CHAPTER 1: The Design and Fabrication of Disposable Sensors: an Overview

Biosensors are devices that are used to quantify the presence or concentration of bioanalyte(s) (viz., biomolecules, biological structures, microorganisms etc.) Usually, such devices contain immobilized biomaterials (enzymes, antibodies, nucleic acids, hormones, organelles or whole cells), transistor components and electronic systems such as signal amplifiers, processors and displays. The immobilized bioreceptors selectively interact with a bioanalyte of interest and the reaction takes place. The reaction is measured using biotransducers that produce measurable physical, chemical or electrical signals. There are a number of biosensors for the detection of various analytes including glucose, lactate, pyruvate, cholesterol, ethanol, creatinine, glutamate, choline and acetylcholine. They have applications in various fields such as healthcare, food, industrial and environmental.^1,2  According to IUPAC, biosensors are defined as devices capable of providing quantitative/semi-quantitative information using biological recognition.^3–5  The role of these biosensors are effective in various fields and particularly in healthcare applications such as monitoring, screening of diseases, pollution control, environmental monitoring etc.^3,6,7 

However, some of the devices are classified as sensors, which are slightly different from biosensors. They are able to produce an output with respect to its specific physical quantity (input) (or) they can convert signals from energy to electrical in the absence of any biomaterial. Some of the common sensor examples are temperature, humidity, light, colour, etc. These are helpful in environmental applications and energy conversion applications.^8–16  However, they are less common in healthcare applications.

The major difference between sensors and biosensors is: (i) sensors are able to produce a signal according to the concentration of a specific compound in a complex system whereas, (ii) biosensors use biologically derived components for the transduction process. (e.g. enzyme electrodes). Recently, however, the term ‘biosensor’ has been applied to any sensor that can measure the chemical concentration of a biological system.¹⁷ 

For a matrix to become a successful electrochemical biosensor, it should obey all the required characteristics of a biosensor which are discussed below.

A biosensor should have a wide linear range for the detection of high substrate concentration.

Sensitivity can be defined as the value of the electrode response per substrate concentration. It should be greater for a better electrochemical biosensor.

An electrochemical biosensor should yield signal selectively for a single analyte. Interference from foreign molecules other than the required bioanalyte must be avoided/minimized to obtain a reliable result.

This is the time requirement of a matrix for having 95% of the response. It should be as low as possible to produce a faster response.

There are a number of analytical tools such as chromatographic methods, spectroscopic analysis, and luminescence available. However, these involve discrete sampling followed by laboratory analysis. These are expensive, time consuming and does not provide high-resolution data. However, electrochemical measurements provide excellent sensitivity and selectivity, continuous reliability and portability. Therefore, they are identified as superior analytical tools.^18–21 

The desired biomolecules such as enzyme, hormones, antibodies etc. are immobilized via physisorption/chemisorption, which will cause an intimate contact with the transducer. The analyte of interest will selectively bind with the biomaterial that produces the measurable electronic response (Figure 1.1).

Based on working principles, electrochemical sensors are classified as follows:

• Potentiometric biosensors

• Amperometric biosensors

• Conductometric biosensors

• Impedemetric biosensors

• Piezoelectric biosensors.

Potentiometric biosensors are traditional sensors used to determine the concentration of the analyte molecule. Ion-selective electrodes are the best examples of potentiometric biosensors. They function based on the working principle of a galvanic cell, which consists of a reference electrode, ion-selective membrane and a voltmeter. The composition of the membrane is designed to produce the potential with respect to the ion of interest. As a result, selectivity can be achieved. The driving force for the analysis is the movement of ions towards the specific site of a membrane due to a concentration gradient. This phenomenon of mobility of ions leads to a potential difference during the analysis. This potential is measured with a standard reference electrode. The difference depends on the activity of a particular ion. The most common potentiometric device is a pH meter. Potentiometric biosensors are also used to transduce a biological reaction into an electrical signal. They are able to produce signals in the concentration range of 10⁻⁸ to 10⁻¹¹ M. The signal is measured at zero current. These biosensors have an enzyme membrane that surrounds the probe from a pH meter.

The equilibrium potential of the electrode can be related to the concentration of the analyte, which is governed by the Nernst equation (eqn (1.1)):

where E_eq is the equilibrium electrode potential, E° is the standard electrode potential, n is the number of electrons involved in the reaction, M ↔ Mⁿ⁺ + ne⁻, [M] is the activity of the analyte ion in the solution, R is the gas constant, T is the temperature in Kelvin and F is the Faraday constant (96 485 coulombs). Since, E°, R, T, n and F are constants, the activity coefficient is also kept constant (eqn (1.2)).

Therefore, potentiometric sensors are very attractive for field operations because of their high selectivity, simplicity and low-cost methods to determine the ionic concentration through potential difference measurements. pH electrodes and other various ion-selective electrodes are examples for potentiometric sensors.^22–24 

Amperometric biosensors work by producing a current when a potential difference is applied between the electrodes.^25,26  These are similar to potentiometric sensors with respect to response time and sensitivity. Clark oxygen electrodes are the simplest form of amperometric biosensors. This experiment follows the reduction of oxygen at a Pt working electrode with reference to Ag/AgCl electrodes at a fixed potential and the sensor is able to produce linear signal concerning the concentration of oxygen.²⁷  Here, the Pt working electrode is immobilized with GOx. The co-factor of GOx, FAD catalyzes the oxidation of glucose. During the course of the reaction, the liberation of hydrogen peroxide will be measured amperometrically and the measured current will be directly correlated with the concentration of glucose (eqn (1.3)–(1.5)).

Amperometric measurements are found to be superior to potentiometric sensors in terms of high sensitivity.^26,28  Therefore, a number of amperometric sensors has been developed for the detection of various bioanalytes such as human chorionic gonadotropin β-subunit (β-HCG) in advanced pregnancy testing²⁹  and adenosine-5′-triphosphate (ATP).³⁰ 

Conductometric biosensors usually measure the change in conductivity during the course of a reaction. Most of the reactions involve a change in composition. Therefore, conductometric biosensors are able to detect a relative change that is taking place in the solution.³¹  Due to the non-specificity and poor signal-to-noise ratio, these sensors are less common in biological applications.³²  However, some researchers have functionalized polymers such as polyaniline (PANI) and polypyrrole(PPy) with biomarkers and used them to analyze cardiac biomarkers such as Myo, cTnI, CK-MB, or BNP and foodborne pathogens.^33,34  During the process of functionalization, there will be a covalent attachment between PANI and antibody, which will cause the change in conductance, capacitance and impedance.^35,36  In addition, many of the enzyme-based reactions like urease and biological membrane receptors are monitored by ion conductometric/impedemetric devices using interdigitated microelectrodes.³⁷ 

Impedemetric biosensors use impedance (Z) or its components such as resistance (R) and capacitance (C). The electrochemical representation of impedance can be given as eqn (1.6):

In these biosensors, two electrodes are used with applied alternating voltage, amplitudes from a few mV to 100 mV. They are helpful in microorganism growth monitoring due to the production of conductive metabolites.³⁸  A false positive result is the major disadvantage of these biosensors. However, there are some promising approaches that are associated with impedemetric biosensors. Hybridization of DNA can be amplified by a polymerase chain reaction that can be monitored by impedemetric analysis.³⁹  Polypyrrole film with captured avidin connected through biotin to anti-human IgG was able to detect antibodies as low as 10 pg mL⁻¹ present in a sample.⁴⁰  The ethanol level in some alcoholic beverages was evaluated using an impedance biosensor with immobilized yeast.⁴¹  The impedance-based commercial device Malthus 2000 was used for an assay of the pathogenic fungus Ichthyophonus hofery⁴²  and Erwinia carotovora rot.⁴³ 

Piezo-electric biosensors are based on the working function known as the piezo-electric effect. The devices use gold to detect the specific angle at which electron waves are emitted when the substance is exposed to laser light or crystals such as quartz. They vibrate under the influence of an electric field. The absorption of the analyte leads to an increase in the mass of the crystal, which can alter the frequency of the oscillations. These biosensors can be utilized for the measurement of ammonia, hydrogen, methane, carbon monoxide, nitrous oxide and other organophosphorous compounds.

Electrochemical measurements are one of the most powerful analytical tools among other methods. This is because of the salient features of electrochemical measurements such as enhanced sensitivity, excellent selectivity, continuous reliability and portability. The major cause of the extraordinary behaviour of electroanalytical tools is the electrodes used in the system. This is because the surface of the electrodes itself can act as an excellent tool for the measurements. They can be made to act as a source/sink of electrons by controlling the potential. Electrons crossing the electrode–electrolyte interface can be determined with great sensitivity using the electrodes.⁴⁴  Therefore, they have been utilized in different fields such as biosensors, batteries, corrosion control and its prevention, food industry, medicine industry, etc.^45,46  For example, Hg was identified as an attractive material for electrochemical measurements. Because of the salient features of Hg, such as high reproducibility, renewable and smooth surface, dropping, as well as hanging Hg electrodes, found their place in polarography and other electrochemical techniques. Afterwards, due to the improvement in technology, and the toxic nature of Hg, the invention of non-mercury electrodes such as glassy carbon and other metal electrodes began. The advantages of such electrodes include low-background current, wide potential window, and chemical inertness. Consequently, they are suitable alternative candidates for electrochemical measurements.

Miniaturization of the electrochemical system/electrodes from the macro electrodes will be a milestone in the area of electrochemical biosensors. This is because it offers a number of advantages such as low sample requirement, commercialization, economical, portability and on-the-spot analysis. Therefore, their research interest has been extended towards the miniaturization of the system. It yields the technique called “thick film technology” where one or more layers of special inks is deposited over inert and flexible supports such as paper, stress ball, wearable cloth etc.^2,47,48  As a result of this technology, people have arrived at a method for the deposition of the film called “screen printing”. In such systems, all the electrodes namely reference, working and counter electrodes are printed on a single platform as shown in Figure 1.2. These electrodes play a vital role in electrochemical sensor applications. They can be inserted into the portable systems and used for on-site environmental monitoring.²  The most critical step in the fabrication of screen-printed electrodes (SPEs) is sensing and the adhesion of the active membrane on the surface of the inert platform.

There are a number of methodologies for the fabrication of the electrodes. These include wet etching, reactive ion etching, conventional machining, photolithography, soft lithography, hot embossing, injection molding, laser ablation, in situ construction and plasma etching. The method selected for fabrication depends on the requirement and availability.

Typically, an SPEs/thick-film biosensor platform can be fabricated on a conducting pad. Since carbon is inexpensive and chemically inert, it is most widely used in the form of an ink or paste along with silver ink in a basic three electrode screen-printed system. The choice of the base material for the fabrication of disposable biosensors varies depending on the requirement. Because of features like plasticity, hydrophobicity, dielectric and insulative properties, thermal stability, etc., flexible polymer substrates like polyimide (Kapton), polyethylene naphthalate (PEN), polyethylene terephthalate, (PET), polytetrafluoroethylene (Teflon) have been used as substrates for the fabrication of these electrodes. In addition to the polymer substrates, carbonaceous material like graphite substrates are also used along with the polymers. Plastic substrates like Mylar, PEN and Kapton were utilized in connection with graphite. Further, paper-based electrodes are a remarkable achievement during the development of disposable biosensors. They have been used for various applications including biochemical analysis, immunoassay, poit-of-care diagnosis, etc. However, in some occasions, gold ink can also be used as the material, since it can facilitate the formation of self-assembled monolayers (SAM). Such electrodes are identified as effective candidates in electrochemical biosensors. To improve the adhesion of the ink on the substrate, mineral binders/polymers are added to the carbon ink.⁴⁹  Therefore, people have focused towards the modification of the electrode surfaces. The main drawback associated with such polymers are that it increases the electron transfer resistance. Hence, the kinetics of the reaction become sluggish.⁵⁰ 

The as-prepared electrodes are not able to produce signal against all the molecules. Therefore, they need to be modified with the suitable mediators. The modification of the electrodes makes the surface suitable for a particular function. There are a number of mediators that can be used, such as carbonaceous materials (nanotubes (CNTs), graphene and its derivatives, carbon black); metal nanoparticles (gold (GNPs), silver (AgNPs) and magnetic beads); and mediators (prussian blue (PB), crown ethers, Meldola's blue and cobalt phthalocyanine) which are used for the modification of redox mediators. Polymers are mainly used as modifiers^1,50  which improve the electrocatalytic property of the electrode. Therefore, the electrodes can be referred to as derivatized, polymer-modified, functionalized and electrochemically bound surfaces. Most probably all the electrodes are conductive. They can make the electron transfer between electrode surface and the substrate that has to be oxidized/reduced. The electrodes can be prepared by different methods, such as:

• Adsorption

• Covalent film forming

• Polymer film coating

• Composites.

In this method, the chemical species is adsorbed on the surface of the electrodes due to the valence force operating on the chemical compounds. The adsorption may either be physisorption or chemisorption. Physisorption is a process taking place at the solid–liquid interface without the occurrence of any chemical reaction which includes electro-deposition of metals and conducting polymers. The phenomenon of chemisorption occurs at the solid–liquid interface by performing a chemical reaction. The layer formed on the surface of the electrode is called the chemical layer. The layer is adsorbed on the electrode strongly and irreversibly. By using this adsorption phenomenon, one can form a monolayer on the surface of the electrode. In another way, the electrodes can be dipped into the solution for the assembling of chemicals on the electrode. These layers are called self-assembled monolayers (SAMs). Aromatic compounds, olefins, long chain aliphatic hydrocarbons can form these kinds of chemically modified electrodes. Extensive research has been carried out for the chemisorption of organic molecules on the electrode surfaces like Pt,⁵¹  Si,^52–54  and SnO₂.⁵⁵  Among them Au and carbon surfaces are able to bring new advancements in molecular electronics and electroanalytical chemistry.⁵⁶  Recently, a number of modification strategies have been attempted for carbon substrates such as activated carbon, graphite, glassy carbon, carbon nanotubes (both single and multi-walled) and graphene due to their abundance and accessibility.⁵⁷ 

The adsorption of sulfides on gold substrate has gained enormous interest because of the simple modification procedures.⁵⁸  A simple pictorial representation of SAM on an Au surface is shown in Figure 1.3. Many attempts have been employed to identify the exact condition for self-assembly like surface topography and surface roughness.⁵⁹  However, a standard procedure for controlling the self-assembly process is still ambiguous. Owing to the latter fact, this method often considers as less controlled approach for attaching organic molecules to the surface. Interestingly in 1998, Rubinstein group showed the feasibility of self-assembling of both mono and disulfides on the Au surface by electro oxidation of the surface.⁶⁰  The interfacial properties of thiol-modified surfaces are highly influenced by packing density, orientation and the tail groups present in the organic chain. To acquire the necessary benefit from the modified films, particularly in electrochemical sensing and electrocatalytic applications, tailoring of the electrode surface with a suitable tail group is mandatory.⁶¹ 

In case of SPEs, most of them are enzyme functionalized and are used for biosensor applications. The enzymes are immobilized via physisorption on the surface. The method for the modification of the electrode has been discussed in detail.⁶²  The electrodes are developed by beginning with a conducting pad (T) where the carbon ink or paste (C) and Pt or other metals will be coated. A mediator (M) or catalyst can be added on the substrate (S). Over the substrate, the enzyme (E) will be immobilized along with the entrapment agent (m) or cross linker (c) and a stabilizer or additive (s). Co-factors can also be added along with the enzymes. The layers are deposited individually and a multiple layer is obtained. It can be represented as follows: S-1TM-2Ecs-3L. Here, the numerical represent the individual steps during the fabrication process. The capital and small letters refer to the principal (enzyme, mediator, transducer, selector layer, etc.) and specific components (cross-linker, immobilization matrix, stabilizer etc.), respectively. Individual layers are separated by a hyphen. More clear information can be given in brackets. Hence, the given notation can be given as S-1TM-2Ec(g)s-3L. It can be explained as follows. At first, TM of the transducer is mixed with mediator and immobilized as a film on the substrate, S. Afterwards, the second layer is 2Ec g s (enzyme E is mixed with the glutaraldehyde cross-linker c g and a stabiliser or additive) is dropped on the first layer. Finally, a selector layer 3L is developed on it.

For example, the notation S-1T(C)M(Ru)-2EChE was used to represent the deposition of choline oxidase onto a screen-printed electrode with ruthenised activated carbon and applied over a polyester flexible film.^63,64  This biosensor was developed for the detection of pesticides.

The formation of layers on the surface of the electrode due to covalent attachment will fall in this kind of chemically modified electrodes. Connecting agents such as organosilanes and cyanuric acid can be used to attach from one to several monomolecular layers of the chemical modifier to the electrode surface. G.H.A. Therese et al., have surveyed most of the electro synthetic routes that control the film thickness and morphology of oxides and hydroxides.⁶⁵  Metal oxides like nickel oxide, copper oxide, ruthenium oxide, manganese oxide and bismuth oxide are reported for their electrocatalytic activity towards sensor applications.^66–69  Among them, the role of bismuth oxide is remarkable in the detection of various analytes^67–68  (Figure 1.4).

Organic/inorganic/organometallic polymer films can be used for electrochemical biosensor applications. The polymer modified electrodes can be functionalized so that the desired surface can be achieved. The process is achieved either by physical anchoring or chemisorption. The polymers can either be conductive or nonconductive. Conducting polymer complexes of tetra-cyano, tetra-oxalato platinates and the kragmon salts charge transfer complexes are known in 1971.⁷⁰  However, research on conducting polymers has been recognized as vital after the discovery of poly(sulfur nitride) [(SN)x] in 1975.⁷¹  The significance of conducting polymers has been known since the discovery of polyacetylene (PA) in 1977.⁷²  Simple doping of oxidizing agents such as I₂, AsF₅, NOPF₆ (p doping) or reducing agents e.g. sodium naphthalide (n-doping) has increased the conductivity of the polymer by 10⁵ S cm⁻¹. Poly-paraphenylene was also identified as an important conducting polymer in 1979.⁷³  Doping of the polymer with AsF₅ increases its conductivity from 10⁻⁵–500 S cm⁻¹. Poly(3,4)ethylenedioxythiophene (PEDOT) is also established as an important conducting polymer for electrochemical biosensor applications.^74–77  In addition to them, polypyrrole (PPy),^78–81  polyaniline (PANI),⁸²  polycaffeic acid,^83,84  etc., have been reported for their extraordinary performance against various (bio)analytes.

Polymer film modified electrodes can further be subdivided depending on their preparation methods. The following methods are involving in the preparation of polymer modified electrodes:

• Dip coating

• Solvent evaporation

• Spin coating

• Electrochemical deposition

• Electrochemical polymerization

• Radiofrequency polymerization

• Cross-linking polymerization.

In this method, the electrode is dipped into a solution of polymer for a definite period of time until the polymer film is coated on the surface. A major drawback associated with this method is that the thickness of the film cannot be controlled.

In this method, the polymer is drop-casted on the surface of the electrode and the solvent is allowed to evaporate. The main advantage of this procedure is that the surface coverage is visible immediately after deposition.

In this procedure, a drop of the required polymer is placed on the surface of the rotating electrode. Any excess of polymer solution is removed and a thin polymer film forms over the surface of the electrode. Multiple layers can be added using the same procedure until the desired thickness is obtained.

Electrochemical deposition is also known as redox deposition. This deposition depends on the stability of the polymer and the stability depends on its ionic state. In this method, an irreversible and stable polymer film is formed.

In this procedure, electrochemical cycling of the electrode in the solution of a monomer leads to its activated state. The activated molecules combine together to form a thin polymer coating on the electrode surface.

In this phenomenon, vapors of the monomer are exposed to radiofrequency (RF) plasma discharge. Different functional groups can be derived on the surface of the electrode due to the chemical damage caused by the highly energetic RF discharge.

Chemical components of a film are combined with the electrode to bring the required properties such as stability, desired permeability and improved electron transfer characteristics. Bi/polyfunctional monomers leads to the formation of cross-linking polymers.

Here, the modifier is mixed with the electrode material as a catalyst along with carbon materials and binder, e.g. carbon paste electrode. In addition to carbon,^85–87  clays,^88–90  zeolites,^91–93  and polymer composites^94–96  were also tried as electrode materials for the detection of various bioanalytes. Therefore, the electrodes can be modified in several ways. They can be utilized for different electrochemical applications such as electrocatalysis, corrosion prevention, electrochemical sensing, etc.

In addition to the mediator modification, pre-treatment is another method that can be used to improve the electrocatalytic properties of the electrode. The method focuses on the removal of contaminants and organic ink constituents. Further, it will improve the surface roughness and functionalities. Therefore, a pre-anodization method, potential cycling method and a two-step pretreatment method which combines chemical treatments and electrochemical treatments has been developed.

Because of its salient features such as low cost, ease of availability, non-toxic nature etc., researchers have concentrated their attention towards carbon materials during the development of screen printing technology. CNTs and graphene are referred to as the primary electrocatalysts for a number of electroanalytical techniques. Compton's group discovered that defects/edge-plane like sites in CNTs enhanced its electrocatalytic nature, which was compared with edge plane pyrolytic graphite electrodes. The role of impurities in/on CNTs was also investigated. Consequently, it was clearly observed that edge plane pyrolytic graphite electrodes produce exact results when compared to CNT.^97,98  Prof. Zen and his group have found that oxygen functionalities on the surface will also affect the electrocatalytic properties of the SPEs along with defects/edge-plane sites.⁹⁹  A comparative study on the role of oxygen functionalities and edge plane sites was done by Prasad et al.¹⁰⁰  As a consequence of the inventions, it is concluded that the pre-anodized SPEs are efficient towards electrochemical sensor applications such as reduction in overpotential, enhancing sensitivity, selectivity, etc.¹⁰¹  Such electrodes are used for non-enzymatic detection of analytes, which are poorly electroactive. The anodization can be performed at the applied overpotential of 2 V vs. Ag/AgCl reference electrode for suitable time in different electrolytes such as phosphate buffer solution (PBS), H₂SO₄ and NaOH under stirring conditions. The electrodes were characterized using X-ray photoelectron spectroscopy (XPS), Raman and scanning electron microscopy (SEM) to confirm the formation of edge plane and oxygen functionalities. Difference in the D band intensities of Raman spectra for screen-printed carbon electrode (SPCE) and oxygen plasma treated screen-printed carbon electrode (OSPCE) confirms the defect/edge plane like sites. Pre-anodized electrodes are found to be effective when compared to OSPCE for the simultaneous detection of ascorbic acid, uric acid and dopamine.⁴⁸ 

Biomolecules/biological molecules are essential organic compounds present in all the living systems. They are the building blocks of the body and are responsible for maintenance and metabolic process. The major classes of biomolecules include carbohydrates, proteins, lipids, nucleic acids, enzymes, hormones, etc. Each class are vital in the day-to-day activities of every living organism. Therefore, periodic monitoring of their levels in human systems is essential. Because of the absence, as well as excess, of biomolecules in human systems, people face a number of biological disorders such as Alzheimer's disease, Parkinson's diseases, diabetes, heart attack, pregnancy complications, osteoporosis, etc. This leads to a decrease in the average life-span of a living system. Therefore, the development of biosensors for the detection of bioanalytes is essential to find out their levels in every human. Hence, disposable electrodes were tried for the detection of various bioanalytes. As a result, it is observed that the pre-anodized SPE was found to be effective in yielding an electrochemical signal with respect to the concentration of various bioanalytes including ascorbic acid, uric acid and dopamine, in human blood samples. It is well documented that these disposable electrodes are able to produce responses against uric acid in human blood samples in the presence of ascorbic acid. The results are well-correlated with the clinical serum analysis by the phosphotungstic acid method.¹⁰²  In addition, the electrodes were found to be an efficient candidate for the detection of creatinine.¹⁰³  They were further used to detect melamine, which is a poor electroactive compound.¹⁰⁴  The role of these electrodes were further extended towards glucose oxidase where the direct electron transfer was improved.^105,106  Disposable SPEs are also reported for their efficient electrocatalytic properties towards lactic acid.¹⁰⁷ 

In addition to biomolecule detection, the estimation of food contaminants are also playing a crucial role in healthcare. This is because people's food culture is changing from traditional to modern. The hygiene and contamination level of modern food must be checked periodically to know the quality of the food. Okadaic acid, brevetoxin, domoic acid and tetrotoxin are some of the low molecular weight marine toxins that are ingested in seafood materials. Such toxins cause poisoning in them. Immunosensors have been developed for their detection using disposable electrodes. It is identified that the biosensors are able to detect the toxins with high accuracy (in ppb level). To monitor the foodstuff, people have targeted the analytes like acetaldehyde, d-lactate and l-lactate.^108,109  Biosensors based on screen-printed electrodes were developed for the detection of lactates and acetaldehyde in wine.^110–112  To monitor the ethanol level in food material like beer and wine, ethanol biosensors were developed.¹¹³  In addition, food allergies also affect the human population to a certain level. The allergens are proteins obtained from eight main groups. They are nuts and tree nuts, fish, shellfish, wheat, soy, eggs and milk. They are the major reason behind number of health risks involving IgE-type antibodies.¹¹⁴  For example, proteins obtained from eggs, milk, wheat or fish gelatin are identified as fining agents in the wine industry, which may produce an allergic reaction in consumers.¹¹⁵  Therefore, monitoring the nutrients and screening of food materials for its quality is essential for food safety and security. Antibody-based sensors, genosensors and molecularly imprinted polymers (MIPs) are exposed for the detection of food allergens. The detection of lysozyme, Ara h 1, gliadin and conglutinin (lupin) allergens are achieved using antibody-based assays as aptamers.^116–121 

Besides the detection of biomolecules and food contaminants, the determination of pesticides in soil and agro products is also vital. The development of agriculture and its technology began from the time of its birth. To achieve improved cultivation, farmers started to use pesticides. In the past, farmers used organic manures, however later, they moved on to the use of harmful synthetic/chemical pesticides. These fertilizers should only be used in minimal amounts (ppm/ppb level) but, are often used in large quantities without caution. This has made agro-products unsafe for consumption where such large quantities of synthetic pesticides are used. The accumulation of pesticides into the vegetables lead to a poisoning effect. These synthetic pesticides also have a direct impact on the soil as well as the agro-products. Continuous usage of the pesticides makes the soil uncultivable and provides poor-quality vegetables and fruits causing a significant effect on human health. Pesticides in the cultivated agro-products can induce a number of diseases such as asthma, diabetes, birth defects, reproductive dysfunction, etc. They do this by suppressing the activity of many enzymes in the human system. Therefore, monitoring the level of pesticides in soil, water and cultivated agro-products is one of the best ways to detect the abuse of pesticides.¹²²  Enzyme modified SPEs are playing an extraordinary role in the detection of pesticides. The immobilized enzymes interact with the substrates and leads to the formation of electroactive species. Commonly affected enzymes are acetylcholinesterase (AChE), butyrylcholinesterase (BChE), organo phosphorous hydrolase (OPH), and Tyr.¹²²  Afterwards, the enzyme comes in to contact with the inhibitors, and its activity is diminished. So, amperometric detection of pesticides depends on the reduced enzyme activity.¹²³  The ability of the sensor platform depends on how the enzyme is immobilized. The methods involved in the immobilization are cross-linking, physical entrapment and sol–gel.^123–127  The main drawbacks associated with the methods are non-renewability of the electrode surface and under harsh conditions, enzymes may get denatured. To overcome the problems researchers have been focused towards the application of magnetic materials in pesticide sensors. This added an excellent improvement such as renewable electrode surface and better microenvironment in the electrochemical sensing of pesticides. A platform containing hybrid core/shell biomagnetic glasses having Fe₃O₄ and a silica shell was used to immobilize AchE.¹²³  A paraoxon sensor has been developed using SPE containing AchE. In addition to Fe₃O₄, Au containg Fe₃O₄ is also employed for the immobilization of AchE. The resulting platform improves the stability of the matrix.¹²⁸  E-coli immobilized SPEs were successfully employed for the detection of methyl parathion.¹²⁹  Cobalt phthalocyanine modified SPEs were used for the detection of organophosphate pesticides.¹³⁰ 

Water is one of the important basic needs of every living organism. Though 76% of the globe is covered in water, the availability of fresh water is only about 1%. The fresh water systems are continuously polluted by human activities such as industrialization, deforestation, and the dumping of trash materials. Because of the pollution to rivers, streams, canals and lakes, people face a number of health risks like malaria, tuberculosis, etc. Dumping of waste causes numerous changes such as change in pH, reducing the dissolved oxygen, introducing chemical contaminants such as nitrites, phosphates, etc.

To identify the pH of aquatic systems, researchers have used glass electrodes for a long period of time. The electrodes are highly unstable and they need continuous calibration. Therefore, researchers are interested in developing an alternate platform for the detection of pH. As a consequence, a successful printing process yields a three electrode system that is capable of pH sensing with improved sensitivity. The application of the electrode was extended towards the electrochemical sensing of hydroxide ions using nickel oxide.^131,132  The concentration of dissolved oxygen is an another parameter that defines the quality of the water samples. Cadmium sulfide modified electrodes have been used as an electrochemical sensing platform for the detection of concentration of oxygen using electrochemiluminescence (ECL) method.¹³³ 

In addition to pH and dissolved oxygen, the determination of nitrites and phosphates in water have also gained reasonable attention due to their toxicity. However, it is very difficult to detect nitrites using conventional electrodes due to the requirement of a large overpotential. Therefore, the extraordinary features of the SPEs were extended towards the detection of nitrites and phosphates in water. Consequently, MnO₂ SPEs were identified for the effective detection of nitrites.¹³⁴  A microelectrode combined with screen printing technology also yielded a novel sensor platform for the detection of nitrites. The platform reduces the iR drop and improves the mass transport. Hence, a rapid response was obtained at a steady current potential.¹³⁵  Without any modification, a screen-printed shallow recessed graphite microelectrode array can act as a better nitrite sensor at lower concentrations in river water.¹³⁶ 

Usually, ion-selective electrodes (ISE) are used for the detection of specific ions in water samples. Because, they can measure various parameters in turbid and coloured samples. Due to their hydrophilic nature, phosphate ions are very difficult to detect. Therefore, three type of electrodes were constructed for the effective detection of the ions. They are screen-printed, carbon paste and conventional PVC electrodes. Among the electrodes, SPE based ISEs are functioning well with a limit of detection of 4 µM L⁻¹.¹³⁷ 

Further, a disposable amperometric biosensor was developed using glutamate dehydrogenase, 2-oxoglutarate and NADH for the determination of ammonium ions.¹³⁸  The electrode was found to be effective in detecting ammonium ions in river water. A similar biosensing platform was commercialized for the detection of ammonium in sewage samples.¹³⁹  Biosensors based on sulfite oxidase with cytochrome-c have been developed for the detection of SO₃²⁻ and SO₂.^140,141 

The presence of phenolic compounds and their derivatives in drinking water is a dangerous issue. This is because phenolic compounds can penetrate the skins of animals and plants, and are toxic.¹⁴²  Therefore, it is very important to develop an electrochemical biosensor for their detection. The investigation of phenolic compounds has been reported using non-pretreated SPEs using direct electron transfer properties.^143,144  Some researchers found the difficulty that some of the oxidized products of phenols will hinder the surface of the electrode and makes it inefficient against the analytes. To overcome this, surfactants have been used. The cationic surfactant, cetyltrimethylammonium bromide (CTAB) was used to determine bisphenol A in water and sewage samples.¹⁴⁴  During the detection of isomers of phenolic compounds and their derivatives with non-pretreated SPEs, the peaks overlap. Hence, the pre-treatment was used. As a result, organic binders in SPEs is removed and graphite particles are exposed with an increased surface. Such pre-treated SPEs were used for the detection of catechol, hydroquinone and aminophenol in river water with well-separated peaks.¹⁴⁵  During the detection of aminophenol, three well-separated peaks were obtained for its isomers present in the mixture.¹⁴² 

In addition to the pre-treated SPEs, modified SPEs are also playing interesting role in the detection of various organic contaminants in water. Because of their enhanced sensitivity and selectivity, they have unique electronic, catalytic and chemical properties.¹⁴⁶  An SPE coated with multi-walled carbon nanotubes (MWCNT) and Au nanoparticles is able to detect three types of dihydroxybenzene isomers.¹⁴⁷  Further, enzymes have also been employed for the detection of phenolic compounds in water.¹⁴⁸  Polyphenol oxidase enzyme, laccase and tyrosinase can be used as catalysts in the reaction where benzenediols and phenols are oxidized to quinones and other radicals.¹⁴⁹  During the development of the enzymatic biosensors, immobilization is very important to prevent the enzymes from denaturing and leaking. To avoid these effects, enzymes are immobilized using the entrapment method. Polyazetidineprepolymer (PAP) and polyvinyl alcohol photopolymer are the immobilizing agents used to immobilize the enzymes. The fabricated SPEs were found to be effective against phenolic compound analysis.^150–152 

Electrodeposition is also another method to immobilize the enzymes. Electrodeposition of polyphenol oxidase (PPO) was done using Bi³⁺ on the MWCNT modified SPE. In this matrix, no binder was used. The matrix was successfully employed for the detection of phenol.¹⁵³  Electrochemical polymerisation and deposition of PPO, 1,2 diaminobenzene (DAB) and a mediator was performed on a screen-printed Pt electrode for the detection of DAB.¹⁵⁴ 

In addition to pH, nitrites, phosphates and other ions, and organic pollutants, the level of heavy metals also defines the quality of water. The presence of excess heavy metals in water is directly related to life-threatening diseases such as mental illness, brain damage, cerebral convulsions, chromosomal damage, etc. Since the human system can easily absorb the metals, their presence in low concentration is also very harmful. Therefore, heavy metal detection is vital. Due to their neurotoxic nature, the detection of Pb²⁺, Hg²⁺, Cd²⁺ and As³⁺ has gained severe attention among other heavy metals.¹⁵⁵  Stripping voltammetric techniques are recognized for the detection of heavy metals. When SPEs were combined with the stripping technique, the combination is efficient in on-the-spot analysis of heavy metals in the environment. The analysis yields rapid, sensitive, inexpensive and accurate results. Modified SPEs are playing a crucial role in their detection. Especially, Au, Hg, Ag, Bi and other materials modified SPEs are effective in terms of sensitivity and selectivity for the detection of heavy metals.^156–161 

To detect Pb with enhanced sensitivity, carbon, bismuth, gold and other materials modified SPEs were proposed.¹⁶²  Since it is ecofriendly, the performance of Bi-modified electrodes was found to be useful in environmental monitoring. They can detect Pb at the ppb level.¹⁶³  Other advantages of the electrodes involve wide negative potential window and not requiring dissolved oxygen.^68,164,165  However, Bi electrodes are associated with some limitations. Bi can easily undergo hydrolysis in neutral or alkaline medium.⁶⁸  In the case of Hg, analysis can be performed in a wide pH range.¹⁶⁶  Since the mercury has high affinity towards metals, the experiments can yield an improved sensitivity with low detection limit. Hence, mercury is used as a modifier for SPEs towards the heavy metal analysis.^166–168  However, its toxicity limits the usage. SPEs modified with the thin film of mercury was utilized for the detection of Cd²⁺.

Mercury is another toxic and bioaccumulative heavy metal. Accumulation of mercury in humans leads to severe health problems in the kidney and respiratory systems, and other organs.¹⁶⁹  Since gold has very good affinity towards mercury, bare as well as modified gold electrodes are efficient candidates for the detection of the metal.¹⁷⁰  However, due to the formation of amalgam, structural changes take place. Therefore, cleaning is highly essential to reproduce the results. To rectify the problem, commercial SPEs are successful towards the electrochemical sensing of Hg.^171,172 

Arsenic is also known for its toxicity. Drinking water containing As leads to poisoning/arsenicosis. Screen-printed electrodes modified with Pt nanoparticles and citrate capped Au nanoparticles are proposed for its effective detection. They can detect As³⁺ without any interference from Cu²⁺.¹⁷³  In addition to the metal nanoparticles, enzymes are also used for the effective determination of the heavy metals.¹⁷⁴  Hence, acetylcholinase immobilized SPEs were successful towards the effective electrochemical sensing of As³⁺.¹⁷⁵  But, the major drawback with the enzymatic detection was interference from other metals like Hg²⁺, Ni²⁺ and Cu²⁺. In addition to the individual metals, simultaneous detection of various metals is becoming interesting. The role of Bi in the detection is inevitable. Various forms of Bi like oxides, nanoparticles, co-deposition of Bi with other metals, in situ/ex situ preparation of Bi along with Hg are better electrocatalysts for the simultaneous detection of the metals.

Because of its salient features, electroanalysis is occupying its own place among the various analytical techniques. Since the measurements are highly sensitive, and selective, they can provide rapid and accurate results. It is proved that the analysis can fulfil the demands required in various sectors including healthcare, agriculture, industrial, and environmental monitoring. Considerable efforts have been made for the successful estimation of various analytes including biomolecules, pesticides, etc. The role of electrodes is crucial during the analysis. The invention of suitable electrocatalysts/modifiers like enzymes, metal nanoparticles, carbonaceous materials, etc. for the detection of a number of analytes is helpful for humans to step forward towards a sophisticated lifestyle. The utilisation of disposable electrodes like screen-printed electrodes, carbon paste electrodes are inevitable in the modern era. Since the electrodes and the electrochemical systems are economical, portable, and providing wide potential range, they are playing an extraordinary role in on-site monitoring. To utilize the effective characteristics of the disposable electrodes, research has to be focused by a wide range of researchers. Hence, people can reach a sophisticated, healthy environment very quickly.

AChE

Acetylcholinesterase

AgNP

Silver nanoparticle

ATP

Adenosine-5′-triphosphate

BChE

Butyrylcholinesterase

β-HCG

Human chorionic gonadotropin β-subunit

CNT

Carbon nanotube

CTAB

Cetyltrimethylammonium bromide

DAB

Diaminobenzene

DNA

Deoxyribonucleic acid

FAD

Flavin adenine dinucleotide

GNP

Gold nanoparticle

GOx

Glucose oxidase

IUPAC

International Union of Pure and Applied Chemistry

MWCNT

Multi-walled carbon nanotubes

OPH

Organophosphorous hydrolase

OSPCE

Oxygen plasma treated screen-printed carbon electrode

PA

Polyacetylene

PANI

Polyaniline

PAP

Polyazetidineprepolymer

PB

Prussian blue

PBS

Phosphate buffer solution

PEDOT

Poly(3,4)ethylenedioxythiophene

PEN

Polyethylene naphthalate

PET

Polyethylene terephthalate

PPO

Polyphenol oxidase

PPy

Polypyrrole

RF

Radiofrequency

SAM

Self-assembled monolayers

SEM

Scanning electron microscopy

SPCE

Screen-printed carbon electrode

SPE

Screen-printed electrode

XPS

X-ray photoelectron spectroscopy