for the identification and monitoring of chemical species are the “targeted approach” and the “non-targeted or fingerprinting technique”. Figure 3 shows the use of fingerprint recording for the species determination of 14 flowers species of Lycoris. Fingerprinting or non-targeted approaches intense techniques based on organic–inorganic plant interactions due to ignorance in understanding the exact mechanisms. Among such a plethora of complex molecules, ‘n’ is the number of phenolic acids, anthocyanins, procyanidins, and flavonoids which have been observed. The genetic profiles of variant species are directly proportional to their chemo-taxonomical studies; this approach is associated with inherited properties of natural products and synthetic analogues. The use of electrochemical approaches involves representations of simple plant extracts, eliminating the concept of prolonged storage under aerobic environments. The approach is quite simplistic due to the redox-active components; many identified chemical counterparts are non-electroactive in nature. Some examples of these phenolic molecules are rutin, vanillic acid, salicylic acid, morin, and quercetin. Isoflavone is one such phytoestrogen or secondary metabolite, particularly present in leguminous plants. Many constituents such as flavonoids, lignans, and coumestrans are fractions of phytoestrogens. These help plants to maintain resistance to diseases such as neurodegenerative disorders, cancer, Alzheimer’s disease, fungal attack, and hypertension. Carotenoids are the most abundant natural pigments and exert substantial pro-vitamin and antioxidant activities. Chlorophyll and its associated compounds have huge antioxidant potential, and thus inhibit lipid peroxidation and even protect mitochondria from oxidative damage [86]. The application of methods can be extended to mammalian tissues and organ domains. These have been further subdivided as follows.

Genomic and metabolomic study: metabolites can vary across a wide range of modifications, making them a complex system to accept as unique biomarkers. Using metabolic engineering could improve the situation by enhancing the synthesis of valuable therapeutic agents. Wang et al. presented a comparative review on plant miRNA biogenesis and degradation, encountering stability issues upon 3’ modifications and studied degradation upon translation [87]. Gao et al. developed a microRNA biosensor based on chemical ligation which involved the concept of electrochemistry for amplified detection [88]. They explained how Northern blots and cloning are the gold standard for microRNA validation; these assays are inappropriate because the consumption of RNA samples is a laborious phase. Subsequent high-throughput and sophisticated instrumentation with highly skilled technicians are necessary for quantification assays.

Electrochemical DNA interpretation involves the use of gold nanoparticles [89,90], the direct oxidation of guanine [91], DNA intercalations [92,93], DNA threading intercalators [94], etc., which were established in 1900 [11,95]. In addition, the sensitivity of metabolic studies alters with response to stress and toxicity investigations. The detection of nucleic acid by various means is an essential part of genomics studies. The use of electrochemical means to mark an effective biomarker involves the stepwise designing, synthesis, and characterization of probe molecules. Many reports have detailed the use of gold surfaces and nucleic acid motif functionalization in investigations of DNA bio-barcodes, DNA hairpins, aptamers, enzymes, etc. [96]. Functionalized sensors for nucleic acid targets focus on a diverse repertoire of chemical readout mechanisms. Commonly, modifications using chromophores, click chemistry reagents, biotin, etc., are vital components of probes. Approaches for the site-specific attachment of thiolated DNA probes are being developed independently.

Transcriptomics: various biological and chemical amplification strategies are being upgraded using enzyme-amplified assays with minute quantities, without the need for PCR [97], such as Au–NP bilayer electrode systems [98], although electrochemical amplification strategies with electro-catalytic redox moieties [94] yielded quite an enhanced sensitivity. There is an ever-increasing demand to develop rapid, sensitive, and selective bioassay methods for various molecular diagnoses and the detection of infectious agents. Methylene blue is one such example of an end-labeled oligonucleotide used as a fluorophore/quencher that relates ‘on’ and ‘off’ stage switches based on the conformational state of methylene blue. The signal is directly proportional to the distance between the quencher and the fluorophone. The use of MB has been accepted in applications in molecular and cellular biology, pathogen detection, and biomedical diagnostics [99].

The use of traditional expensive instrumentation poses a setback in the research field due to the unavailability of instrumentation. The analysis protocols are time-consuming with tedious pre-concentration steps before the investigation. Conventional systems require complex processing steps performed by highly skilled technicians.

4. Use of Electrochemistry

Electroanalysis reflects the fingerprints of pigments, plants, and cultural relics based on recordings of electrochemical signals [83,100]. The electrochemical detection of biological species requires reactions consisting of bio-recognition steps involving current or potential, impedance, redox kinetics, etc., or non-electrochemical properties involving van der Waal interactions, intercalation with matrix due to electrical fields, mass transportation beyond the Helmholtz’s double layer, conformational changes, etc., inducing fluctuations in electrical responses. Miniaturized biosensors, due to their compatibility the advanced use of semiconductors, are combinations of accurate and inexpensive platforms. These sensors offer electron transfer reactions without labelling for ultrasensitive DNA detection [101]. Domenech-Carbo et al. used squarewave voltammetry to characterize tomato plants based on six diverse varieties [102]. They placed a glassy carbon electrode directly into fresh tomato varieties and estimated the ratios of vitamin C to total phenolic compounds [103,104,105]. Many other such plant constituents have been analyzed, and are presented in Table 1. A plot for use of Differential pulse voltammetry as shown in Figure 4 has been highlighted to determine the relation between species.

Many methods have been proposed for the application of electrochemistry as a tool to understand plant feedback mechanisms to stress via assessments of the redox activity of phytocompounds. The initial condition for determining species involves replicating experimental procedures on the chosen species type, followed by studies of voltametric curves with increasing peak resolutions. Voltammograms display different values for different species based on the multiplication of current values (I) with potential (E), involving ‘i’ solvent extracts having ‘j’ electrolytes. Hence, the matrix dimension are I × (ij), referred to as PCA diagrams for wave voltammograms or dendrograms. We can distinguish and segregate species signatures in various phylogenetic classification based on electrochemical approaches; however, few similar species diverge from genetic similarities. However, overall, satisfactory correlations with phylogenetic trees at the level of taxonomical orders can be judged [53]. Fu et al. used a DPV technique for the electrochemical fingerprinting of Lycoris petal species of several varieties and confirmed the consistency of the PCA diagram with the dendrogram correlating the infragenic relationships between native Lycoris species [57]. As explained by Carbo et al., ramifications in the phylogenic tree concerning the Rosaceae family lead to complications related to voltammetric analysis. The authors explained that the differences might be attributed to evolutionary tendencies due to the diversification of polyphenolics in them. In addition, they also related the advent and disappearance of the signals as temporary sequences due to genetic changes that help us in acknowledging the modifications; hence, this corroborates other classifications of phylotaxonomy [106,107]. The concept can be further extended to the subcellular analysis of plants, with outstanding data enabling further manipulation of the essential constraints for improving health and product outcomes using plants. Leon et al. compared electrochemical and conventional techniques for the determination of antioxidants used in the monitoring of DPPH radical capture [108]. On comparison between spectrophotometric and chronoamperometry techniques, sample dilution and pretreatments steps were easily eliminated in cases of amperometry without any issues of concentration and absorbance uncertainty. Table 1 shows the culmination of various electrochemically based sensor systems and the scope of their applications.

The voltammetry of immobilized particles using solvent extraction (ethanolic and methanolic) approaches uses the preferred parts of plants [55]. Cyclic voltammetry (CV) enables non-faradaic contributions that result in non-steady signal outputs. Hence, constant potential with amperometry or chronoamperometry is one such technique that ensures a steady state response. Scanning electrochemical microscopic analysis involves the direct imaging of plant systems and their internal transformations/modifications based on the electroactivity of phytonutrients.

5. Electrochemical Profiling of Biochemicals

Phenolic acids, flavonoids, and carotenoids are considered to be key compounds due to their electrochemical profiles [109]. The accessibility of techniques involving easy operation via portable in-field equipment with simple sampling protocols that ensure immediate analysis without prolonged maceration/extraction is highly beneficial. Most importantly, the intrinsic ability to select molecules such as sugars, cellulose, or carotenoids can be excluded because this refers to electrochemically silent moieties; only a limited number of components are active in a similar way to phenolic moieties. These non-conducting ingredients consume key parts of plant tissues, making it difficult to analyze the conducting counterpart ingredients. An embedding plant strategy has been designed to ensure selective and amplified responses using a matrix or polymers [25,110]. It is a relatively tedious job to summarize the protocols and resolve the varieties and species domains in the Plantae kingdom; hence, conventional systems are not applicable in real-world analysis. Considering electrochemical techniques, only voltammetric profiles did not exhibit any alterations in condition when studied. Differential pulse voltammetry is one such technique, which can be used to establish ‘p’ values for MANOVA tests: this helps in maintaining normalized wave signals of a particular species group. However, the ‘p’ values can be quite close to 0.5 for inter-species attributes; for the case of intra-species analyses, p >> 0.5 thus helps in establishing significant relationships [57]. This analysis can further be used for the detection of food and medicinal attributes exerted by phytonutrients [111,112,113]. The concept of correlating species with their successive voltammetric attributes has previously been studied [114].

Thin film cyclic voltammetry represents the optimal choice for the evaluation of P450 chromosome enzymes. This approach has momentum, especially in the field of deciphering electroactive constraints wherein specifically engineered enzymes are fused in conjunction with electroactive nucleotide chains. These fused molecules act as an interface component of electrodes modified with biomimetic systems; further techniques are employed for biosensing applications [115,116].

Nurmi et al. reported the profiling of phytoestrogens using hyphenated HPLC electro-array methods. These techniques have shown good sensitivity and reproducibility over various assays [117]. Similarly, Klejdus et al. initiated an electrochemical platform for isoflavone profiling by adsorptive transfer stripping voltammetry in soyabean plants at pH 9.2 [118]. Based on successive investigations, Gil et al. reviewed the use of electrochemistry for flavonoid profiling, with in-depth knowledge of electrode platforms and their buffer media [119].

An innovative approach for the identification of plant species based on the fingerprinting of petal tissue was reported by Fu et al.; the use of electrochemistry was emphasized, although the comparison of infragenic identification was based on a traditional botanist approach. Electrochemical fingerprints were analyzed after various solvent extractions and the contents of electroactive molecules were controlled by respective genes [57].

6. Conclusions and Future Prospects

This paper presents a culmination of all the different techniques and their probable outcomes for the profiling of plants. The basic comparison between other classical and electrochemical techniques shows that electrochemically based platforms are optimum pathways because they are relatively rapid and simple. The results for profiling are comparable with other techniques. The main conclusions and future scope of this study focus on the identification of molecules produced within plants, and obtaining/extracting them on an industrial scale with the scope of using them further as selective pesticides. Based on the electrochemical behavior of plants, a lower potential is more suitable, i.e., the possibility of establishing an ‘electrochemical taxonomy’ will be soon realistic. The report by Fu et al. can be extended to other plant varieties and compared with other techniques to develop the PCA diagrams. The electrochemolomic methodology is a potential set of analysis techniques for assessing different vegetal species in order to establish taxonomical groups; it can be used as a complementary technique to molecular phylogeny, in tandem with existing genetic and chemical assays. The apparent potential of this technique may be an assistive technology for phytogenic studies in many other fields of application. The importance of preserving biosphere diversity is highlighted through the correct development of science and technology. The use of a rapid practical and reliable methodology with good analytical performance and suitable selectivity indicates the contemporary need for profiling systems. Clinical applications of these sensors are still distant, because major selectivity/specificity and performance issues need to be addressed.