Development of screen-printed enzyme electrodes for the estimation of fish quality

Enzyme electrodes for the determination of biogenic amines have been developed using monoamine oxidase (MAO) from Aspergillus niger and putrescine oxidase (PO) from Micrococcus rubens. Determination is based on the electrochemical oxidation of enzymatically produced H2O2 at screen-printed platinum electrodes. The enzymes are immobilized on silanized electrodes by cross-linking with glutaraldehyde. Compositions of the immobilization mixtures are optimized with respect to stability, sensitivity and selectivity of the sensors. The electrodes using MAO as the biochemical component respond to several amines including histamine, an important amine in the determination of fish freshness. The PO electrodes show a significant response not only to putrescine and its homologue cadaverine but also to tyramine, an electrochemically active amine. The optimal buffer for both types of amine oxidase electrodes is Clark and Lubs (C+L) buffer pH 8.5. Simultaneous determination of the substrates of both enzymes can be accomplished by immobilizing PO and MAO onto different working electrodes of the same sensor. The sensors have been used to monitor the freshness of mackerel and codfish in storage. As expected, sensor signals increase with storage time of the fish, indicating the production of biogenic amines. During storage of mackerel, mainly histamine is produced, which leads to an increase in the signals obtained with the MAO electrodes. On the other hand, the putrefaction process of codfish during storage is detected mainly by the PO electrodes. All results are confirmed by comparison with HPLC data.