Review—Environmental Biosensors for Agro-Safety Based on Electrochemical Sensing Mechanism with an Emphasis on Pesticide Screening

Climate change directly affects all aspects of the environment and accounts for changes in our surroundings seen or unseen. With the growing need for accountability in the agricultural commercial and regulatory spheres, there is a widely accepted consensus that there needs to be quantitative information on the different environmental entities to understand and solve challenges posed towards food production and agricultural activities. The main purpose of the creation of this article is to propagate an era of quantitative metrics to better assess environmental health which can adversely impact human health. This review assesses 3 different environmental vectors prone to pollution and in high contact with human entities. This review also discusses different electrochemical sensing platforms used as biosensors for the detection of a wide myriad of pollutants with an adverse effect on human health. Furthermore, the breakdown of each section includes a survey of the evolution and design of various sensor platforms that are aimed towards a comprehensive monitoring platform for pollutant screening with an emphasis on agro-safety and environmental health. Detailed pesticide screening case-studies are also explored for a better understanding of the current electrochemical biosensors that exist for the sole purpose of environmental monitoring.


Background
Heavy metals, toxins and pesticides are some of the most common issues that plague current agro-safety and regulation. Heavy metal pollution has spread all over the world and poses serious health hazards to consumers. The United States is one of the world's largest consumers of pesticides. Glyphosate, one of the world's most used pesticides, is an organophosphate pesticide and belongs to Monsanto. 1 The United States is said to account for around 19% of the world's consumption of Glyphosate and millions of tons of pesticide are sprayed on U.S. crops every year. However, with the rise of pesticide-resistant pests and weeds, there is a marked increase in the usage of pesticides over the last several years. Annually in the United States, almost 80 million pounds of organophosphate-based pesticides are utilized on millions of acres of food crops. 2 The global agricultural demand is expanding due to the growing population and food production. 3 Heavy metals, toxins and pesticides can spread through the environment through a variety of vectors such as food media and water. More importantly, pesticides, toxins and heavy metals can seep into groundwater and cause soil and water pollution. There is also the association between consumption of foods tainted by pesticides, toxins and heavy metals and their inevitable deleterious effect on the human body. Pesticide levels exceeding widely known tolerance levels can cause acute toxicity in the human body and lead to other life-threatening conditions. 4 Organophosphates -one of the largest classes of pesticides -are AChE inhibitors. 2 AChE is an enzyme that degrades the neurotransmitter acetylcholine into choline and acetic acid. Continued inhibition of AChE can cause neuroinflammation, neuropathy and muscular weakness. 2 Consumption of heavy metals such as As, Pb, Cd and Hg is highly dangerous in many aspects and considered in the top 20 list of dangerous substances by the United States Environmental Protection Agency. 5 Heavy metals such as Cu, Fe and Zn can adversely affect metabolic processes and is linked to the metabolic functioning of biota. 5 With increasing urbanization and industrial practices, contamination of soil and food media by heavy metals is growing more prevalent. Medicinal plants, greenhouse vegetables and open field vegetables all show heavy contamination of heavy metals when grown near industrial areas. 5 In tandem with the increasing heavy metal contamination of the environment, the increasing consumption of pesticides in human society is a public health concern that needs to be addressed immediately. However, current analytical methods for the detection of pollutants are all laboratory-based and don't allow for on-site field testing. Biosensors, however, can fill this gap. There is a large need for biosensors in the agricultural sphere. Current laboratory analytical methods such as GC-MS, LC-MS and other spectroscopic methods require stringent pre-processing of samples, expensive equipment as well as trained personnel. For field-deployment in low-resource environments, there is always the threat of a lack of infrastructure. Due to these issues, new analysis tools are necessary to obtain rapid and reliable information about the presence of a particular analyte in a sample. 6 Biosensors provide reliable and consistent performance comparable to their gold standard counterparts while being able to be translated into a portable, point-of-care diagnostic device. A preferable subset of modern biosensors is based on-electrochemical detection, providing a sensing platform that serves as a signal transduction pathway of chemical reactions into electrical signals that can be deciphered by a myriad of devices and software. Current solutions for biosensors are generally immunobased, requiring antibody-antigen interactions or surface reaction based i.e., chemical reaction at the sensor surface to provide a valid signal. When it comes to field testing for pesticides and other toxins in food crops, select samples of crops must be sent to a laboratory for testing. However, this can be of a significant monetary burden to the farmer or commercial interest. Creating a biosensor that can provide reliable results in real-time with no noticeable delay can change the landscape of food safety regulation and detection. Research and development into affinity-based biosensors has advanced greatly due to their simplicity and heightened sensitivity. 6 In biosensors, there are a wide multitude of nanomaterials that are used to improve the operational efficiency of the sensing system. The functionalized nanomaterials are used as catalytic z E-mail: Shalini.Prasad@utdallas.edu tools, immobilization platforms, or electroactive labels that improve the biosensing performance by a wide and noticeable margin. 6 In this article, the usage of biosensors for the detection of analytes of wide importance in the agriculture sector will be thoroughly analyzed. Biosensors fall into two large categoriesbioreceptor-free and assay subsets for electrode modification. Bioreceptor-free modification generally uses inorganic composites to enhance signal transduction. Several electrochemical approaches for the detection of pesticides have been reported 7 utilizing receptor free modification. Several catalyst nanocomposites have been used to improve the selectivity and sensitivity of enzyme detection. 8 Some examples of these nanocomposites used to increase and improve the efficiency of pesticide detection are Cu-BTC MOF, 9 rGO -CuNPs composites, 10 Cu-(poly)pyrrole composite, 11 MMWCNTs-IL/CuO nanoparticles, 12 SPE/Chi/CNO/TYR, 13 and lastly n-Si/PANI/HRP. 14 These nanocomposites, when utilized in tandem with an electrochemical sensing platform, have increased operational performance of biosensors in pesticide detection. Just as there are many nanocomposites that improve the performance of the electrochemical biosensors, there are different types of biosensors depending on the biomolecules immobilized on the electrode surface. This falls under the category of bioreceptor-based modification of the electrode surface. This is because there's multiple moving steps in adhering a biomolecule to an electrode surface and optimizing target-receptor interaction to facilitate signal transduction of the desired analyte. Affinity biosensors use the selective and strong binding of biomolecules such as antibodies, membrane receptors or oligonucleotides with a target analyte to produce an electrical signal. 15 The high affinity and specificity of the biomolecule and its ligand make these sensors sensitive and selective. 15 Immunosensors, based on antibody-based receptor elements where the detection of the analyte or the antigen is induced by its binding to an active site on the Antibody. 15 These affinity-based biosensors show the theoretical strength of the capacity of these biosensors and the subsequent importance of biosensors for usage in on-site situations. These electrochemical biosensors are characterized through different techniques or modalities such as voltammetric, potentiometric methods or Electrochemical Impedance Spectroscopy and Chronoamperometry. These electrochemical measurement techniques allow for efficient optimization of the biosensors and are capable of being miniaturized. The synergy of these characterization processes makes biosensors a viable electroanalytical diagnostic since they can match gold standard laboratory methods and consume less resources. This article will especially delve into measurement of pollutants such as pesticides, toxins and heavy metals within a food medium utilizing an affinity-based electrochemical sensing platform illustrated in Fig. 1. This is a particularly troublesome obstacle since food media contains interfering analytes that can wreak havoc on the detection mechanism. Some current electrochemical-based sensing methods involve the reduction of nitro compounds formed on the electrode surface, immunoassay peroxidase-based sensor on the modified polymer-gold substrate, graphite epoxy electrode modified with multiwalled carbon nanotubes and modified metal electrodes such as gold, copper, silver, and platinum which are known to have high sensitivity because of the electrodes' transfer property. 16 However, these electrochemical sensors can only operate in a neutral pH, making them unable to be used for translational studies such as field deployment and unable to be used as a suitable replacement for laboratory gold standards.
These environmental pollutants spread through a variety of vectors such as soil, food crops and freshwater sources. Biosensors need to be able to reliably detect pesticides, toxins and heavy metals in food media and freshwater media in order to be a strong force in consumer-driven detection and regulation. This article will show the strength of current electrochemical-based biosensors in the realm of toxin and pesticide detection as well as the future of these biosensors with regards to miniaturization, IOT and point-ofcare analytics.

Soil Quality Assessment
Introduction.-Environmental pollutants that generally have adverse effects on human, animal and plant life generally emanate from three main vectors: soil, water, and air. Among these vectors, the pollutant groups which have the highest direct and indirect effects on food and agricultural production are soil and water contaminants. The objective of this chapter is to provide some examples of work done in assessing the presence and quantification of soil pollution designed for Point-of-Use (PoU) understanding of soil quality. The pollutants generally permeate the soil environment through direct penetration or percolation at different soil depths. Over time, these pollutants accumulate in the soil environment becoming environmentally dangerous, and this has a collinear impact on the ecosystem balance and the food chain. 17 Within this class of soil pollutants-there are different groups including fossil fuel based petrochemical products and industrial waste, heavy metals and an overuse of pesticide products which increasingly contribute to the pollution and degradation of the soil and land environments over time. 18,19 With mounting concerns regarding the maintenance of quality and safety in agro-food production, there is a prime need for determining how soil is affected by contaminants and how this polluted soil affects the food safety and quality? The emergence of robust biosensors and chemosensors enable non-destructive, realtime sampling of analytes. E-nose and E-tongue sensors are powered by techniques such as optical, spectroscopic, acoustic, mechanical, and most importantly, electrochemical modalities. Electrochemical solutions are heavily desired due to their ease-of-use and practicality for in-field deployment and testing. Additionally, electrochemical methods also make non-destructive analysis, robust sensor performance with high degree of specificity and sensitivity viable while being economical and reliant on minimal reagents and sample processing. Most importantly, the premise of scalability in screening in a high-throughput manner for contaminants in samples proves to be indispensable. 20,21 Understanding potential pollutants and their effects in soils is largely underwhelming since soil quality has been looked at from a local agro-ecosystem perspective with regards to productivity and environmental quality/biodiversity. 22,23 Soil behaves uniquely in that in deviates from its normal characteristic behaviour in accordance with deviant land management techniques. Due to these concerns and the increasing difficulty in soil screening, there has been a noticeable surge in sensor-mediated soil quality assessment. The inclusion of electrochemical biosensors in soil assessment will allow us to study how soil inherently affects and modulates crop growth, determine the agriculture ecobalance and concurrently sets the landscape for food security and production. [24][25][26][27][28][29] Case-study: rapid electroanalytical sensor device for pesticide screening in soil run-off.-Pesticides refer to a class of chemical compounds that curb pest activity in different agricultural environments. The umbrella term "pesticides" is used for chemical agents that inhibit the growth of different lifeforms such as insects, virus/ bacteria, rodents, weeds, etc. Some common pesticides include herbicides, fungicides, insecticides, disinfectants, and antimicrobials among others. With over a thousand different pesticide types available, the global usage of pesticides is around 6 billion pounds (weight) per annum while the United States uses more than 1 billion pounds (weight) worth of pesticides in different agriculture-related industries. 30 The impact of human activity on the environment has been profound with findings displaying the correlation between public health concerns and land use, emergence of genetically modified crops, and industrial activities leading to pollution. Exposure to pesticides is one major aspect of how the food industry is closely tied to modern health issues. The routes of exposure can be classified into two areas -occupational contact methods through agriculture and industrial use and non-occupational sources from everyday activities and consumption like food and water ingestion. Many peer-reviewed scientific studies focus on the health concerns associated with pesticide usage and exposure, including possible carcinogenicity, endocrine disruption and hormone modulation/ imbalance, non-alcoholic fatty liver disease, reproductive issues, birth related complications and celiac disease as well as others. [31][32][33][34][35][36][37][38][39][40][41][42] From this, it is understandable that both routes of pesticide lead to a situation where 80%-85% of pesticide residues end up in the human body from food sources and it is even more crucial to understand that 95% of global food production is produces on soils. 43 Therefore, soil is a major system in terms of pesticide traces that contribute to human health.
In this section of the review paper-we discuss a robust sensor system that can be directly deployed in a portable manner for ondemand quantification of pesticides in soil run-off as depicted in Fig. 2 below. 44 Soil run-off is defined as water that comes out of soil systems that are irrigated. This is a viable and representative testing methodology since pesticides in soil leach into the soil horizons and thereby into groundwater. As little as 0.1% influences its intended target. [45][46][47] Electrochemical sensors are most suited for in situ analysis of pesticide detection in soil run-off systems.
From different studies, it can be gathered that the sensor electrode geometry has an influence on the overall sensor performance in the case of biosensor design and feasibility. Interdigitated and spiral designs are preferential for this type of affinity mediated sensing mechanisms to capture and measure analyte levels by incorporating bioreceptors on the sensor surface. 48 The electrochemical transduction methodology is summarized as such: metallized electrodes electroplated on a hard PCB substrate are conjugated with a pesticide specific antibody via a thiol linkage mediated using Dithiobis (succinimidyl propionate) dubbed "Lomant's reagent." This can be visualized through a layer-by-layer (LBL) immunoassay stack that can recognize and detect pesticide analytes in samples. 49 The sensor was tested with two widely used pesticides: Glyphosate and Atrazine in soil run-off, which have half-lives of 47 days and 60-75 days, respectively. The charge of the pesticides was also found to be different with glyphosate possessing an overall negative charge while atrazine's inherent charge was closer to zero. 50,51 Seen in Table I, atrazine uncharged has a positive Log K ow of 2.61 (range-2 to 2.6) indicative of more hydrophobic properties whereas glyphosate possess a net negative charge and has a negative Log K ow of −3.4. Hence, this universal pesticide sensor framework was tested with targets of different physiochemical nature in a particularly complex matrix. Hence, the postfunctionalized sensor surface was tested in a label-free environment i.e., no use of redox tags or additional reagents to act as surrogate markers or indicators and no sample processing prior to testing. To accomplish this target of direct determination of pesticide levels in soil run-off, non-faradaic electrochemical impedance spectroscopy (EIS) was utilized for probing the sensor-sample interface and extracted subtle binding information between the pesticide targets and the receptor molecules.
Impedimetric techniques are an electroanalytical technique fundamentally based on electrical impedance spectroscopy. Impedance is a resistance in alternating current circuit. Ohm's law postulates that current obtained I in an electrical circuit is proportional to the bias voltage having a proportionally constant R known as resistance of the circuit. Similarly, application of sinusoidally varying bias voltage across a circuit induces an alternating current and this gives an AC analogue as per equation below: Here, Z corresponds to impedance and the modulus suggest that these quantities are basically a time-dependent component. Z has the unit of ohms (Ω). In an EIS experiment, the impedance of a sample is measured over a wide range of frequency using a frequency analyzer, also termed as phase-gain or signal-response analyzer. The analyzer applies a small alternating voltage and measures the alternating current induced. From these data, the impedance Z* and the phase angle θ between the current and voltage is calculated. These steps are performed over a range of frequencies and from these values of Z* and θ, the components Z′ and Z″ are determined and represented as Nyquist plot. Whereas the modulus of Z can also be plotted against frequency and depicted as a Bode plot. Analysis of the impedance data is performed by fitting the impedance data into an equivalent circuit consisting of resistors and capacitors in general and resistance, capacitance, and inductance occasionally. 52 The components of an electrochemical cell are assumed as resistors and capacitors and are organized in such a way that the impedance behavior in the Nyquist plot matches the behavior of the simulated circuit. The arrangement of resistor and capacitor placed in parallel, also called as RC element, is more common in electrochemistry and corresponds to the electric double-layer. The RC element produces a Nyquist plot with a semicircular arc of diameter R. The Nyquist plot of different RC circuits have been represented in Fig. 3.
In an electrochemical setup, the electric double layer acts as a plate capacitor and generates double-layer capacitance, C dl . Simultaneously, there is a resistance to the flow of electrons termed as the resistance to charge transfer, R ct . C dl and R ct has a behavior of arrangement in parallel and hence produces a semicircular arc in the Nyquist plot. The double layer represented by the RC element has    although they possess complex behavior, are superior in terms of sensor development which will be employed for real time detection. An electrical double layer (EDL) is formed between the treated or functionalized immunoassay stack and the analyte system. This can be visualized as a dielectric system like a parallel plate capacitor where opposite charges are aligned on two plates separated by a distance. Therefore, as the interface is modulated by changes in the analyte under test-these can be seen as a function of capacitive variations to the EDL. This implies that-Non-faradaic EIS is ideally suited for this task since it is a comprehensive tool for surveying the subtle chemical interactions at the electrode-electrolyte interface. As a result, using this interfacial probing mode can yield great sensitivity to the target analyte. With increasing concentrations of pesticide in the sample, the overall level of binding between the target and the antibody receptors will be different and this difference causes an impact on the double layer (length and charge variation across the diffuse layer). It is this modulation to the double layer that is recorded as impedimetric signal changes corresponding to pesticide levels in soil run-off.
To further explain this occurrence, consider the following: When a 10 mV AC voltage is supplied to the electrode, it causes the solid-liquid contact to be perturbed. Due to binding between the antibody and the pesticide molecule, EDL capacitance fluctuates. The dielectric permittivity of the system is varied due to the doublelayer structure and length of disruption in this model, which causes capacitance modification.
The impedance spectra are plotted in a tri-functional manner with the Nyquist (Zreal vs Zimg), Bode phase (Freq vs Zph), and Bode magnitude (frequency vs Zmod) plots. In Figs. 4A-4B, it can be gathered that as a function of pesticide concentration addition to the sensor surface-there is an equivalent bending of the curved lines towards the x-axis signifying reduction in the radius of curvature for both atrazine and glyphosate pesticides. Additionally, it was also noted that the curved lines in the Nyquist plot is depicted as an incomplete semi-circle which implies the infinitely large charge transfer resistance (R ct ) component in the impedimetric results. 53 This Nyquist behavior electrochemically confirms the binding characteristics between the pesticide analyte and its respective antibody receptors. The graph (C-D) was zoomed in to the 10 Hz region to better visualize the trend in impedance shift with increasing doses for both atrazine and glyphosate, and to better visualize the bending of the Nyquist curve corresponding to target-receptor binding.
Next, the electrokinetic rate of antigen-antibody binding is extracted and summarized from the Bode plots for atrazine and glyphosate. It was noted that from the bode-magnitude plot and bode-phase-there was a functional impedimetric parameter change seen with dose additions as: Zmod (magnitude of impedance) change and Zph (phase-impedance) movement from −90 degrees (capacitive) towards 0 degrees (resistive) was indicative of more binding interactions causing a layer of charged screening. It was gathered from this sensor that the effect of the sample matrix (soil-water slurry or run-off) being particulate in nature was causing the overall system response time to be at 10 min or higher.
An analogous electrical circuit termed as a modified Randle's circuit was used to view and simulate the entire EDL structure as well as the impedance parameters associated with the EIS spectra. This simulation was done conjunctively with experimental findings to derive which electrical component was indicative of the binding interactions that occurred at the electrode-electrolyte interface. The impedance modulations recorded by this technique are triggered by antibody-antigen binding and the concentration of analyte molecules present in the sample. From the parametric-fit and simulation run, it was determined that the analyte-to-signal ratio is visualized as changes to the capacitive components namely: CDL (double-layer capacitance) or CPE (pseudo-capacitance).
This EIS analysis was important to map where in the bode characteristic plot the antigen-antibody binding effects at the electrode interface can be tracked while minimizing "cross-talk" or signal noise from bulk solution behavior and non-specific binding.
The Zmod values at 10 Hz largely represented the capacitive dominant component of impedance signal to extract sensing performance from the system. Thereby, Calibration dose response curves for atrazine and glyphosate respectively as seen in Fig. 5 (left-top and bottom) was calculated by plotting the (x-axis) differential signal change from baseline i.e., sensor signal when no pesticide analyte is present in the system versus the (y-axis) concentration of pesticide in ng ml −1 . The regression factor denoted by R 2 values of almost 0.97 and 0.98 for atrazine and glyphosate respectively shows the linear behavior of the sensor system in soil run-off. To further evaluate and quantitatively capture the sensor system performance, the calibration curve equation was tested with validation samples to see what the recovered concentration value would be against the dose response behavior. A Pearson's correlation test yielded r values >0.999 cementing the sensor robustness from spiked concentration against recovered or estimated concentration values.
To establish and test the PoU capability and on-demand measurement, this developed sensor is further integrated to build a pesticide sensing ecosystem. A portable potentiostat system was used as an on-board miniaturized test platform along with integrated machine learning functionality for categorization of pesticide contamination levels. The study produced detection limit levels for atrazine at 0.001 ng ml −1 while for glyphosate it was 1 ng ml −1 from the direct sensor data perspective and post-hoc training-test with the ML algorithm whose performance metrics is seen as an ROC curve and confusion matrix plot in Fig. 6 and yielded an accurate prediction rate of 80% in real soil samples. This work was discussed in detail to understand different levels of biosensor design and testing including-sensor anatomy breakdown, mode of transduction and signal processing and data analysis cum feedback. More work on sensor platforms and microdevices are vital to ensure that soil pollution and characteristics are well characterized for an overall holistic perspective on agro-food assessment.

Water Quality Assessment
Introduction.-Water is considered a prime source of life on this planet and all the living species depend on water for existence. Statistics indicate that roughly 72% of the planet is covered with water. The interesting fact is that only 3% of all water is freshwater and only 1.2% is drinkable which illustrates the severity of water pollution. Assessment of water quality is a major task for many governments as well as health agencies. 54 There are many factors including pollutant chemicals which are the major cause of water pollution. Pollutant chemicals include industrial waste, toxic chemical spill, heavy metal pollution, pesticides etc. Electrochemistry is a branch of chemistry that deals with the transformation of chemical energy into electrical energy. Electrochemistry is a unique topic and equally intriguing to the research community for its unique applications. One of the key applications in this field is the development of Figure 5. (A) Calibrated dose-response (CDR) curve with semi-log fit and linearity >0.95 plotted as dZ which is the change in Zmod (for each dose) from baseline signal against the concentration of atrazine in ng ml −1 measured using the prototype device. (B) Correlation plot given as atrazine concentration recovered using the lab instrument on the x-axis (i.e., performance of benchtop instrument reference) vs concentration recovered using a proto device on the yaxis (test performance of electroanalytical PoU system). The table on the right shows actual vs recovered concentrations behaviour for laboratory instrument (column 2) and proto device (column 3). (C) Calibrated dose-response (CDR) curve with semi-log fit and linearity >0.95 plotted as dZ which is the change in Zmod (for each dose) from baseline signal against the concentration of glyphosate in ng ml −1 measured using the prototype device. (D) Correlation plot given as glyphosate concentration recovered using the laboratory instrument on the x-axis (i.e., the performance of benchtop instrument reference) vs concentration recovered using a proto device on the y-axis (test performance of electroanalytical PoU system). The table on the right shows actual vs recovered concentrations behaviour for laboratory instrument (column 2) and proto device (column 3). novel electrochemical sensors for molecular and biosensing applications. [55][56][57] Electrochemical sensors have several advantages including specificity, sensitivity, miniaturization, device integration, IoT capability, on-field operational ability which makes it superior compared to its other sensor platforms. Electrochemical sensors consist of 3 different parts: analyte, transducer, and electronics. Electrochemical transducer transduces chemical interactions to a meaningful electrochemical signal upon interaction with the analyte and the integrated electronics help to get the data output. 58 Electrochemical sensing platforms, powered by electroanalytical techniques integrated with proper electronics, can offer specificity, sensitivity, reliability, reproducibility, along with the ability to perform on-field operation. We have described three distinct examples for the quantification of harmful pollutant chemical species: cyanide, Cu (II) and atrazine, responsible for major water pollution. In each method, different electrochemical techniques have been used such as in situ conductometry for the detection of cyanide, potentiometry for the detection of Cu (II) and impedimetric techniques for the detection of atrazine. All of these detection strategies and results have been described in different sub sections.
Case-study: in-situ conductometric sensor for the detection of trace level cyanide in water sample.-Materials with fatal toxicity have an immense effect on the environmental and biological world. Among many of such materials present in our environment, cyanide ion is known to be the deadliest due to its extreme hazardous chemical property. It has been found out that this species has a stronger affinity towards cytochrome-c oxidase. Accumulation of high concentration of cyanide can reduce the cellular respiration in human by suppressing the oxygen transport in cell, resulting in cardiac arrest, coma and even death within a few minutes. 59,60 World Health Organization (WHO) have advised that if the amount of cyanide present in water is greater than 10 ppb, it is considered polluted and equally harmful for consumption. Other reputed environmental agencies like USEPA also have similar threshold limitations of cyanide concentration to be 12 ppb. 61 Knowing the harmful effect of cyanide to the environment, practice of cyanide utilization is still prevalent in electroplating, metallurgy, and the mining industry. As per the statistics, there is a huge production of cyanide -about 1.4 million tons each year worldwide -which is a major environmental concern. The greater the use of this chemical, the higher the probability of prolonged and toxic exposure to the environment. Moreover, the acute toxicity of cyanide causes nuisances not only for humans but for all other living organisms including plants. One of the deadliest examples of cyanide spilling from a gold mine company in Romania in 2008, caused the death of huge number of fishes due to water pollution in Hungary and Yugoslavia. 62 Considering the harmful effects of cyanide, its monitoring seems to be a priority for many agencies and even governments. There are conventional analytical techniques to quantify cyanide in water including conventional spectroscopy, inductive coupled plasma etc. 63 These techniques are costly, require sophisticated instrumentation and trained personnel which inhibits them from on-field sensing capability.
Conductivity is an intrinsic property of material that can be evaluated by normalizing the conductance with area. There are mainly two types of conductivity: electrical and electrolytic. The molar conductivity of an electrolyte solution can be found by measuring equivalent conductivity and the solution resistance, governed by the equation: where R depicts solution resistance and m Λ is the equivalent conductivity which also has a direct dependency with the concentration of the electrolyte, governed by the famous Kohlrausch's law: Λ is the limiting molar conductivity specific for each ion. 64 There are two different protocols to measure conductance by I-V method: ex situ and in situ. The Ex-situ method is based on two/four probe Van Der Pauw operation while the In-situ method is used for the materials whose conductivity is a function of its electrochemical state. The conductance is measured in situ by maintaining the electrochemical state of the material by using a Bipotentiostat. This configuration is reported as CHEM-FET and is also depicted in Fig. 7. In a typical experimental setup, it consists of two microelectrodes bridged with a one-dimensional conductor, whose conductance is a function of electrochemical potential (conducting polymer). 65 Conventional ex situ conductivity measurements include the external polymerization of the conducting polymer and directly scratching it from electrode surface, following by a measurement of conductivity using the Van Der Pauw method to determine sheet resistance. This approach has several limitations: the surface of the conducting polymer is infinite and scratching this surface causes a destabilization of the synthesized material rendering this conductivity measurement irreproducible.
Paul et al. reported the proof of concept for a simple dip and read in situ conductometric sensor for cyanide ions using polyanilinereceptor modified electrode in aqueous media. The cyanide specific receptor: 2-phenyl-1H-anthra-[2,3-d]-immidazole-5,10 dione, was directly immobilized onto the surface of electrochemically synthesized polyaniline matrix to fabricate this conductometric device. The proposed transduction mechanism is supported by chemical and computational analysis, suggesting that the charge nullification of the polyaniline species, due to deprotonation, resulted in a decrease in the in situ conductivity of polyaniline. The work has been depicted in Fig. 8 which illustrates the sensor fabrication, electrochemical response, and the theoretical explanation.
The obtained sensory response was measured in three concentration segments of cyanide: low (0.1 nM to 100 nM), medium (100 nM to 1 μM) and high (1 μM to 1 mM). The experimental data suggests that the fabricated device is less sensitive in the low concentration range of cyanide. Moreover, the sensitivity was found to be increased in the medium concentration range, which depicts that the device acted as a detector. Finally, the device showed substantial sensitivity in the high concentration range with high proportionality. The limit of detection for cyanide was obtained to be 10 nM (2.6 ppt), which is significantly lower than the threshold limit of cyanide contamination in wastewater. The obtained sensory responses were also examined under various experimental conditions such as the effect of pH and the electrochemical state of the conducting polymer. The reported sensor shows superior sensitivity at pH 7.4 and at a bias voltage of ∼400 mV vs Ag/AgCl. 66 Case-study: potentiometric ion selective electrode for the detection of Cu (II) in wastewater.-Heavy metals are a threat to nature and the environment due to their harmful side effects upon contamination. This water pollution directly affects living organism including humans. 67 Copper, a very common heavy metal, is readily found in the Earth's crust and is widely used in households, agriculture, and a variety of other industries. Copper stands after iron and aluminium in terms of its global usage and this shows a potential problem regarding its misuse and pollution. Copper is a high electrical conductor; hence it is utilized extensively in the electronics industry. Other important properties of copper include superior ductility, flexibility, thermal stability, and corrosion resistivity, which make it a valuable heavy metal. Along with the standard metal properties, copper also possesses unique chemical properties which make itself extremely useful in the chemical industry. Some of the complex procedures that involve copper are froth floatation of sulphide ores, preparation of wood preservatives, ECS Sensors Plus, 2023 2 024601 Figure 7. A schematic of a typical electrode setup usually used for in situ conductance measurement. electroplating industry, dye industry, and the manufacture of copper compounds. 68,69 This suggests that the demand of copper is huge globally which brings the argument regarding its regulation. World Health Organization (WHO) advises that the maximum allowed limit of Cu (II) adulteration should not exceed 1.5 ppm in drinking water. Moreover, as per WHO, the threshold for intake of copper for a normal adult should not surpass 10-12 mg/day in body and should not surpass 100-150 μg dl −1 in blood. Surpassing this limit of copper intake is toxic for the human body and has short and longterm implications including gastrointestinal, catarrh, hypoglycaemia, and dyslexia. Excessive accumulation of copper in the human body can also cause Wilson's disease. 70 Due to such adverse effects of copper in human health and environment, its regulation is one of the prime priorities. The regulation includes monitoring of trace level Cu (II) in wastewater and environmental samples. The current conventional tools to quantify trace amount of Cu (II) in aqueous samples include atomic absorption spectrometry (AAS), inductively coupled plasma-optical emission spectroscopy (ICP-OES), anodic stripping voltammetry, chromatography, gravimetric detection, or photometry. [71][72][73][74] On-field real time analysis of trace level Cu (II) can be an intriguing solution to this nuisance. These conventional techniques require sophisticated and heavy instrumentation, trained personnel and a state-of-the-art laboratory which inhibits their usage for on-field analysis. Ion-selective electrode, a subclass of potentiometric sensor, can be an excellent alternative to solve this problem due to its miniaturized architecture, specificity and sensitivity and its ability for on-field testing with Internet of things (IoT) capability.
Potentiometry is an ancient electrochemical technique to measure activity of ions by obtaining the equilibrium cell potential, in the absence of any input current. The technique utilizes a working electrode, also known as an ion selective electrode, made of a selective membrane to allow ions to migrate from inner to outer solution, creating a potential gradient in the junction. This potential gradient can be further measured as a potential difference with reference to a standard reference electrode. The potential difference generated due to the diffusion of the target ion is specific for that analyte and hence a calibrated dose response can be obtained accordingly. Moreover, one should be reminded that this technique is utilized to quantify activity of the ions which may be further correlated to the concentration sought to get a sensory output. As discussed previously, the ISE is made of a selective membrane which is specific to the target analyte and its basic architecture is depicted in Fig. 9. The sensor is sealed with the selective membrane, containing a specific ionophore which binds with the specific target ion and allows for the migration of ions from the inner solution to the outer solution through the membrane channel. This creates a concentration gradient, followed by a potential gradient, which can be measured by a conducting wire. The migration of ions from inner solution to the outer solution is due to the instability of the ionic activity. The ionic activity of the inner solution (a i inner ) is known and fixed, whereas the target ion activity (a i outer ) is unknown, present in the outer solution generates an electrical potential gradient, represented as: The potential difference is often termed as Donnan potential, can be measured with an additional pair of electrodes, namely the reference electrode, until the equilibrium (Donnan equilibrium) is reached. The potential difference measured across the working electrode, measured with the reference to a reference electrode is then dependent on the concentration of the outer solution through the fundamental Nernst equation seen above.
As depicted in Fig. 9, both of the electrodes contain a chamber which has the same inner solution. The conducting wire for ISE can be any conductor but preferably be Ag|AgCl, whereas the reference electrode conductor is Ag|AgCl as this couple is inert to any chemical changes in the analyte. The voltmeter measures the potential difference E n or the emf, depicted as: The equation suggests the Nernstian slope for monovalent cation is +59 mV/decade at 25°C whereas for a monovalent anion, it is −59mV/decade at 25°C. If the cation or anion is divalent, the ideal slope will be ± 29.5 mV/decade. 75 Paul et al. reported a novel Cu (II) selective membrane in polyvinyl chloride matrix prepared by mixing with 2-Nitrophenyloctylether (NPOE), as a plasticizer and sodium tetraphenylborate (NaTPB), as solvent mediator and a mechanochemically developed: Rhodamine 6 g hydrazide as Cu (II) specific ionophore. Several bulk membranes are fabricated by varying the composition for optimized sensor response. The design of the experiment and sensor responses are depicted in Fig. 10. Results obtained from the sensor response shows that the sensor is working efficiently with a superior Nernstian response of 29.38 ± 0.55 mV/ decade. The effect of pH has been studied and it has been found that the sensor response is independent of pH 3-7.5, which suggests the superiority of the optimized ion selective electrode platform. We have also calculated the limit of detection of the sensor, and it is found to be 5 μM of Cu (II), which is significantly lower than the amount of Cu (II) which can cause heavy metal pollution. The sensor also possesses a fast response time, extended shelf life and superior selectivity which makes itself highly applicable for on-field real time sensor application to detect Cu (II) in aqueous solution. The fabricated ion selective membrane is characterized by various physico-chemical techniques including Scanning Electron Microscopy to visualize the surface morphology. It has also been found that the sensor surface is reversible in nature and can be regenerated upon treatment with EDTA. The fabricated sensor has been utilized for real sample analysis after achieving a good response with the spiked sample. Real samples are prepared by digesting electronic waste such as copper wired circuit boards by acid treatment. The fabricated ion selective electrode shows good sensory response for Cu (II), present in the real sample, duly correlated with ICP-OES. The result is depicted in Fig. 10, which shows the sensor can perform in real samples with good selectivity and sensitivity and can be readily employed for the analysis of trace level Cu (II) in environment sample. 76 Case-study: impedimetric sensor for trace level detection of Atrazine in water.-Pesticides are one of the key ingredients of agricultural world. They are primarily employed towards agricultural sectors, mainly for the protection of crop from pests, insects, and other harmful entities. Pesticides plays a significant role in the crop growth and eventually the production of food by increasing the agricultural yield. Pesticides are generally chemical species and hence considered to be a toxic pollutant for both human and environmental entities. It is proven that the accumulation of pesticides in the human body has acute and chronic health implications. The most problematic feature of cheap pesticides are these species remain in soil for a long time and eventually contaminate ground water. [77][78][79] The most prominent way of intaking these species is through the vegetables and plants we consume directly. Due to these adverse effects of pesticides to human health and environment, many developed countries have implemented strict regulations to control the usage of pesticides. There are many developing countries who have banned pesticides in agriculture, but it is a cheaper way to achieve substantial production of crop. It was believed that there are more than 1000 different varieties of pesticides, currently used in agricultural purpose and each of them have its own advantage and toxicological effects to human health and the environment. 80 Among many of commercially available pesticides in the market, Atrazine (subclass of triazene) is a popular herbicide, extensively used to avert endangered broadleaf weeds, found in maize (corn), sugarcane crops and on conventional turf. Atrazine is a toxic chemical in terms of human consumption, but the use of Atrazine is very popular in major countries including the USA and it has been found that this pesticide contributes significantly to the water pollution. Due to these adverse effects of pesticides, many efforts have been made by the scientific community to detect and quantify pesticides in drinking water. 81 Most of those reports include analytical techniques which require sophisticated instrumentation and trained personnel which not only increases the cost but also inhibits its application towards on-field sensing. 81 Electrochemical sensors can be a wise choice in this aspect due to its unique properties including sensitivity, selectivity and most importantly, onfield accessibility.
Prasad et al. utilized the non-faradaic impedance technique described in previous sections to fabricate a novel biosensor platform for label free trace level detection of Atrazine in real samples. The author has used nano porous alumina membrane coated over a customized gold screen printed electrode to develop an on-chip impedimetric platform for the detection of Atrazine. Using nano porous alumina coated over the electrode surface inherently increases the effective surface area, eventually amplifying the impedance output of the fabricated sensor which helps to detect trace level of atrazine in real samples by leveraging the size-based immobilization of atrazine's small molecules. The sensor was calibrated using phosphate buffered saline and utilized for trace level detection of Atrazine in real samples such as river water and bottled drinking water samples. The result obtained from the modified electrode stack shows superior selectivity and sensitivity towards Atrazine with a limit of detection in the range of femtogram ml −1 (fg ml −1 ) (parts-per-trillion) level. The sensor also shows a  rapid response and a wide detection range from 10 fg ml −1 to 1 ng ml −1 (0.01 ppt to 1 ppm), depicting the superiority of the fabricated sensor compared to other reported sensors for atrazine. Moreover, another very common pesticide: Malathion has been utilized to understand the selectivity of the fabricated impedimetric sensor and the result shows the sensor is susceptible towards malathion with a low detection range of fg ml −1 in all the cases. 82 The work has been depicted in Fig. 11, comprising sensor design, equivalent circuit, and dose dependent responses.

Food Quality Assessment: A Portable Electrochemical Approach
Introduction.-When it comes to the food quality and its assessment, the first question that concerns to the consumer is the chemical contaminants in the food? With the alarming growing human population, requiring intensive agricultural and farming, maintaining high food quality has become a global issue and a challenge in maintaining the sustainability of the food or agro-industry. 83 The sources being the food contaminant occurring naturally or unintentionally during the food handling, during processing including use of preservatives or additives, or during the storage through the leaching of the metal containers subsequently affecting the quality of the food and the human health. 83,84 One of the most serious issues currently is about the food contaminants arising through the extensive use of pesticides for the protection of the agricultural plants. This has been associated with growing food toxin due to the pesticide's residue affecting human health. 85,86 To date, the qualitative and quantitative testing of the numerous pesticide's residue/toxins in food have been done by the gold standard analytical methods such as triple quadrupole LC/MS, GC/ MS with the high accuracy, and the detection time as fast as in ∼5-6 min. 85,87 Despite the quality data analysis, the main disadvantage of the existing analytical method is the tedious sophisticated experimentation requiring expertise in the sample processing. The methods include the extraction of the analytes from the bulk matrix and separation, which is time consuming and costly for an average consumer on a day-to-day basis. 85 Considering this, the best step would be to minimize the testing approach through miniaturization of the test system and development of portable devices which can trace a myriad of pesticides in one platform. The basic requirements of the biosensors are the capability for regular monitoring, low cost, on-field and rapid screening for food contaminants. 84 In terms of advancement in the field, the electrochemical methods are among the most widely used in deployable portable devices. 84 Electrochemical biosensor technology integrates a bioreceptor or biocomponent such as an enzyme, immunoassay or aptamer, and the response in the form of electrical signal is measured as a function of the specific interaction between the target molecule/antigen and biocomponent. 7,84 The advantages of the system's implementation are that it is highly sensitive, rapid detection with no pre-sample preparation step or training, and relatively low-cost to be utilized by an average consumer.
The enzyme-based and the affinity-based electrochemical biosensors are the two important classes of electrochemical biosensor for testing food contaminants. Biosensors are based on the inhibition of the enzyme and are reported in the detection of pesticides or mycotoxins. The inhibition activity of the enzyme in the presence or absence of the inhibitor is measured as a function of the concentration of the inhibitor or the target. 88 The commonly used enzyme includes -Acetylcholine esterase (AChE), alkaline phosphatase, tyrosinase or butyrylcholine esterase (BChE). [88][89][90] Other types of the enzyme -based biosensors include the determination of the substrate. The enzyme catalyzes the substrate chemical transformation and the generated by products are directly detected in the form of the signal. Oxidases being the most common for the detection of the biogenic amines. The bio-amines detection is highly important for assessing the quality and the freshness of the wide variety of protein foods such as fish, meat, wine, vegetables, and fruits etc. 91 Similarly, the electrochemical lactate biosensor uses the L-lactate oxidase to detect the food freshness and nitrite detection using the nitrite reductase widely used in the food industry as preservatives. All of these are examples of the biosensor based on enzymes as an alternative to the conventional laboratory-based detection method, for their low-cost, rapid detection and ease of use in on-field applications. 92 Other important class of the biosensor are the affinity-based electrochemical biosensor due to the high selectivity in terms of the complex food matrix. It uses a recognition based on the antigen -antibody, receptor -hormone and response are measured as function of the non-faradaic interaction of the target antigen to the antibody modified electrochemical transducer surface. 93 The electrochemical method employed, and signal output could be either through commonly used electrochemical impedance spectroscopy 94 or nonfaradaic chronoamperometry. 7,95 Case-study: electrochemical teardown of a trace-level pesticide detection sensor in food samples.-Based on a dive-in detail into the affinity based portable sensing approach and advancement for assessing the food contaminant or toxins into different food matrices, our previous works highlight the usage of the electrochemical affinity testing approach to detect the pesticides residue in buffer and food matrices. 7,16,94,96 The receptor-based sensing platform consists of the interdigitated gold (Au) modified with a Glyphosate antibody (Glyp-Ab) linked through a thiol-based crosslinker as shown in Figs. 12A, 12C. The advantage of using affinitybased sensors allows the recognition of the specific antigen as a function of the binding of the antigen to antibody modified electrode surface. The non-faradaic methods such as Electrochemical Impedance Spectroscopy (EIS) and Chronoamperometry (CA) helped to map the interfacial binding phenomenon as a function of the modulation of the double layer. 7,16,93 There are advantages of using affinity-based (immunoassay-based) as compared to affinityfree sensing in terms of the selectivity and sensitivity towards target antigen when application is to use at larger matrices variation. 86 In one of our earlier works, we reported the proof of feasibility study for the detection of Glyphosate and Atrazine (pesticides of two different polarity) using an affinity based sensor and was compared with non-affinity-based sensor as shown in Figs. 12C and 12D.
The biorecognition receptor-based sensing platform consists of the interdigitated gold (Au) modified with a Glyphosate antibody (Glyp-Ab) linked through a thiol-based crosslinker while the affinity-free platform consists of the poly(3,4-ethylenedioxythiophene) modified interdigitated Au substrate. Higher specific signal outputs with greater sensitivity in greater dynamic range from 0.5 ng ml −1 to 10 μg ml −1 were achieved through affinity-based sensor as compared to the affinity-free method.
As a proof of feasibility study for the immunoassay-based sensor, 16 our recent work was extended to the pesticide sample in real oat-meal sample as shown in Figs. 13A, 13B, were focused was on the detection of Glyphosate in presence of Glufosinate, a structural similar herbicide electrochemically. The signal output measured in terms of the non-faradaic chronoamperometry (CA) was tested successfully in real time oatmeal matrix which could detect the spiked glyphosate in three concentrations variations from Low (10 ng ml −1 ), Medium (1 μg ml −1 ) and High (15 μg ml −1 ) selectively responded to the target antigen in presence of Glufosinate (as shown in Fig. 13B).
In another study by our group, with the idea of expanding into field deployable device, the recptor-based sensor was tested for the pesticide spiked in various food matrices (as data shown in Fig. 13D) such as strawberry, apple, bell pepper and carrot. A sample matrix preparation protocol was developed by crushing the food samples and mixing the produce pulp to the 1:4 equivalents of the PBS solution. The pesticide spiked into the food matrices were first analyzed using the laboratory benchtop method at three concentration variations as low, medium, and high. The spiked samples data were then compared with the data generated using portable sensor sensing devices for all similar concentration variations. Both the systems demonstrated the dynamic response range from 0.01ppm to 5ppm with the limit of detection (LOD) of 0.01 ppm as shown in Figs. 13A-13D. This study demonstrated the validity of the affinitybased portable sensing which could be potentially implemented in an on-field qualitative testing device for the routine pesticide detection in our food matrices.

Limitations and Future Scope
While the different solutions proposed within this review framework holds the potential to be deployed in situ as a first-response to screening and solving for soil, water, and food quality indices and pollution/contamination understanding and thereby boosting agricultural throughput and security-there lies an immediate step that prior to commercialization and eventual deployment-the technology needs to be tested in real field/world samples since food-based products and environmental samples are complex in nature and tend to vary in terms of location and environment. This step would be needed to factor in the variability for universal usage.
There is a broader need for an AI (Artificial intelligence) and ML (machine-learning) integrated synergistic approach to create a more robust screening and quality ecosystem enabled via-a smart sensor system that can provide intuitive understanding of environmental factors for users based on a geo-spatially tagged contextual database and an on-demand quality of life "ONE HEALTH" index based on different ecological triggers (Eg: soil, water, air and food sources, among others).

Conclusions
The different works discussed in this review featured multiple sensor ecosystems designed to examine different environmental triggers using electrochemical sensing strategies that form the basis of this section on environmental biosensors. The environmental quality can be holistically assessed by monitoring quality and contamination of the following focus areas that together represent the major entities of the global ecosystem: 1. Soil system 2. Food system 3. Aquatic system Within each area of study, the intent to create a sensor framework that has been extensively probed and tested towards the use-case scenario has been discussed with topic areas including: • Design of an electrochemical sensor towards the use-case scenario in a point-of-use (PoU) manner.
• Physical and chemical characterization of the Electrochemical Sensor and feasibility study to determine capability of platform.
• Study of Electrochemical Response in various field-samples tested using the sensor device. Therefore, to summarize-this review identifies the impact of novel smart biosensors to survey environmental quality with more modern breakthroughs involving integration of IoT devices and Machine Learning (ML) feedback to traditional sensor frameworks to amplify and boost biosensor robustness in performance. This overall sensing ecosystem can be potentially deployed to assess realtime environmental impacts and quantify an environmental metric to shape future governmental policy. Comparison of the Glyphosate dose response obtained using non-faradaic chronoamperometry (CA) method generated through the laboratory-based benchtop method (X-axis) and compared to the portable device at Y-axis (ElectrochemSENSE). The signal response corresponding to both the measurements were recorded at low, MRL, and high doses for all samples matrices apple, strawberry, bell pepper and carrot samples respectively. 95