An electrochemical flow analysis system for putrescine using immobilized putrescine oxidase and horseradish peroxidase

An electrochemical flow analysis system was optimized together with immobilized putrescine oxidase and horseradish peroxidase for putrescine measurement. Four coupling agents, suberic acid bis(N-hydroxysuccinimide ester), gamma-maleimidobutyric N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and glutaraldehyde, were used for immobilizing the two enzymes on porous aminopropyl glass beads to form a bienzymic detection column. Although the glutaraldehyde crosslinking procedure offered the highest response, the immobilized bienzyme system was responsive to putrescine, spermidine (123% of the putrescine response at 250 muM) and cadaverine (98% of the putrescine response). In contrast, the enzymes immobilized on the glass beads using suberic acid bis(N-hydroxysuccinimide ester) offered significantly better selectivity towards putrescine at the same concentration. For comparison, cadaverine and spermidine only provoked a response of 4.7% and 27.5% of the putrescine signal. The response to cadaverine and spermidine was further suppressed by lowering the detection pH from 8 to 7. At 250 PM, the response obtained for cadaverine and spermidine was only 1.5% and 3.9%, respectively, of the signal obtained for putrescine. A linear response to putrescine was obtained from 5 to 75 muM (0.629 muAs muM(-1), R-2= 0.997) with a detection limit of 5 muM (S/N= 3). The amperometric response retained 75% of its initial value after 600 repeated injections. The immobilized PUO/HRP (putrescine oxidase/horseradish peroxidase) was successfully demonstrated for measuring putrescine in fish extracts as an indicator of fish spoilage.