*3.3. Impedimetric Biosensor*

An impedimetric biosensor is constructed by immobilizing biological recognition elements onto an electrode surface. It reports, through measurement and/or monitoring, the targeted analyte through the output of an electrical impedance signal made proportional to the analyte activity. The electrochemical biosensor in which the variation in impedance is used to detect the analytes or biological entities is called impedance biosensor. The most common technique used in this method is Electrochemical impedance spectroscopy (EIS). Using EIS, the properties of the bulk electrode as well as the processes taking place at the interface of the electrode can be determined easily [74]. The EIS spectrum is obtained as Bode or Nyquist plots, and both are a function of the frequency. The Nyquist plot comprises a semi-circular region on the axis representing the electron-transfer process followed by a straight line depicting the diffusion process. When the transfer of electrons is a quick process, the Nyquist plot only shows a straight line, whereas slow electron transfer is shown by a large semi-circular region [75]. Here, the resistance of the electron transfer is equal to the diameter of the semicircle. On the other hand, Bode's plot is a type of logarithmic plot where the log of phase(Φ) and impedance (Z) is plotted against frequency (logν). In the EIS, there is a very minute variation in the amplitude of the signal. Moreover, the measurement of impedance does not depend on the presence of redox couples [76], since the biosensor measures a biological event using agents such as antibodies, enzymes, bacteria, viruses, etc. [3,77]. In impedimetric biosensors, the focus is to produce amino, carboxyl, and similar groups on the surface of the electrodes so that the antibodies will be entrapped. This is the most important part while developing an impedimetric biosensor, as it ensures the permanency and repeatability of the sensor. In addition to this, nanomaterials have also been used for such purposes. When the antibody is detected using antigens, the resistance of electron transfer would tend to increase, which eventually decreases the capacitance [4]. The nanoparticles of gold can be used for entrapping antibodies as they do not affect their activity and performance [5]. Canbaz et al. [6] detected HER-3 using the impedimetric technique. Kim et al. [7] used EIS to determine the concentration of bacteria in a fermentor used at the laboratory. The biosensor consists of a PDMS polymer, silicon wafer coated with gold, and a borosilicate glass tube. The EIS was performed for bacteria E. coli for 13 h at a frequency of 0.01 MHz AC. The biosensor successfully detected the presence of E. coli in the fermentor. Seven et al. [78] used EIS to detect MCF-7 cancer cells. On a polypyrrole-NHS electrode, anti-c-cerbB-2 was entrapped using covalent linking. The sensor successfully detected the cancer cells showing the sensitivity of 100–10,000 cells per mL. Ankan et al. [79] investigated the sensing of E. coli bacteria (O157:H7) using an

antigen–antibody binding mechanism. The antibody was linked covalently on the surface of PANI film and an EIS study was performed to study the sensitivity and performance of the sensor. The change in impedance upon increasing the concentration of bacteria was measured and recorded. The biosensor was highly sensitive towards E. coli and can be used in labs. Rushworth et al. [80] developed a biosensor for sensing Alzheimer's amyloid-beta oligomers. By increasing the flow of current through the biosensor, the binding of the oligomer increases, hence decreasing the impedance of the biosensor. The EIS study, CV, and a diagram showing increase in the surface conductivity of the biosensor upon oligomer binding is shown in Figure 8.

**Figure 8.** (**A**,**B**) EIS plots. (**C**) CV study of the biosensor towards AβO. (**D**) Diagram showing an increase in the surface conductivity due to AβO. Reproduced from Reference [80] with permission. Copyright 2014, Elsevier.

In another work, Mao et al. [81] induced SWCNTs into gold electrodes (employing surface modifications) and the electrochemical biosensor was used to detect the DNA sequencing via impedance study. Shirsat et al. [82] reported glucose sensing using multilayer film of polypyrrole and SWCNT on Pt as an electrode. The biosensor displayed excellent sensitivity of 7.06 uA/mM towards glucose.

#### *3.4. Potentiometric Biosensors*

A potentiometric biosensor works on the principle of potential difference between working and reference electrodes. The measured species are not consumed like in the amperometric biosensor. Its response is proportional to the analyte concentration by comparison of its activity to the reference electrode. The great advantage of potentiometric biosensors is their sensitivity and selectivity when a highly stable and accurate reference electrode is used. Potentiometry, a mostly used electrochemical technique in the field of sensors, is cost-effective and used over a wide range of ion concentrations. The sensors developed using potentiometric methods are mostly available in the market. These sensors can be fabricated easily and a reduction in their size does not affect their performance. The use of potentiometric tools in the field of biosensors has opened many new doors for diagnosis and sensing. The two main advantages of using potentiometric biosensors

are: the signal produced is in the form of potential and the biochemical component used is the part of the receptor [83]. To monitor the perspiration in humans, potentiometric biosensors in the form of tattoo has been developed [84]. The potentiometric biosensor fabricated by coating polypyrrole on a gold electrode and using mat horseradish peroxidase as a biochemical agent has been developed for detecting tumour, hepatitis B, digoxin, and troponin I [85]. Recently, a potentiometric biosensor with a gold electrode and extended FET transistor was used to detect interleukin with LOD 1 pg mL−<sup>1</sup> [86]. The acetylcholinesterase was used as an antibody that produced thiol which was adsorbed on the electrode. Mishra et al. [87] fabricated a tattoo shaped biosensor for detecting G-nerve agents using potentiometric methods. In this biosensor, the design of the tattoo sensor was in the form of a skull with one eye working as a reference electrode and the other as a working electrode. The concept, design, printing, and application on human skin are represented diagrammatically in Figure 9.

**Figure 9.** Diagrammatic representation of the tattoo-shaped potentiometric biosensor. (**A**) The concept of the biosensor. (**B**) Designing of the tattoo biosensor. (**C**) The printing process of the biosensor on paper. (**D**) Successful transfer and removal of the tattoo on human skin. Reproduced from Reference [87] with permission. Copyright 2018, Elsevier.

Canovas et al. [88] developed a method for detecting glucose in human blood serum without dilution. The sample can be tested as received requiring no pre-treatment procedures. A Pt electrode printed on paper was used along with a polyelectrolyte called aquivion. The polyelectrolyte helps in entrapping the glucose enzyme and reducing interference and potential instability. Seema et al. [89] prepared nanoparticles of urease from jack beans and developed a potentiometric biosensor to detect urea in human urine samples. The biosensor displayed an excellent sensitivity of 23 mV/decade and a low LOD of 1 µM/L.

To summarize, various electrochemical methods that have been used for biosensing applications are presented in tabulated form in Table 1.


**Table 1.** List of electrochemical biosensors along with their applications.


**Table 1.** *Cont.*
