# Electrochemical Biosensors Based on Conducting Polymers: A Review
## 2 Preparation of Sensitive Materials
### Strategies for immobilizing Biological Sensing Elements into Conducting Polymers  
Biological sensing element immobilization plays a fundamental role in the performance characteristics of biosensors since biomolecules must be directly attached to the surface of the biosensor to obtain a good sensitivity and a long operational life. The most commonly used methods to immobilize biomolecules to polymers are physical adsorption, covalent attachment and entrapment (Figure 1). The choice of immobilization strategy mainly depends on the type of biological element. Indeed, antibodies and ssDNA are preferentially immobilized by adsorption or covalent binding onto the surface of the conducting polymer films to facilitate the access of the analyte to these biorecognition molecules when entrapment is generally used to immobilize oxidoreductase within the polymer film to facilitate the electron transfer from the enzyme's redox center to the analyte solution surrounding the conducting polymer and the rapid redox reaction of electroactive species such as hydrogen peroxide generated by enzymatic catalysis. The method of covalent immobilization uses the functional groups of biomolecules (such as -COOH, -NH2, or -SH) for binding with a conducting polymer. Thus, a biomolecule containing amino groups has the capacity to form amide bonds with a conducting polymer bearing carboxylic groups. For example, [PERSON] et al. have developed a glucose biosensor with a conducting electrosynthesized  
Figure 1: Strategies of immobilization of biomolecules in/on conducting polymers: advantages and drawbacks. poly(terthiophene benzoic acid) bearing benzoic acid groups which allow the immobilization of glucose oxidase (GOx) through amide bond formation [67]. Similarly, [PERSON] et al. electrosynthesized the conducting polymer 4-(2,5-di(thiophene-2-yl)-1H-pyrrol-1-yl) benzenamine to immobilize GOx through amide bonds [68]. Moreover, covalent attachment of biomolecules is frequently achieved by initial synthesis of functionalized monomers with an amino side group, followed by electrochemical polymerization of these functionalized monomers leading to conducting polymer films with interfacial attachable side groups that can be covalently bound to biomolecules containing the corresponding groups. To facilitate the formation of covalent bonds between biomolecules and polymers, crosslinking agents such as glutaraldehyde [69; 70] or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) [71; 72] are commonly used. The covalent immobilization method has the benefit of providing low diffusional resistance, giving strong binding force between biomolecule and polymer, thus reducing loss of biomolecule. Therefore, these electrodes are more stable in time even if it may be difficult in some cases to retain the biomolecule activity. The adsorption method is very simple and only consists in the physical adsorption of the biomolecule on the polymer surface. Sometimes, the presence of opposite charges into the conducting polymer and the biomolecule facilitates the immobilization of the biomolecule. Thus, negatively charged glucose oxidase was successfully adsorbed onto positively charged polyaniline-polyisoprene films at pH 4.5 to provide a material sensitive to glucose concentration changes [73]. This method has the benefit of providing small perturbation of the biomolecule native structure and function and so generally leads to very sensitive responses. However, a strong drawback is that direct physical adsorption of biomolecule on a surface generally leads to poor long-term stability of the sensor because of biomolecule leakage from the surface when changes in the environment arise (pH, ionic strength) even if the modification of the surface by a polymer film can slow this leakage [74; 75]. Entrapment is another method widely used for the immobilization of enzymes [76; 77], antibodies [78] or DNA [79]. It involves the preparation of an electrolyte solution containing both monomer and biomolecule, followed by the electropolymerization of the whole solution.