Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification

A bienzyme electrode based on the amplification of a signal has been developed which allows the determination of picoto nanomolar concentrations of p-aminophenol. The active element of the sensor comprised of coimmobilised laccase and glucose dehydrogenase enzymes coupled with an oxygen electrode. Laccase catalyzes p-aminophenol oxidation by oxygen to give p-iminoquinone. The latter is reduced by excess of glucose in the presence of glucose dehydrogenase and results in recycling of the substrate. The detection is realized by measuring the decrease in oxygen concentration. The detection limit for p-aminophenol is 100 pM. The feasibility of the determination of a number of other substrates (polyphenols, polyamines, ferrocene derivatives) in the nanomolar range has been demonstrated. A significant background signal has been found for p-aminophenylphosphate. This background is probably caused by the ability of laccase to catalyze the oxidative dephosphorylation. In the presence of phosphate ions this background is practically completely eliminated. 50 pM of alkaline phosphatase could be determined after a 2 min incubation in p-aminophenylphosphate solution by determination of the p-aminophenol formed as the result of hydrolysis. The whole analysis time does not exceed 5 min. The new technique is suitable for application in alkaline phosphatase based enzyme immunoassays.