*1.2. Electrochemical Sensors*

Electrochemical sensors have aroused great interest in providing fast and highly sensitive detection of proteins, which include the antibodies generated during the immune response against infections. Recently, electrochemical sensors have found widespread applications in clinical diagnostics, food safety, food quality, biological analysis, and environmental monitoring. Electrochemical sensors are sensors that change the effect of an electrochemical reaction of target species on the electrodes into useful signals which demonstrate alterations in potential and conductivity. With the utilization of biofunctionalities in the electrochemical sensor such as recognition and catalysis, these sensors are known as "biosensors" [21]. A typical biosensor is a combination of a transducer and certain biological elements. The biosensor can also be described as an electrochemical, optical, thermal, or piezoelectric biosensor depending on the type of transducer used.

In order to functionalize the biological element during the development of the biosensor, the immobilization step is a crucial step. The most popular biosensor utilizes enzymes as the biological substance with an electrode functioning as a transducer. The enzyme is directly immobilized onto the surface of the electrode, or is immobilized onto the electrode using a suitable matrix. In most cases, there are four strategies for enzyme immobilization that can be considered. These strategies, derived from the chemistry of immobilization, are shown in Figure 2. The first strategy is covalent bonding to the electrode or matrix (Figure 2A). The second is physical adsorption to the electrode or matrix (Figure 2B). The third is entrapment involving the enzyme and the electrode (Figure 2C). The final is affinity, which uses a specific biochemical interaction (Figure 2D). However, these strategies cannot each be considered as the perfect strategy because the optimum biosensor still needs to be selected depending on the specific enzyme and transducer [22].

**Figure 2.** Schematic representation of strategies for enzyme immobilization: Covalent bonding (**A**); Adsorption (**B**); Entrapment (**C**) and Affinity (**D**). E, enzyme; M, matrix [22]. Reproduced with permission from [H. Muguruma], [Biosensors: Enzyme Immobilization Chemistry]; published by [Elsvier], [2018].

The capture of a target analyte onto a bio or non-bioreceptor immobilized on a CP will generate a measurable analytical signal which is then converted into an electrical signal. The electrical signal can be identified using three main types of sensing modes such as potentiometric (membrane potential change); impedimetric (impedance change); and voltametric or amperometric (change of current for an electrochemical reaction with the applied voltage in the former, or with time at a fixed applied potential in the latter) [23]. It is desirable to strategize the development of an electrochemical sensor in order to provide low detection limits, high selectivity, and utilize a limited amount of indicator species. The impedimetric mode measures the targeted analyte through the output of an electrical impedance signal that is proportional to the analyte activity [9,24]; whereas in the voltametric mode, data about an analyte are obtained by measuring the current while applying a working electrode potential. The electrochemical reaction on the electrode surface and the electrode/electrolyte interface layer produces the final current [25]. The voltametric strategies include linear sweep voltammetry (LSV), cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV). These strategies provide a detailed measure with a broad range of effectiveness and a low detection limit. Meanwhile, in the amperometric mode, a consistent potential is connected to the working electrode, resulting in a current over time measurement. The difference between the amperometric and voltametric modes is the use of a potential step instead of a potential sweep. Using the amperometric mode will provide more selectivity and sensitivity since the reduction or oxidation potential utilized in the determination is normal for the analyte sample [26].

In this review, the discussion of sensing modes for the electrochemical sensor will be focused on impedimetric and voltametric modes. It will be start with an introduction followed by a description in detail, then the practical impacts of these modes on the electrochemical sensors will be discussed. We aim to present and highlight how these sensing modes can contribute to rationalizing the optimization of PPy based conducting polymers in electrochemical sensor applications.
