**1. Introduction**

An analytical device that provides information about a biological process through a transducer is called a biosensor. The information can both be semi-measurable and measurable. The biochemical receptor is used to collect information about the biological process and then transferred to the transducer. An ideal biosensor should be temperature and pH-independent, recyclable, and specific. It was Cammann who first invented the term "biosensor" and the definition of the biosensor was introduced by IUPAC [1]. Biosensor fabrication is a multidisciplinary field as it involves chemistry, biology, physics, electronics, material science, engineering, etc. [2]. Recently, various new elements, such as molecularly imprinted polymers, aptamers, nanomaterials, etc., have been developed in the field of biosensors [3–7]. The growth of nanotechnology has provided strong benefits to the biosensor field as the nanomaterials have a large surface-to-volume ratio that helps in achieving higher sensitivity and efficiency [8]. In most of the biosensors developed so far, electrochemical detection has been used in one way or another, i.e., amperometric, voltammetric, and impedimetric detection [9]. Moreover, the catalytic biosensor that uses

**Citation:** Singh, A.; Sharma, A.; Ahmed, A.; Sundramoorthy, A.K.; Furukawa, H.; Arya, S.; Khosla, A. Recent Advances in Electrochemical Biosensors: Applications, Challenges, and Future Scope. *Biosensors* **2021**, *11*, 336. https://doi.org/10.3390/ bios11090336

Received: 24 July 2021 Accepted: 31 August 2021 Published: 14 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

some kind of enzymes covers half of the production of biosensors, whereas the affinity biosensors using antibodies represent the other half [10]. The affinity biosensors are mostly used in monitoring the quality and safety of food products along with DNA (de-oxyribose nucleic acid) detection in reactions (hybridization) [11]. The electrochemical biosensors consist of three electrodes, i.e., reference, counter, and working. The electron generated or consumed during the catalysis of enzymes is used for producing a signal that helps in the detection process [12]. These are further classified as potentiometric, amperometric, voltammetric, and impedimetric biosensors. Recently, computers have made a walk-in in every field of science and technology. Machine learning is an important aspect of the computer language program and has been used in monitoring and refining various data outputs. While sensing complex structures and entities, large data are obtained, and it becomes difficult to manually interpret all the data. Machine learning helps in interpreting large sensing data and has been used in various applications. Moreover, the data obtained sometimes are not clear and possess some noise and disturbance. Machine learning can help in analyzing such data and obtain the required results. In the case of biosensors, the presence of impurity affects the performance of the sensor and machine learning helps in removing the signals obtained from the contaminants to obtain a high sensitivity. Moreover, the interpretation of the data becomes quite easy and effective by using machine learning in the biosensing process [13–15]. In this review, various machine learning tools and techniques that are used for biosensing are discussed briefly along with their applications and limitations.

#### **2. Types of Biosensors**

## *2.1. Catalytic Biosensors*

The development of classical first-generation enzyme electrodes has started some five decades ago. The procedure involves depositing enzyme on the surface of the transducer and the signal produced depends upon the products produced on the transducer surface after the reaction and diffusion of the target on the substrate. To improve efficiency and lessen the enzyme electrode's cost, different types of electrodes are specifically modified using nanomaterials and sometimes replaced by carbon electrodes. Cinti et al. [16] developed a cholesterol biosensor using screen-printed electrodes that were modified using nanoparticles of Prussian blue. The hydrogen peroxide produced due to the oxidase enzyme can be detected with such a setup. Sannini et al. also explained this by developing a lactate biosensor that can be used to monitor fermentation in an interferent-rich matrix-like wine [17]. There has been the detection of spermine and spermidine, which are polyamines, which can help in monitoring food safety and human health. This method employs newly designed oxidase, i.e., spermine and polyamine. The oxidase is captured in the gel of polyvinyl alcohol, and carbon-based electrodes that are modified with Prussian blue are used for carrying out the reduction process [18]. Another method is to create a barrier on the electrode surface and entrapping the enzyme on the layer of the electrode surface by physical entrapment or cross-linking. For a selective membrane, an ideal contender is overoxidized polypyrrole. A Pt electrode having lysine oxidase entrapped on it is also used as a biosensor for detecting maturation [19]. The biosensors based on glucose oxidase have been mostly used for glucose sensing. Strambini et al. developed a microneedle-shaped biosensor that can monitor glycaemia present in the intestinal fluids [20]. They mounted a glucose biosensor on the back of the needle that can detect glucose present in the intestinal fluid with an accuracy of ±20% only in 30 s. The biosensor worked on the capillary action and can be used for real samples [21]. Figure 1a shows the design of the biosensor where the glucose sensor is mounted on the back of a needle-shaped chip. The glucose sensor comprises graphite electrodes having glucose oxidase entrapped into the pores. Figure 1b shows the calibration curves and Figure 1c,d shows the experimental data depicting that the sensor can be used to detect concentrations as low as 0 to 35 mM with a sampling time of 5, 30, and 100 s.

**Figure 1.** (**a**) Representation of the biosensor having glucose sensor attached on the back of the microneedle; (**b**) Calibration curves for the concentration between 0–35 mM; (**c**) Calibration curves of developed biosensors and stand-alone biosensors in ISF, and (**d**) Calibration curves for developed biosensor for concentration 0–35 mM at 5 s, 30 s, and 100 s of sampling times. Reproduced from Reference [20] with permission. Copyright 2015, Elsevier.

The second-generation enzyme electrodes that have been developed by depositing gold on PC membranes have also been used in manufacturing electrochemical biosensors. By using the mediator as (Ferrocenylmethyl)trimethylammonium, the limit of detection (LOD) of the glucose was 36 µM. The fouling of the electrode surface can also be recognized due to oxidation which does not exist in the first-generation electrodes. Thus, first-generation electrodes can be used to prevent their fouling. Malvano et al. [22] used alcohol dehydrogenase and entrapped it into an SPE electrode customized with poly(2 acrylamido-2-methyl-1-propane) sulfonic acid polymer doped in polyaniline which helped in preventing the fouling. Functional parameters can also be calculated using enzyme electrodes, as the food contains antioxidants (produced by chemicals in the food). The enzyme-based biosensors have been used for detecting the antioxidants produced from yogurt and berry juices [23]. Moreover, these biosensors have also been used to monitor antioxidants and ascorbic acid in orange juice, blueberry, and kiwi using modified graphite and fullerenes as shown in Figure 2a [24]. In Figure 2b, the CSEM (Cryo scanning electron microscopic) images of the biosensor are shown, whereas Figure 2c,d represents the schematic diagram and working of the biosensor, respectively. Due to the oxidation of the enzyme, there was a decrease in the current which helps in monitoring and measuring the ascorbate present in the sample.

**Figure 2.** (**a**) Pseudoreference, working, and auxiliary electrodes; (**b**) CSEM images of biosensor surface modified with different materials. (**c**) Diagrammatic representation of the sensors with the modified surface; (**d**) working of the sensor. Reproduced from Reference [24] with permission. Copyright 2015, Elsevier.

It must be noted that enzyme-based electrodes have been used for sensing pesticides, but with advancement and technology, they can be used for monitoring food safety. Atrazine is an important component of pesticides. The activity of the enzyme tyrosinase can be inhibited by the atrazine chemical. The electrode-based on tyrosinase has been used for detecting atrazine with 0.3 ppm LOD in the water bodies [25]. Similarly, an enzyme-based electrode of bilirubin oxidase has been used to monitor the oxygen level of a microbial fuel cell having a 0–0.08 mW power range [26]. The enzymes such as urease, d-lactate dehydrogenase, acetylcholinesterase, and β-galactosidase have been used in catalytic biosensors for optical detection of milk quality and safety [27].

#### *2.2. Affinity Biosensors*

The term affinity biosensor in this article refers to a device incorporating immobilized biological receptor molecules that can reversibly detect receptor–ligand interactions with a high differential selectivity and in a non-destructive fashion. In affinity biosensors, antibodies are used as biochemical agents for detecting the biological process. Immunosensor is an important class of affinity biosensors. In the last four decades, the focus in the development of immunosensors has been reducing incubation time, better design, signal amplification, label-free detection, and control over antibody placement. When it comes to the sensitivity and production cost of such immunosensors, the enzyme-linked immunosorbent assay (ELISA) technique has been used most widely. A label-free immunosensor based on optical fiber was fabricated using a thin film of titania–silica dip-coated over the sensing area of the fiber [28]. The analyte sensing was done by measuring the wavelength at the initial and endpoint of the fibre. The fabricated immunosensor was successfully used for detecting Immunoglobin (IgG) and anti-IgG in human serum with very low LOD. In another example, Tardivo et al. [29] reported a sensor having thousands of micropillars and columnar resonators arranged in an area of 1 mm square. The sensing was done using image and software analysis capturing images at 15 fps. Since there are various sensors incorporated, thus the variation obtained in the frequency measurement by a single sensor

is neglected. Figure 3a,b shows an optical image and the process of measuring the intensity of the image as a function of frequency, whereas Figure 3c represents 53 traces obtained for 53 pillars, and Figure 3d,e shows the tracking of the parallel pillars at a resonant frequency in real-time.

**Figure 3.** (**a**) T-shaped pillar array and its optical image; (**b**) Process of measuring the intensity as a function of frequency; (**c**) 53 traces corresponding to 53 different pillars as a function of actuation frequency. Actuation mode, (**d**) single-mode, and (**e**) multiple modes. Reproduced from Reference [29] with permission. Copyright 2015, Elsevier.

For detecting a prostate antigen called interleukin, which is an inflammatory cytokine, an immunosensor with a detection range from 0.3 to 100 nM was fabricated using an electrolyte-gated organic field-effect transistor (EGOFET) [30]. Moreover, ELISA which is a complete and simple detection format, can be easily used with mobile phones, with mobile phones acting as a detector. In Figure 4, Zhao et al. proposed an improved screenprinted carbon electrode (SPCE) for multiplexed label-free amperometric immunosensor.

It is observed that the limit of detection was 5.5 pg mL−<sup>1</sup> and the sensitivity of this immunosensor was 0.83 µA (lg(ng ml−<sup>1</sup> ))−<sup>1</sup> [31].

**Figure 4.** Multiplexed label-free amperometric immunosensor based on SPCE modified by Aupd NPs and some polymeric matrices for the detection of some biomarkers. Reproduced from Reference [31] with permission. Copyright 2018, Elsevier.

Aptamers can also be used for detecting antibodies. The aptamers are ribonucleic acid (RNA) and DNA molecules (single-stranded) that are highly selective and bind to particular antibodies, thus helping in their efficient detection. Different aptamers have different affinities based on which they can be used in developing biosensors with low LOD value. Castillo et al. [32] used a dendrimer structure for developing a voltammetric biosensor for sensing a mycotoxin called AFB1 (aflatoxin B1) in food products. The biosensor had a LOD of 0.40 nM for concentrations of 0.1–10 nM of AFB1. For the case study, the biosensor was tested on polluted peanut extract and peanut snacks. Scarano et al. [33] fabricated a piezoelectric material-based biosensor to detect this protein in human blood serum. Tintore et al. [34] used two aptamers with gold-coated magnetic nanoparticles for detecting single methylation in the DNA of aptamer using α-thrombin protein and plasmonic detection. The protein usually causes the agglomeration of nanoparticles resulting in the variation of color and UV of the solution. However, in this case, one of the aptamers prevented the agglomeration of the nanoparticles via single methylation of the DNA. Thus, the biosensor developed can be used to detect DNA modifications. Ravalli et al. [35] developed a biosensor based on an impedance study for detecting human epidermal growth factor receptor 2 (HER2). The antibody used for the detection of analyte was a protein produced using biotech engineering. The biosensor can detect a 0 to 40 µg/L concentration of HER2 with an LOD of about 6 µg/L. The albumin of the rat and human serum can bind with bicalutamide. In one such work, a transistor was developed where the gate of the transistor was modified with an odorant protein [36]. Pietrantonio et al. [37] deposited an odorant protein such as porcine, bovine, and double-mutant bovine protein on the surface of a wave resonator using the LIFT technique. The biosensor successfully distinguished between the carvone and octanol molecules that are present in the molds. Thus, the biosensor can be used for monitoring food products against molds. Cennamo et al. [38] detected butanal using an odorant protein called porcine. The biosensor selectively detected the butanal with concentrations ranging from 20–1000 µM. Compagnone et al. [39] developed a biosensor using gold-modified quartz crystal arrays. The biosensor was able to detect 95% of the off-flavored chocolate sample using the PLS-DA analysis technique.

Zuccaro et al. [40] developed a graphene-based biosensor that can monitor the behavior of the enzyme topoisomerase IB present in the human DNA. The interaction between the enzyme and the graphene substrate was analyzed by determining the field-effect characteristics of the biosensor. The biosensor provided real-time analysis and can be used for screening drugs in the future. The hybridization of DNA is an important feature whose detection is very helpful in various fields of biology. The biosensors with high selectivity and sensitivity can be used for detecting DNA hybridization. Mariani et al. [41] reported a method for detecting genomic human DNA (obtained from lymphocytes) without using the polymerase chain reaction (PCR) amplification process. The nanostars of gold were arranged in a sandwich-type model depending upon the analyte to be detected in the DNA and surface Plasmon resonance (SPR) imaging tool was used as a detector. A LOD of 10 pM was obtained with this method, which is low as compared to other biosensors developed, i.e., 6.1 nM. In another published work, Karel Lacina et al. [42] observed decrease in the impedimetric response (as shown in Figure 5) due to the binding of positively charged proteins. These assumptions were confirmed using gel electrophoresis which seems promising as a simple tool for such applications.

**Figure 5.** (**A**) Schematic visualization of the processes on the electrode. (**B**) Corresponding gel electrophoresis proving the charge of utilized compounds upon experimental pH. Reproduced from Reference [42] with permission. Copyright 2018, Elsevier.

Electrochemical methods can be used for sensing the hybridization of DNA. In one work reported, the electrochemical biosensor with an enzyme was used to detect the modified and unmodified DNA in PCR samples [43]. In another work, gold nanoparticles were incorporated as dots on a reduced graphene oxide electrode to produce an aptamerbased electrochemical biosensor [44].

#### **3. Electrochemical Biosensors**

The electrochemical biosensor is a special type of biosensor where a biological entity is detected by converting the information into an electrical signal, i.e., voltage, current, impedance, etc. [45,46]. The first electrochemical biosensor was developed by Clark to monitor the glucose level in human blood serum. The electrochemical biosensors have been developed to detect various biological entities such as enzymes, proteins, viruses, antibodies, etc. [47]. The electrode is the most vital component of an electrochemical biosensor as it controls the flow of electrons and bioagents. Various electrochemical biosensors that have been developed will be discussed in this section.

#### *3.1. Amperometric Biosensor*

An amperometric biosensor is a self-contained incorporated device based on the amount of the current ensuing from the oxidation offering exact quantitative analytical information. Generally, these Biosensors have reaction times, energetic ranges, and sensitivities comparable to the Potentiometric biosensors. In amperometric biosensors, the output current of the sensor is analyzed and used for the sensing process. The sensitivity of the amperometric biosensor is determined by comparing the current obtained for the different analyte concentrations. Such biosensors utilized only two electrodes, one for applying the voltage and the other for measuring the current flowing through the device. The amperometric biosensors do not utilize optical or electrochemical devices, but rather depend only on the current measurements. The development of biosensors started from glucose sensing and most of the glucose-sensing involves the catalytic reaction of the enzyme with the glucose oxidase [48,49]. The sensitivity of such glucose sensors is affected by the variation in temperature and pH. In amperometric glucose sensors, cyclic voltametry (CV) and electrochemical impedance spectroscopy (EIS) are the two electrochemical techniques that are used during the sensing process. The sensitivity of an amperometric glucose sensor is measured by the change in current per mM concentration in an area of one-centimeter square, i.e., µA mM−<sup>1</sup> cm−<sup>2</sup> . The sensitivity of an amperometric glucose sensor ranges from 1 to 15,000 µA mM−<sup>1</sup> cm−<sup>2</sup> . This is another important parameter while detecting glucose that determines its minimum and maximum concentration to detect the glucose successfully. The range of glucose in the blood (clinical) is between 4.4 and 6.6 mM. Tucci et al. [50] developed an amperometric biosensor for detecting herbicide using Anabaena variabilis cyanobacteria. Diuron and atrazine were used as target herbicides to be detected. The photocurrent produced in the biosensor due to the oxidation of water was found to be dependent upon the concentration of the herbicides. The current starts to decrease with an increase in the concentration of the herbicides. The sensitivity of the biosensor towards atrazine was found to be around 24.6 mA mM−<sup>1</sup> cm−<sup>2</sup> with an LOD of 0.56 mM. The mechanism of the inhibition is shown in Figure 6.

**Figure 6.** A diagrammatic representation of the photosynthesis inhibition by herbicides. Reproduced from Reference [50] with permission. Copyright 2019, Elsevier.

The biosensor was selectively used to detect glucose in uric acid, fructose, lactose, ascorbic acid, and mannose. The uric acid in the blood is in the range of 140–430 µM, and any changes in this level lead to kidney stones and gout [51]. The monitoring of uric acid in human blood is very important and biosensors are used effectively for this purpose [52]. Urea is another waste product produced in the kidneys and again variation in its level leads to serious kidney-related diseases [53,54]. The nanorods of ZnO were used to monitor the concentration of urea [54]. An array of ZnO nanotubes was deposited

on a silver/silicon substrate to detect cholesterol levels [55]. The sensor showed a sensitivity of about 79.40 µAmM−<sup>1</sup> cm−<sup>2</sup> with 0.5 nM LOD. Similarly, ZnO nanorods in a FET transistor, ZnO and multiwalled carbon nanotube (MWCNT) coated Pt/Au electrode, and a composite of CuO and ZnO have been successfully used to detect the cholesterol with good sensitivity and low LOD [56–58]. Fang et al. [59] successfully detected the presence of creatine kinase (an enzyme produced during muscle damage of brain or other body muscle) using an electrochemical biosensor made of gold. Cai et al. [60] reported an electrochemical immunosensor with gold and polythionine (PTh-Au) acting as an electrode. The immunosensor had good sensitivity and an LOD of 2.2 pg/mL towards the analytes. Both Fe3O<sup>4</sup> and Ag@Au show good reducing properties towards the hydrogen peroxide. Zhang et al. [61] fabricated an electrochemical biosensor using nanospheres of Fe3O<sup>4</sup> with nanorods of Ag@Au as an electrode. The biosensor demonstrated exceptional and superior behavior towards the reduction of hydrogen peroxide as compared to Fe3O<sup>4</sup> and Ag@Au. Xu et al. [62] developed an amperometric biosensor for the detection of glucose in blood serum. In their work, they self-assembled glucose oxidase and Pt nanoparticles (encapsulated with dendrimers) on a multi-wall carbon nanotube (MWCNT). The biosensor displayed good sensitivity and a low LOD towards glucose. Using a nanocomposite of reduced graphene oxide and gold nanoparticles (rGo/AuNP), Han et al. [63] reported a label-free electrochemical biosensor with enhanced conductivity.

#### *3.2. Voltammetric Methods*

Voltammetry belongs to a category of electro-analytical methods, through which information about an analyte is obtained by varying a potential and then measuring the resulting current. The electrochemical biosensors in which voltammetric techniques such as differential pulse voltammetry (DPV), CV, SWV, and LSV are used for sensing are known as voltammetric biosensors. The voltammetric methods are widely used in sensing platforms due to their low cost, good selectivity, and high sensitivity. Gupta et al. [64] developed a biosensor based on DPV and CV methods to detect anti-iTG antibodies in humans. The biosensor was built by modifying gold nanoparticles with GQD/PAMAM and then embedding them into MWCNT. The biosensors detected successfully the concentration of the antibody as low as 20–50 fgmL−<sup>1</sup> and are shown diagrammatically in Figure 7 below.

**Figure 7.** Diagrammatic representation of the biosensor based on DPV and CV to detect anti-tTG in the human celiac disease. Reproduced from Reference [64] with permission. Copyright 2017, Elsevier.

In another published work, Neves et al. [65] fabricated a nano immunosensor working on the CV technique to detect anti-tTG antibodies in the human serum. A carbon electrode that was modified with Au nanoparticles and CNT was taken and onto which the anti-tTG antibodies were printed using screen printing. The sensor showed excellent performance

and sensitivity towards the detection of antibodies and can be used for clinical trials. Hianik et al. [66] developed the first aptasensor that worked on voltammetric methods. In this sensor, the DNA aptamers were used to detect thrombin using the DPV technique. The aptamers were deposited on an Au electrode and methylene blue was used as an indicator. The aptasensors can be based on amperometric, potentiometric, voltammetric, conductometric, and impedimetric techniques [67,68]. The voltammetric aptasensors mostly use DPV, CV, and square wave voltammetry (SWV) techniques to detect the antibody. The voltametric biosensor is based on the detection of analyte using the variation in current according to the applied potential. Such biosensors detect both variations in potential and current. The potential is scanned over a wide range and the corresponding result in current and potential are measured. The current produced is directly proportional to the concentration of the target in the electrolyte [69,70]. The voltammetric methods produce low noise and can be used to detect multiple species with different peak potentials in a single scan [71]. The CV technique is used to study the stability, repeatability, and reproducibility of the biosensor [72]. Ly et al. [73] used a CNT electrode modified with bismuth to detect the DNA of Helicobacter pylori present in the gastric tissue of a patient. The biosensor successfully detected the DNA of Helicobacter pylori with a LOD of 6 µg/mL. The response time of the sensor was just 2s and can be used for clinical trials.
