Redox polymers, biosensors

Functionalized conducting monomers can be deposited on electrode surfaces aiming for covalent attachment or entrapment of sensor components. Electrically conductive polymers (qv), eg, polypyrrole, polyaniline [25233-30-17, and polythiophene/23 2JJ-J4-j5y, can be formed at the anode by electrochemical polymerization. For integration of bioselective compounds or redox polymers into conductive polymers, functionalization of conductive polymer films, whether before or after polymerization, is essential. In Figure 7, a schematic representation of an amperomethc biosensor where the enzyme is covalendy bound to a functionalized conductive polymer, eg, P-amino (polypyrrole) or poly[A/-(4-aminophenyl)-2,2 -dithienyl]pyrrole, is shown. Entrapment of ferrocene-modified GOD within polypyrrole is shown in Figure 7. 

P.P. Joshi, S.A. Merchant, Y. Wang, and D.W. Schmidtke, Amperometric biosensors based on redox polymer-carbon nanotube-enzyme composites. Anal. Chem. 77, 3183—3188 (2005). 

Revzin A, Sirkar K, Pishko M. Glucose, lactate, and pyruvate biosensor arrays based on redox polymer/oxidoreductase nanocomposite thin films deposited on photo-lithographically patterned gold electrodes. Sensors and Actuators B 2002, 81, 359-368. 

Sirkar K, Pishko M. Amperometric biosensors based on oxidoreductases immobilised in photopolymerised polyethylene glycol) redox polymer hydrogels. Analytical Chemistry 1998, 70, 2888-2894. 

Figure 1. Structures of redox polymers used as electron relay systems in flavoenzyme-based biosensors. Shown are siloxane (top), ethylene oxide (middle), and branched siloxane-ethylene oxide (bottom) polymers.

Table I. Apparent Michaelis-Menten Constants and Maximum Current Densities for Redox Polymer Based Glucose Biosensors(a)...

One area where the relationship between the structure of the polymer matrix and the physical processes of the thin layer has been studied in detail is that of electrodes modified with polymer films. The polymer materials investigated in these studies include both conducting and redox polymers. Such investigations have been driven by the many potential applications for these materials. Conducting polymers have been applied in sensors, electrolytic capacitors, batteries, magnetic storage devices, electrostatic loudspeakers and artificial muscles. On the other hand, the development of electrodes coated with redox polymers have been used extensively to develop electrochemical sensors and biosensors. In this discussion,... 

The example considered is the redox polymer, [Os(bpy)2(PVP)ioCl]Cl, where PVP is poly(4-vinylpyridine) and 10 signifies the ratio of pyridine monomer units to metal centers. Figure 5.66 illustrates the structure of this metallopolymer. As discussed previously in Chapter 4, thin films of this material on electrode surfaces can be prepared by solvent evaporation or spin-coating. The voltammetric properties of the polymer-modified electrodes made by using this material are well-defined and are consistent with electrochemically reversible processes [90,91]. The redox properties of these polymers are based on the presence of the pendent redox-active groups, typically those associated with the Os(n/m) couple, since the polymer backbone is not redox-active. In sensing applications, the redox-active site, the osmium complex in this present example, acts as a mediator between a redox-active substrate in solution and the electrode. In this way, such redox-active layers can be used as electrocatalysts, thus giving them widespread use in biosensors. 

Acetylcholineesterase and choline oxidase A glassy C electrode surface was modified with osmium poly (vinyl-pyridine) redox polymer containing horseradish peroxidase (Os-gel-HRP) and then coated with a co-immobilized layer of AChE and ChO. A 22 pL pre-reactor, in which ChO and catalase were immobilized on beads in series, was used to remove choline. The variation in extracellular concentration of ACh released from rat hippocampal tissue culture by electrical stimulation was observed continuously with the online biosensor combined with a microcapillary sampling probe. Measurement of ACh and Ch was carried out by using a split disc C film dual electrode. 

A more complex biosensor for acetylcholine has been developed by Larsson et al. [154]. Three enzymes, AChE, ChOX, and HPR, have been coimmobilized in an Os-based redox polymer on solid graphite electrodes. After a careful optimization of the immobilization procedure, the biosensor, inserted into a flow cell of very small volume, was integrated into a flow injection system, and some samples of microdialysate, taken from rat brains before and after stimulation with KCl, were analysed. Even if a clear increase in signal could be noted, it was not possible to distinguish whether it was due to an increase in choline or in acetylcholine, since the biosensor responded to both metabolites. 

Co-extensive with the development and application of redox enzyme wired with redox pol5nner to amperometric glucose biosensors by Degani and Heller, Okamoto, Skotheim, Hale and their co-workers reported a similar approach but using different redox polymer to prevent free mediator diffusion into the bulk media [3]. 

Enzyme immobilization methods other than using the hydrogels (A) Since appUcations of the redox hydrogels (A) to glucose sensors was first reported by Pishko et al. in 1991, various other methods have been reported to immobilize redox polymers to construct biosensors as indicated (see Section 3.3.3.2). Other recent examples are as follows. 

When ferrocene-containing polysiloxane proved to be an efficient electron-transfer relay system, further modification of this type redox pol3uner was investigated to develop optimal enzyme biosensors. Attempts were made to synthesize redox polymers with different mediators and/or different polymer backbones and/or different side chains through which mediators are attached to the polymer backbone. Resulting redox poisoners were tested to construct different types of enzyme sensors. 

As was indicated in Section 3.3, an issue to be addressed before glucose or other biosensors is a commercially practical sensor fabrication. An easier and simpler sensor fabrication method was recently investigated using ferrocene modified redox polymer hydrogels. Sirkar and Pishko reported amperometric biosensors based on oxidoreductase immobilization in UV-photopolymerized... 

When constructing biosensors, which are to be used continuously in vivo or in situ, maintaining sensor efficiency while increasing sensor lifetime are major issues to be addressed. Researchers have attempted various methods to prevent enzyme inactivation and maintain a high density of redox mediators at the sensor surface. Use of hydrogels, sol-gel systems, PEI and carbon paste matrices to stabilize enzymes and redox polymers was mentioned in previous sections. Another alternative is to use conductive polymers such as polypyrrole [123-127], polythiophene [78,79] or polyaniline [128] to immobilize enzymes and mediators through either covalent bonding or entrapment in the polymer matrix. Application to various enzyme biosensors has been tested. 

Habermtiller, K., Reiter, S., Buck, H., Meier, T., Staepels, J., and Schuhmann, W. (2003) Conducting redox polymer-based reagentless biosensors using modified PQQ-dependent glucose dehydrogenase. Microchimica Acta, 143 (2-3), 113-121. 

The interest in these polymers has been accelerated by their applicability in the area of chemically modified electrodes (Abruna 1988 Kaneko and Wohrle 1988). Especially biosensor applications are very interesting applications for redox polymers... 

However, the use of these ionic and redox polymers was rather limited to applications, such as molecular electronic devices, or sensors and biosensors (using immobilized enzymes), where the need for fast electrochemical reactions was not essential, because of the limitation of the reaction rate by a slow charge transport process inside the polymer (see Section 2). This excludes large-scale applications, as in fuel cells or in organic electrosynthesis. 

Since the mediators should be mobile in order to provide the electron flow between the enzyme catalytic centre and electrode, they are usually soluble in the electrolytic medium, and therefore could be lost during the repetitive measurements. This, in combination with inactivation of enzyme, due to its denaturing leads to the loss of sensitivity of the biosensor with time. The possible solution to this problem is using an excess of enzyme and mediators in the measurement. Recendy, a new technique has been developed where redox polymers were used in dual function as immobilisation matrix and as materials facilitating electron-transfer. In materials such as these, the mediator redox... 

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