**5. Intrinsically conducting polymers in biosensors**

#### **5.1. Types, structures and features**

It is generally recognized that the modern study of electric conduction in conjugated polymers began in 1977 with the publication describing the doping of polyacetylene (PA). Conductive polymers or, more precisely, intrinsically conducting polymers (CPs) having unique conju‐ gated π-electron backbone system, are organic conjugated polymers that conduct electricity and are one of the more promising biocompatible materials [48]. Professor Alan Heeger along with Prof. Alan G. MacDiarmid and Prof. Hideki Shirakawa shared the 2000 Nobel Prize in


OE: oxygen electrode; PCS: porous carbon sheet; AGCE: aminated glassy carbon electrode; ERMM: enzyme reverse micelle membrane; GOD: glucose oxidase; ASOD: ascorbate oxidase.

**Table 1.** Amperometric sensors using PMS as a stabilizer

Although, it should be mentioned that the free enzyme exists in the outer surface of micelle may lose its activity due to the hydrophobility of chloroform reagent, which was used to dissolve PS and PMS, thus there is partly loss of enzyme activity during the immobilization, the strong adsorption ability of PS membrane makes the enzyme micelle based biosensors particularly attractive for on-line analytical systems which need high stability and good endurability due to the long-time and continuous determinations in a flow system during

Furthermore, PMS exhibits a strong affinity specific to a variety of hydrophobic materials such as polystyrene, polyethylene and polyetheretherketone due to the styrene moiety. Then PMS is considered to be an ideal stabilizer for both covalent bonding enzyme and hydrophobic affinity to PS film [30−32]. The application of PMS as a convenient immobilization reagent is summarized in table 1. PMS appears to be effective and promising for the maintenance of

It has been found that the urease reverse micelle membrane exhibits good sensitivity after stored in a phosphate buffer solution (0.1 M, PH 5.5) for one month compared with its initial sensitivity. The ideal immobilization process should be easy, quick, and enzyme friendly, result in high loading surface. In our previous study, enzyme micelle membrane, which is onestep immobilization, has been proved to be an excellent immobilization method to preserve enzyme conformation resulting in good activity and stability. To form enzyme micelle, hydrophilic PMS covalently bonded-enzyme (via both exposed and buried sulfhydryl groups and amino groups of enzyme and maleimide groups of PMS) was dispersed in hydrophobic PS solution. PMS-bonded enzyme aggregated to form micelle with enzyme inside the structure and PMS outside. The enzyme micelles were immobilized on the surface of glassy carbon electrode (GCE) utilizing PS, which has admirable properties of biocompatibility and strong adsorption ability. PMS also possess good biocompatibility, the PMS-bonded enzyme exhibits good activity due to PMS is a significantly excellent stabilizer for enzyme. On the other hand, it is amazing that the free enzyme exists in the inner part of micelle enhanced the stability of enzyme. The applications of PMS as a function polymer have been published [34-40] and were

It is generally recognized that the modern study of electric conduction in conjugated polymers began in 1977 with the publication describing the doping of polyacetylene (PA). Conductive polymers or, more precisely, intrinsically conducting polymers (CPs) having unique conju‐ gated π-electron backbone system, are organic conjugated polymers that conduct electricity and are one of the more promising biocompatible materials [48]. Professor Alan Heeger along with Prof. Alan G. MacDiarmid and Prof. Hideki Shirakawa shared the 2000 Nobel Prize in

experimentation.

76 State of the Art in Biosensors - General Aspects

biological activity as well as long time stability.

**5. Intrinsically conducting polymers in biosensors**

**4.2. Application of PMS to biosensors**

summarized in table 1.

**5.1. Types, structures and features**

Chemistry for their seminal contribution to the discovery and development of conductive polymers. A substantial account of the electronic properties was studied. From the point of versatility of synthesis techniques, properties, and broadness of the scope of application, CPs have raised a great deal of scientific and technological interest and have led the research in materials science in a new direction. The last comprehensive reviews devoted to CP were published and are excellent summary of earlier work [49-52].

Since the beginning of conductive polymer research, it has witnessed the emergence of CPs as an intriguing class of organic macromolecules that offer high electrical conductivity and optical properties of metals and semiconductors and, in addition, have the processability advantages and mechanical properties of polymers, in particular, are especially amenable to be further exploited to develop a new form of electrochemical biosensor either as sensitive components or as a matrix for providing biomolecule immobilization, signal amplification and for rapid electron transfer for the fabrication of efficient biosensor devices. A variety of monomers can be electropolymerised on an electrode surface and under correct conditions form stable conductive films. Structures of some CPs commonly used in biosensors are described in Fig. 9.

CPs like polypyrrole (PPy), polyaniline (PANI), polythiophene (PT) can be obtained by electrochemical polymerization either potentiostatically, galvanostatically or by means of multi-sweep experiments. The thickness of the polymer films can be defined by measuring the charge transferred during the electrochemical polymerization process and by controlling parameters like temperature, monomer concentration, polymerization potential or current, as well as the concentration and nature of the supporting electrolyte. Moreover, CPs films exhibt interesting properties concerning the decrease of the influence of interfering compounds due to their size-exclusion and ion-exchange characteristics.

The π-electron backbone which is an extended conjugated system having single and double bonds alternating along the polymer chain is responsible for their unusual electronic properties such as electrical conductivity, low energy optical transitions, low ionization potential and high electron affinity [53]. Scientists from many disciplines are now combining expertise to study organic solids that exhibit remarkable conducting properties. A key requirement for a polymer to become intrinsically electrically conducting is that there should be an overlap of molecular orbitals to allow the formation of delocalized molecular wave function. Besides this, molecular orbitals must be partially filled so that there is a free movement of electrons throughout the lattice [54]. The electronic conductivity of conducting polymers changes over several orders of magnitude in response to changes in pH and redox potential of their environment. The electrical properties can be fine-tuned using the methods of organic synthesis and by advanced dispersion techniques. Polymeric material containing interesting electrical properties is a step forward for research in materials. CPs has the ability to efficiently transfer electric charge produced by the biochemical reaction to electronic circuit. Moreover CPs can be deposited over defined areas of electrodes. The study of unique property of CPs has resulted in fundamental insights into the understanding of the chemistry and physics of this novel class of materials, and it has been exploited for the fabrication of amperometric biosensors.

#### **5.2. Application of CPs in biosensors**

Polymers are being discarded for their traditional roles as electric insulators to literally take charge as conductors with a range of novel applications. The electronic CPs has an organised molecular structure on metal substrates, which serves as proper and functional immobilizing platforms for biomolecules. These matrices provide a suitable environment for the immobili‐ zation and preserve the activity for long duration. This property of the conducting polymer together with its functionality as a membrane has provided opportunities to investigate the development of biosensors. Application of organic CPs in biosensors has recently aroused much interest as potential candidates to enhance speed, sensitivity and versatility for electro‐ chemical biosensors due to their easy preparation methods along with attractive unique properties such as high stability at room temperature, good conductivity output and facile polymerization and being compatible with biological molecules in a neutral aqueous solution. Moreover, the CP film provides a suitable environment for the immobilization of biomolecules. Thus, CPs have been studied extensively for the development of biosensors. The electrochem‐ ically prepared conducting polymers used for the biomolecule immobilization are polyacety‐ lene (PA), polypyrrole (PPy), polythiophene (PT), polyaniline (PANI) etc because of their good electrical properties, environmental stability. Many applications of conducting polymers including analytical chemistry and biosensing devices have been reviewed by various researchers [55, 56].

Enzyme immobilization onto the electrode surface is a crucial step in assembling amperometric biosensors. The CPs have attracted much interest as suitable matrices for biomolecules due to that the extended conjugation along the polymer backbone provides unusual electrochemical properties such as low energy optical transitions, high electrical conductivity, low ionization potential, high electronic affinities. Polymer matrices can be used either in the sensing mechanism or in the immobilization of the bioelement responsible for sensing the analyte. The

**Figure 9.** Structures of some conducting polymers commonly used in biosensors.

such as electrical conductivity, low energy optical transitions, low ionization potential and high electron affinity [53]. Scientists from many disciplines are now combining expertise to study organic solids that exhibit remarkable conducting properties. A key requirement for a polymer to become intrinsically electrically conducting is that there should be an overlap of molecular orbitals to allow the formation of delocalized molecular wave function. Besides this, molecular orbitals must be partially filled so that there is a free movement of electrons throughout the lattice [54]. The electronic conductivity of conducting polymers changes over several orders of magnitude in response to changes in pH and redox potential of their environment. The electrical properties can be fine-tuned using the methods of organic synthesis and by advanced dispersion techniques. Polymeric material containing interesting electrical properties is a step forward for research in materials. CPs has the ability to efficiently transfer electric charge produced by the biochemical reaction to electronic circuit. Moreover CPs can be deposited over defined areas of electrodes. The study of unique property of CPs has resulted in fundamental insights into the understanding of the chemistry and physics of this novel class of materials, and it has been exploited for the fabrication of amperometric

Polymers are being discarded for their traditional roles as electric insulators to literally take charge as conductors with a range of novel applications. The electronic CPs has an organised molecular structure on metal substrates, which serves as proper and functional immobilizing platforms for biomolecules. These matrices provide a suitable environment for the immobili‐ zation and preserve the activity for long duration. This property of the conducting polymer together with its functionality as a membrane has provided opportunities to investigate the development of biosensors. Application of organic CPs in biosensors has recently aroused much interest as potential candidates to enhance speed, sensitivity and versatility for electro‐ chemical biosensors due to their easy preparation methods along with attractive unique properties such as high stability at room temperature, good conductivity output and facile polymerization and being compatible with biological molecules in a neutral aqueous solution. Moreover, the CP film provides a suitable environment for the immobilization of biomolecules. Thus, CPs have been studied extensively for the development of biosensors. The electrochem‐ ically prepared conducting polymers used for the biomolecule immobilization are polyacety‐ lene (PA), polypyrrole (PPy), polythiophene (PT), polyaniline (PANI) etc because of their good electrical properties, environmental stability. Many applications of conducting polymers including analytical chemistry and biosensing devices have been reviewed by various

Enzyme immobilization onto the electrode surface is a crucial step in assembling amperometric biosensors. The CPs have attracted much interest as suitable matrices for biomolecules due to that the extended conjugation along the polymer backbone provides unusual electrochemical properties such as low energy optical transitions, high electrical conductivity, low ionization potential, high electronic affinities. Polymer matrices can be used either in the sensing mechanism or in the immobilization of the bioelement responsible for sensing the analyte. The

biosensors.

researchers [55, 56].

**5.2. Application of CPs in biosensors**

78 State of the Art in Biosensors - General Aspects

empolyment of promising CPs in electron transfer as an appropriate surface for enzyme immobilization provides rapid response encourages the coexistences of biomolecules and raises the stability of the biosensors. Numerous papers have been published indicating organic CPs as a convenient component, forming an appropriate environment for the immobilization of enzyme at the electrode surface. Stable immobilization of macromolecular biomolecules on conducting microsurfaces with complete retention of their biological recognition properties is a crucial problem for the commercial development of miniaturized biosensor. Most of the conventional procedure for biomolecule immobilization such as cross-linking, covalent binding and entrapment in gels or membrane suffer from a low reproducibility and a poor spatially controlled deposition.

Due to that CPs have considerable flexibility in the available chemical structure, which can be modified as required, CPs have attracted much interest to serve as good matrices for the immobilization of enzymes. The techniques of incorporating enzymes into electro-depositable conducting polymeric films permit the localization of biologically active molecules on electrodes of any size or geometry and are particularly appropriate for the elaboration of multianalyte micro-amperometric biosensors. Another advantage offered by CPs is that the electrochemical synthesis allows the direct deposition of the polymer on the electrode surface, while simultaneously trapping the protein molecules. In addition, the electrochemically prepared CPs can be grown with controlled thickness using lower potential and they also provide an excellent enzyme-entrapping property. The polymerization through electrochem‐ ical oxidation provides greater control over the process and enables control over the thickness of the polymer layer and even small electrode substrate to be coated. This technique offers a suitable way to make a homogeneous film that adheres strongly with the electrode surface.

Another important advantage of using CPs is that the biomolecules can be immobilized onto the nanowire structure in a single step rather than the multiple steps that are required when other non-polymeric materials are used. Nanostructured conjugated polymers and their nanocomposites represent new advanced materials that are key issues for the development of new devices and structures offering the association of the various properties required in advanced applications. As conducting polymer nanomaterials are light weight, have large surface area, adjustable transport properties, chemical specificities, low cost, easy processing and scalable productions, they are used for applications in nanoelectric devices, chemical and biological sensors [57]. There are extensive studies in the literature concerning the synthesis, characterization, and application of these CPs.

Among the CPs, PPy is one of the most extensively used conducting polymers in design of bioanalytical sensors. PPy and its derivatives play a leading role due to several interesting properties such as electroactivity, ionic exchange properties, supercapacitors for energy storage, secondary batteries, and elastic textile composites of high electrical conductivity, as well as good stability. PPy have the most versatile applicability for the construction of different types of bioanalytical sensors. The background presented illustrates that PPy is a very attractive, versatile material, suitable for preparation of various catalytic and affinity sensors and biosensors. The immobilization of biologically active molecules into PPy can be obtained during electrochemical deposition during which either some undesirable electrochemical interactions can be prevented or the electron transfer from some redox enzymes can be facilitated. The developments in nano-structured conducting polymers and polymer nano‐ composites have large impact on biomedical research. Significant advances in the fabrications of nanobiosensors/sensors using nano-structured CPs have been reviewed [58]. Recent advances in application of PPy in immunosensors and DNA sensors and recent progress and problems in development of molecularly imprinted PPy have been presented. The use of PPy in conjunction with bioaffinity reagents has provident to be a powerful route that has expanded the range of applications of electrochemical detection and its future development is expected to continue [59].

Among various CPs, PANI which can be directly and easily deposited on the sensor electrode and has controlled, high surface area, chemical specificities, long term environmental stability and tuneable properties has attracted much attention to be a suitable candidate to be used in various applications in biosensors due to its unique and controllable chemical and electrical properties, its environmental, thermal and electrochemical stability, and its interesting electrochemical, electronic, optical and electro-optical properties. PANI has gained much popularity in biosensor applications, partially due to its favourable storage stability, simple synthetic procedures with good processibility, rapid electron transfer and direct communica‐ tion to produce a range of analytical signals and new analytical applications. Efforts have been made to discuss and explore various characteristics of PANI responsible for direct electron transfer leading towards fabrication of biosensor interfaces and can also be used as a suitable matrix for immobilization of biomolecules [60, 61]. Moreover, PANI exhibits two redox couples in right potential range to facilitate an enzyme–polymer charge transfer and thereby acts as self-contained electron transfer mediator. In particular, PANI's transport properties, electrical conductivity or rate of energy migration, provide enhanced sensitivity. In addition, Nano‐ structures of PANI can offer the possibility of enhanced performance and also helps to overcome the processibility issues associated with PANI. In a conclusion, the various remark‐ able characteristics of PANI matrix make it a novel platform for fabrication of variety of biosensors interface.
