## 4 Conductive Polymers
### Immobilization by Electrochemical Polymerization of CP Monomers  
Electrochemical polymerization is a method in which CP monomers and enzyme molecules are present in an electrolyte solution. By applying a voltage, CP monomers undergo oxidation to generate oligomers or polymers, which then co-deposit with enzyme molecules onto the electrode surface, achieving the immobilization of enzyme molecules [138; 139]. The oxidation potential of polymers is always lower than that of monomers. Therefore, during the electrochemical polymerization process, monomers form radical cations on the electrode surface under the applied potential. These radical cations undergo dimerization through deprotonation, forming dimers that are subsequently oxidized again to form cations. Coupling reactions then occur between the cations and monomer radical cations, leading to chain propagation [140; 141]. As a result, the main chain of the polymer carries a positive charge [142], which is neutralized by introducing anions from the electrolyte. Enzymes located near the electrode surface can be physically embedded into the polymer [141]. This has proven to be an effective way to capture enzymes in situ within organic polymers, providing a new means for the preparation of a variety of novel composite materials. This method is the most direct approach for enzyme immobilization, offering the following distinct advantages [129; 142]: (1) Preservation of enzyme activity and stability: immersing enzymes in conductive polymers helps maintain good enzyme activity and higher stability. (2) One-step process with fast immobilization: the entire process is completed in a single step, resulting in a fast immobilization rate. (3) Uniform distribution of immobilized enzymes: the immobilized enzymes are evenly distributed, independent of the geometric shape and size of the electrode. (4) Precise control of membrane thickness: the membrane thickness can be accurately controlled by regulating the amount of charge involved in the deposition steps. Deposition can be achieved using either constant potential or constant current methods [143]. [PERSON] and colleagues employed an in situ electrochemical immobilization process by mixing a solution containing aniline monomers and enzymes (GOx or Ur) to prepare a polymerized aniline film embedded with enzymes. This resulted in glucose and urea biosensors incorporating immobilized GOx and Ur [135]. Additionally, [PERSON] and colleagues developed a novel biosensing electrode by simultaneously electro-polymerizing pyrrole and co-depositing graphene oxide (gRGO) and cholesterol oxidase (CHOx). Compared to other reported cholesterol biosensors, this one-step-manufactured biosensor exhibited superior sensitivity, a broader linear response, and a lower detection limit. The excellent porosity of this composite material improved the efficiency of cholesterol diffusion, facilitating enzyme-catalyzed reactions [144; 145]. PPy itself has a relatively low affinity for enzymes, leading to lower efficiency when using PPy as a CP monomer for enzyme immobilization. Enzymes are prone to release from the PPy matrix [146]. To address these issues, in recent years, various nanomaterials and other substances have been used in the enzyme immobilization process to enhance the efficiency, stability, and sensitivity of biosensors [147]. [PERSON] and colleagues reported a one-step electrochemical method to prepare an electrode composed of PPy, polydopamine (PDA), and enzymes (such as glucose oxidase (GOx) or lactate oxidase (LOx)) (Figure 6). The addition of a small amount of PDA helped improve the efficiency of enzyme immobilization during the PPy polymerization process [138]. [PERSON] and collaborators used a one-step electro-polymerization approach to prepare a cholesterol nanobiosensor based on a Pt electrode, incorporating gold nanoparticles (AuNPs), cholesterol oxidase (COX), cholesterol esterase (CE), PPy, and K\({}_{4}\)Fe(CN)\({}_{6}\). The addition of Fe(CN)\({}_{6}\)4- and AuNPs enhanced the sensitivity of the cholesterol biosensor.