**2. Construction of enzyme-based biosensors**

thus, a support judiciously chosen can significantly enhance the operational performance of the immobilized system. It is widely acknowledged that analytical sensing at electrodes modified with polymeric materials results in low detection limits, high sensitivities, lower applied potential, good stability, efficient electron transfer and easier immobilization of enzymes on electrodes. In recent years, there has been growing concern in using polymeric materials as supports for their good mechanical and easily adjustable properties [11]. Of the many carriers that have been considered and studied for immobilizing enzymes, conducting polymer (CP), redox polymer (RP), sol-gel and hydrogel materials, chitin and chitosan are of interest in that

Theproperuseofdifferentcompositionsofbinderandimmobilizationmatrix,electrontransport mediators, biomaterials and biocatalysts, and solid supports as electron collectors in the construction of enzyme electrode is critical to generate optimum current from the enzymatic redox reactions. In essence, design and fabrication of advanced materials coupled with good understandingoftheirbehaviorswhenincorporatedasinterfacialortransducerelementswould be of paramount importance. The recent advancement of polymer materials is greatly influenc‐ ingthe redox reactions andelectrontransportkineticsofthe enzyme electrodes.Toachievehigh specificity, high sensitivity, rapid response and flexibility of use, it is clear that the research continues to focus on new assembly strategies. Polymers are becoming inseparable from biomolecule immobilization strategies and biosensor platforms. Their original role as electri‐ cal insulators has been progressively substituted by their electrical conductive abilities, which opens anew andbroadscope of applications.This chapterhighlights recent contributions inthe incorporationofpromisingpolymericmaterialswithinbiosensors, specialemphasiswasplaced ondifferentclassesofpolymericmaterialssuchasnanomaterials,sol-gelandhydrogelmaterials, conducting polymers, functional polymers and biomaterials that have been used in the design of sensors and biosensors. We want to remind our readers that this chapter is not intended to provide comprehensive coverage of electrochemical biosensor development but rather to provide a glimpse of the incorporation of polymers within biosensors. These materials have attracted much attention to their potentials for interesting applications, broad applicability as well as tunableproperties accordingto applicationsneeds.Inaddition,the criticalissues related to the fabrication of enzyme electrodes and their application for biosensor applications are also highlighted in this article. Effort has been made to cover the recent literature on the advance‐ ment of polymers to develop enzyme electrodes and their potential applications for the

Analyzer bioreceptor signal transducer

light mass change

electroactive substance

electric signal pH change

they offer most of the above characteristics [12, 13].

68 State of the Art in Biosensors - General Aspects

construction of biosensors [14-15].

**Figure 1.** Principle of biosensor

#### **2.1. Immobilization methods of enzyme for biosensors**

Enzyme immobilization is one of the most important subjects for any enzyme-based biosensor research. Considerable efforts have been invested in this topic for a number of years [16-18]. The biosensing process of the immobilized enzyme is regarded as a heterogeneous phase reaction; thus, the main consideration for enzyme immobilization is to achieve stable and high enzymatic activity with low mass-transfer resistance. Enzymes electrodes have the longest tradition in the field of biosensors. They are one of the most intensely investigated biosensors due to highly selective and fast response. One of the key factors in developing a reliable biosensor is the immobilization of enzymes at transducer surfaces. The method of enzyme immobilization plays an important role on the performance of an enzyme electrode, such as lifetime, linear range, sensitivity, selectivity, response time, stability and anti-interferent.

Enzymes may be immobilized by a variety of methods (Fig. 2), which may be broadly classified as physical approaches and chemical approaches. To the physical methods belong: (i) physical adsorption on a water-insoluble matrix based on hydrophobic, electrostatic and van der Waals attractive forces; (ii) entrapment enzyme in sol-gel, or hydrogel, or a paste, confined by semipermeable membranes; (iii) microencapsulation with a solid membrane; (iv) encapsulation, containment of an enzyme within a membrane reactor; (v) formation of enzymatic Langmuir-Blodgett films or self-assemble monolayer which are the spontaneous and uninstructed structural reorganizations that form from a disordered system.

The chemical immobilization methods include: (i) covalently binding enzyme to support materials immobilizing enzyme into a membrane matrix or directly onto the surface of the transducer; (ii) crosslinking enzyme employing a multifunctional, low molecular weight reagent based on the formation of strong covalent binding between the transducer and the biological material using a bifunctional agent and (iii) electrochemical polymerization based on electrochemical oxidation of a given monomer from a solution containing the enzyme obtaining a conducting or non-conducting polymer layer and (iv) Micelle: The molecule must have a strongly polar/ hydrophilic "head" and a non-polar/hydrophobic "tail". When this type of molecule is added to water, the hydrophilic head of the molecule presents itself for inter‐ action with the water molecules on the outside of the micelle, and the hydrophobic tails of the molecules clump into the center of a ball like structure, called a micelle. Enzyme micelle membrane presented here is an innovative way and will be a well-developed biosensor technology to provide rapid and reliable measurements of food, water pollution and clinical analysis.
