*4.1. Enzyme-Based Bio-Recognition Materials*

Enzym electrodes have been widely studied for the superior catalytic activity of inclusiveness of the enzymes and are commercially accessible. Regarding the electrochemical detection of water pollutants with enzymes, in some cases, they are converted to indirect detection of corresponding substrates by inhibiting enzyme activity. The biotransformation

of various compounds can catalyze these enzymes through oxidation–reduction reactions (REDOX). The amperometric biosensors' electrical responses of Oxidase/Peroxidase have electrical responses to a specific substrate that can be measured either by two different methods, such as direct or indirect methods [58]. Enzymes can be immobilized by physical adhesion or entrapped by the process of electrochemical techniques. According to the Michaelis–Menten equation, the enzymatic sensor's detection limit depends on the enzyme's activity. Thus, accommodating proper conditions (desirable temperature and buffer pH, etc.) for the development of the biosensor throughout the experiment is significant.

Ayenimo et al., rapidly developed a reliable, sensitive amperometric glucose biosensor to rapidly determine Hg2+, Cu2+, Pb2+, and Cd2+. A conductive ultrathin polypyrrole (PPy) film where the thickness was 55 nm thick was utilized to entrap glucose oxidase (GOx) with a fast response time. Upon exposure to trace metals, a more robust inhibition of GOx activity led to reduced glucose amperometric response [59]. Messaoud et al., have used fixed potential amperometry to determine bisphenol A (BPA), based on xanthine oxidase (XOD) enzymatic inhibition hypoxanthine as enzyme substrate, as is shown in Figure 1a,b. The mechanism of enzyme inhibition was estimated from the Cornish–Bowden and Dixon plots that were reversible with the competitiveness. An extremely low detection limit of 1.0 nM was achieved with excellent repeatability and reproducibility. The biosensor water samples' selectivity, stability, and practical tests were also investigated [60].
