D-alanine is considered as important marker of bacterial contamination of food products. Usually it is determined using various modes of HPLC with chiral selectivity (see e.g. [1]) or GC after suitable derivatization. The aim of this work was to investigate the possibility of design of screen-printed amperometric biosensors using D-amino acid oxidase (DAAOx) and examine its selectivity for determination of D-alanine. DAAOx oxidizes D-alanine to puryvate and produces hydrogen peroxide from the oxygen reduction. It results in regeneration of enzyme to oxidized form. Hydrogen peroxide is a source of analytical signal, based on transition of the Prussian Blue from reduced to oxidized form.

The three electrode screen-printed sensors used in this study were produced in Department of Chemistry, University of Florence, Italy. The graphite working electrode was first pretreated by anodic polarization in phosphate buffer and then modified by Prussian Blue, precipitated in-situ from mixture of hexacyanoferrate (III) and ferric chloride on the electrode surface [2]. The carefully dried surface of modified working electrode was covered with cation exchange protective layer produced by evaporation of Nafion solution. Then DAAOx was immobilized on this surface by cross-linking with bovine serum albumin and choline oxidase using glutaraldehyde. It was found necessary to purify the commercial preparation of DAAOx by dialysis in order to remove Tris salts interfering in immobilization. The observed higher stability of biosensor response in buffer containing FAD indicates that cofactor is being removed during dialysis or storage of biosensor in solution. The biosensor optimization included selection of the suitable buffer, pH of measurements, composition of the enzyme layer and the way of its preparation and storage. Sensitivity and the life time has been also examined.

A fast and linear response of developed biosensor was observed in static measurements for D-alanine in the concentration range from 5 to 200 mM. The excellent enantioselectivity towards D-amino acids was found. The complex matrix of natural milk samples had no influence on the response of biosensor.

Multiwalled carbon nanotubes have been used for modification of working electrode. The effect of modification for the sensitivity of biosensors has been checked.

[1] K. Voss and R. Galensa, Amino Acids, 18 (2000) 339.
[2] F. Ricci, A. Amine, G. Palleschi, D. Moscone, Biosens. Bioelectron., 18 (2003) 165.
[3] M. Trojanowicz, A. Mulchandani, M. Mascini, Anal. Lett., 37 (2004) 3185.

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