Reagentless enzyme electrode

Fig. 55. Schematic representation of a reagentless enzyme electrode using electrochemical regeneration of NAD for the determination of reduced dehydrogenase substrates.

S. S. Razola, E. Aktas, J.-C. Vire, J.-M. Kauffmann, Reagentless Enzyme Electrode Based on Phenothiazine Mediation of Horseradish Peroxidase for Subnanomolar Hydrogen Peroxide Determination. Analyst, 125 (2000) 79-85. 

Blaedel W.J. and Engstrom R.C. (1980) Reagentless enzyme electrodes for ethanol, lactate and malate. Anal. Chem., 52, 1691-1697. 

Conducting polymer-based amperometric enzyme electrodes towards the development of miniaturized reagentless biosensors. Synthetic Metals, 61 (1-2), 31-35. 

Schuhmann, W., Huber, J., Kranz, C., and Wohlschalger, H., Conducting polymer-based amperometric enzyme electrodes. Towards the development of miniaturized reagentless biosensors, Synth. Met., 61. 31-35 (1994). 

Future work will be directed towards the entrapment of mediator-modified enzymes into mediator-modified polymer layers. As a final goal, reagentless am-perometric enzyme electrodes with low working potential, decreas influence of the oxygen partial pressure and interfering compounds should be envisaged, simultaneously taking into account the need for mass production and miniaturization. 

A virtually interference-free and reagentless approach is immobilizing the redox enzyme on a suitable electrode surface in such a way that the protein-integrated redox site can directly exchange electrons with the electrode (enzymes with direct electron transfer contact). 

This review is a survey of the research on the direct electron transfer (DET) between biomolecules and electrodes for the development of reagentless biosensors. Both the catalytic reaction of a protein or an enzyme and the coupling with further reaction have been used analytically. For better understanding and a better overview, this chapter begins with a description of electron transfer processes of redox proteins at electrodes. Then the behaviour of the relevant proteins and enzymes at electrodes is briefly characterized and the respective biosensors are described. In the last section sensors for superoxide, nitric oxide and peroxide are presented. These have been developed with several proteins and enzymes. The review is far from complete, for example, the large class of iron-sulfur proteins has hardly been touched. Here the interested reader may consult recent reviews and work cited therein [1,19]. 

The general advantages of reagentless biosensor structures can be summarized as follows. Since all components of the assay are securely immobilized on the electrode surface, there is no or just a negligible loss of redox mediators, cofactors, and/or enzymes over the time of operation. This is of importance for the performance and safety of a device because the impact of free-diffusing possibly toxic substances is minimized. Therefore, reagentless biosensor architectures are often used for in vitro and in vivo measurements as outlined in Sec lion 1.4.5. 

One successful strategy to improve ET rates between enzyme and electrode is the modification of conducting polymers with redox mediators in order to obtain reagentless biosensors [11, 270, 271, 292-299]. The drawback of electropolymerization of conducting polymers is that the reaction is sensitive to oxygen, which complicates fabrication at the industrial scale. 

Schuhmann, W., Zimmermann, H., Habermtiller, K., and Laurinavicius, V. (2000) Electron-transfer pathways between redox enzymes and electrode surfaces reagentless biosensors based on thiol-monolayer-bound and polypyrrole-entrapped enzymes. Faraday Discussions, 116, 245-255. 

In a similar way to mediators, enzymes may be covalently bound to electrode surfaces, thus giving enzyme-chemically modified electrodes (ECME). When enzymes and mediators are coimmobilized, addition of auxiliary substances during the measuring process can be avoided a reagentless measuring regime becomes feasible (Fig. 19). 

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