Amperometric Carbon Paste Biosensors Based on Horseradish Peroxidase and Oxidases

Abstract: Amperometric carbon paste peroxide sensors based on horseradish peroxidase (HRP) were developed. In the first approach, HRP was chemically modified with polyethylene glycol to improve the stability of the enzyme in the organic paste matrix and compared with native HRP electrode. In the second approach, HRP was "wired" with a redox polymer containing a covalently bound phenoxazine type mediator, toluidine blue O (TBO), acting as a two-electron donor. Here comparison was made between mediated vs. non-mediated electron transfer and also the type of the paste binders, paraffin oil vs. paraffin wax. The peroxide sensor based on TBO was subsequently miniaturised and integrated with a glucose oxidase immobilised micro reactor in a micro flow cell for the analysis of glucose. Amperometric carbon paste biosensors based on oxidases (alcohol oxidase, glucose oxidase and L-lactate oxidase) co-immobilised with HRP were developed. The biosensors were designed based on two different approaches, i.e. direct or mediated electron transfer. For non-mediated systems, the enzymes were admixed with both additives previously shown to be efficient and also some new activators/stabilisers, such as polyethylenimine, lactitol, DEAE-dextran, polymeric dialdehyde, gluteraldehyde and a polyester sulfonic acid cation exchanger (Eastman AQ-29D) which was either mixed in the paste or applied as a protective membrane on the electrode surface. The mediated alcohol biosensor was based on an osmium containing three-dimensional redox hydrogel, (poly[1-vinyl imidazole osmium (4,4´-dimethyl-bipyridine)2Cl])+/2+, which was used to "wire" the HRP before co-immobilising the alcohol oxidase. The sensors were characterised regarding the sensitivity, selectivity, pH optima, operational and storage stabilities in a flow injection mode at a working potential of -50 mV vs. Ag/AgCl (0.1 M KCl). The developed sensors were applied under real operating conditions, such as, the alcohol sensor was used as a detection unit in column liquid chromatography for the determination of ethanol and methanol in biological fluids, while the L-lactate and glucose sensors were housed in a dual flow-through cell for the simultaneous monitoring of glucose and L-lactic acid during a fermentation process in an aqueous two-phase system by on-line FIA with microdialysis sampling.