*5.1. Voltammetric/Amperometric Biosensors*

The method is based on the current detection technique, either by ramping up the working electrode's potential at a given rate or keeping the potential constant compared to the reference electrode. The system's response would be observed in both methods [65]. An amperometric biosensor (Figure 2) is based on the current generated from any electrochemical oxidation and reduction mechanisms of any electroactive species. It consists of a three-electrode system where a time-dependent excited potential is applied to the working electrode-changing the potential which is also relative to the fixed potential to the reference electrode. A current flows between the working electrode and the auxiliary electrode (nA to μA), where it is correlated with a bulk concentration of the electroactive species or the construction and expenditure rate within the adjoining biocatalytic layer. Platinum wire can be used as auxiliary electrode and an Ag/AgCl electrode can be used as reference electrode.

**Figure 2.** (**a**) Schematic design of the catalyzed oxidation on the catechol (analyte) electrode surface by laccase. (**b**) The proposed calibration curves of the catalytic currents vs. catechol (analyte) concentrations; and (**c**) calibration curve (Adapted from [98]) based on amperometric responses; (**d**) Schematic design of surface functionalization, where the Thiol-modified aptamers are bonded covalently and immobilized on the surfaces; (**e**) The Bode plots of the functionalized sensors; (**f**) The calibration curves are obtained for pesticides, such as acetamiprid (reproduced with the permission of [57]).

In 1956, Leland C. Clark introduced the oxygen probe, the simplest form of an amperometric biosensor. The oxygen probe measures the dissolved oxygen during the electrochemical reduction of oxygen. The associated electrolyte current is considered a response signal. This method can suit affinity sensors, which provide the electrochemically active compound as the recognition material and electrochemical labelling. Some electrochemically active

nucleobases are included in the nucleic acid structure and are used for monitoring the recognition of hybridization. A new design [99] of biosensor strips was integrated with a conducting copper track and a graphite–epoxy composite for pesticide analysis. It was applied by screen-printing, and the enzyme (AChE or BChE) was immobilized manually by crosslinking with glutaraldehyde. Micaela Badea et al. [100] have reported modified platinum electrodes with a cellulose acetate membrane to fabricate rapid amperometric detection of nitrites and nitrates in water. The developed method is simple, fast, and does not need an extra reagent for nitrite detection.

Biagiotti, Vanessa, et al. [101] have reported a platinum electrode modified by electropolymerized films and polymer nanotubule nets. They tried several analytical parameters; among them, poly(1,3-DAB) film showed the best performance for nitrite detection in drinking water. The electrode was characterized electrochemically by cyclic voltammetry and amperometry coupled to flow injection analysis (FIA). It has shown the linear range of concentration (10–1000 μM), LOD (2 μM), and good reproducibility (R.S.D.%: 0.4). Stoytcheva, Margarita, et al. [102] undertook a work to determine the enzymatic phenols by developing polymer film formation on the working electrode. Pan, Yanhui, et al. [103] developed an electrochemical biosensor that was constructed by nitrogen-doped graphene nanoribbons (NGNRs) and ionic liquid (IL). The molecularly imprinted polymer (MIP) was used to develop the composite film to determine 4-nonyl-phenol (NP), and the determination of concentration range was 0.04–6 μM. They obtained satisfactory results from real samples with high sensitivity, selectivity, and stability.


**Table 3.** Various types of transducers

 and

characteristics.

#### *5.2. Impedimetric Biosensors*

An impedimetric biosensor (Figure 2) was fabricated by immobilizing the bio recognition elements onto the surface of the electrode. Different bio-recognition elements can detect nutrients, heavy metals, or waterborne pathogens. The targeted analyte can be measured through the output of an electrical impedance signal made proportional to activity of the analyte. It is a two-electrode system where the alternating voltage can be applied with a few to 100 mV amplitude. The impedance (Z), or the components of resistance (R) and capacitance (C), can be changed due to the behaviour of the material. The applied voltage frequency can be scanned over various frequencies to get the corresponding impedance and characterize the sensor for specific material. The equivalent circuit parameters are also used for impedance spectra for characterization purposes. For developing an impedimetric biosensor, the prerequisite condition is the reproducible ability of the immobilizing bio-recognition molecules onto the sensor surface with the possession of their biological activity [109].

The impedance spectrum can be displayed in Nyquist or Bode plots. The plot is a semicircle region lying on the axis, followed by a straight line. Usually, electrochemical impedance spectroscopy (EIS) is used to investigate the properties of bio-recognition events at the modified surface.

An impedimetric biosensor was reported [110] with highly conductive tantalum silicide (TaSi2) to detect and quantify *E. coli* O157:H7 in drinking water. The developed biosensor shows a linear response with a concentration of 101–105 CFU mL−<sup>1</sup> and a sensitivity of 2.6 ± 0.2 kΩ. It can avoid interference which also confirms the excellent selectivity. The developed biosensor can be used multiple times with good repeatability. Hnaien, M. et al. have reported [111] a bacterial impedimetric biosensor for trichloroethylene (T.C.E.) detection in drinking water. Gold microelectrodes were used with single-wall carbon nanotubes, further linking with anti-Pseudomonas antibodies. It also showed a good linear response with the T.C.E. concentration up to 150 μg L−<sup>1</sup> and a low L.O.D. (20 μg L−1). It also showed excellent stability and recovery in real sample water. Lin, Zhenzhen et al. [112] have reported a biosensor for simultaneous detection of metal ions, such as Pb2+, Ag+, and Hg2+ in lake water. The DNA-based bio-recognition element was immobilized on the working gold electrodes. The developed biosensor had high sensitivity and selectivity, which were evaluated using the charge transfer resistance (RCT) difference before and after the immobilized DNA interactions with Pb2+, Ag+, and Hg2+. Madianos, L. et al. [113] developed a biosensor to detect acetamiprid and atrazine (pesticides) in natural water. The e-beam lithography technique deposited platinum nanoparticles (Pt NPs) between the interdigitated electrodes (IDEs) to create a bridge structure. The aptamer was chemically used to functionalize the Pt N.P.s on the sensing surface. The developed biosensor was highly sensitive and selective and also showed excellent linear response in the range of 10 pM to 100 nM for acetamiprid and 100 pM to 1 μM for atrazine.

#### *5.3. Capacitive Biosensors*

Capacitive biosensors consider be the group of affinity biosensors that operate by the direct binding between the surface of the sensor surface and the target molecule. It measures the variations in the dielectric properties and/or the thickness of the dielectric layer at the electrolyte/electrode interface location. A conventional electrical plate capacitor contains two conductive metal plates with specific dielectric properties separated by a certain distance. The following relations can express the:

$$\mathbb{C} = \frac{\mathbb{C}A}{d} \tag{1}$$

where € is the permittivity of the dielectric material, *A* is the area of the plate, and *d* is the distance between them. Therefore, when there is a change in the properties of the materials, a change in capacitance can be measured by the above equation. The second type of capacitive biosensor depends on the theory of electrical double-layer. The electrodes submerged in an electrolyte solution can resemble a capacitor for storing charge where an insulating layer covers the surface. The specific biorecognition element can be immobilized on top of this layer. The solvated ions and water molecules create a capacitance near the electrode surface.

N. V. Beloglazova et al. [114] reported a capacitive biosensor to detect benzo(a)pyrene (BaP) in river water. MIPs and monoclonal antibodies (mAb) are used as recognition elements on the electrode. The sensor is validated in a contaminated water sample from different places in Ghent, Belgium. Graniczkowska et al. [115] reported the development of a capacitive biosensor to monitor an amphetamine as a trace amount in water samples. The gold sensing electrode is immobilized with MIPs for creating sensing elements. Samuel M. Mugo et al. [116] reported a pathogen imprinted polymer for detecting *Escherichia coli* in water. The conducting electrode is based on multi-walled carbon nanotubes (CNT), and nitrocellulose (CNC) films, which were integrated with polyaniline (PANI) doped phenylboronic acid (PBA). The proposed sensor used both the capacitive and impediometric method for detecting the *E. coli* with a rapid response of ≤5 μmin.
