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 d eveloped which allows the determination of pico-to nanomolar concentra tions of p-aminophenol. The active element of the sensor comprised of coimmobilised laccase and glucose dehydrogenase enzymes coupled with a n oxygen electrode. Laccase catalyzes p-aminophenol oxidation by oxyge n to give p-iminoquinone. The latter is reduced by excess of glucose i n the presence of glucose dehydrogenase and results in recycling of th e substrate. The detection is realized by measuring the decrease in ox ygen concentration. The detection limit far p-aminophenol is 100 pM. T he feasibility of the determination of a number of other substrates (p olyphenols, 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 o f the p-aminophenol formed as the result of hydrolysis. The whole anal ysis time does not exceed 5 min. The new technique is suitable for app lication in alkaline phosphatase based enzyme immunoassays.