Amperometric measuring cell for the determination of putrescine oxidase activity


A microfabricated, flat form, amperometric microcell (microchip) is used in a simple, two-electrode arrangement for putrescine oxidase enzyme activity determinations. The cell contains a platinum microdisk working electrode and an Ag/AgCl reference electrode covered by a porous, hydrophilic membrane. An electrochemically-prepared size-exclusion layer is applied on the working electrode surface, to avoid the effect of electroactive interferences in the sample. The hydrophilic membrane, resting on the bottom of the cell, is soaked with a small volume of buffered substrate solution and a few μl enzyme containing sample solution is dispensed over the electrodes. During the enzyme activity measurement a catalytic reaction takes place in the membrane-supported liquid film over the working electrode surface. The hydrogen peroxide produced in the reaction is detected amperometrically. The amperometric current-time curves are used for evaluation. In our work putrescine was used as a substrate to determine the unknown putrescine oxidase enzyme activity of the sample. Elevated diamine oxidase enzyme activity in the vaginal milieu can indicate premature rupture of the amniotic membrane. Results with membrane discs, containing all the necessary chemicals in solid or lyophilized form, are very encouraging with respect of a single use, 'reagentless' biosensor for home care.

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