ANALYTICA zyxwvut CHIMICA zyxwvutsrqpo ACTA zyxwvutsrqpon ELSEVIER Analytica Chimica Acta 304 (1995) 25-31 Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification A.L. Ghindilis a, A. Makower b, C.G. Bauer b, F.F. Bier b, F.W. Scheller b3 * zyxwvutsrqpo ” Department of Biosensors, Research Centre for Molecular Diagnostics, Simpheropolskii blvd. 8, 113149 Moscow, Russian Republic b Department ofAnalytical Biochemistry, University of Potsdam, Potsdam, Germany Received 1 June 1994; revised 19 September 1994; accepted 8 November 1994 Abstract A bienzyme electrode based on the amplification of a signal has been developed which allows the determination of pico- to nanomolar concentrations of p-aminophenol. The active element of the sensor comprised of coimmobilised lactase and glucose dehydrogenase enzymes coupled with an oxygen electrode. Lactase 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 lactase to catalyze the oxidative dephospho- rylation. 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. Keywords: Immunoassay: Biosensors; Substrate recycling; Lactase; Glucose dehydrogenase 1. Introduction The detection of the reaction products formed by alkaline phosphatase is used in different schemes of immunoassay. The spectrophotometric detection of p-nitrophenol (product of the enzymatic hydrolysis of y-nitrophenylphosphate) is commonly used. * Corresponding author. Tompson et al. [1,2] pioneered the use of p- aminophenylphosphate (PAPP) as a substrate of al- kaline phosphatase in combination with amperomet- ric measurement of p-aminophenol (PAP). In this case the detection limit of 7 nM PAP [2] is approxi- mately 20 times better than the spectrophotometric method. The determination of low concentrations using recycling enzyme systems has been described for a number of analytes [3-111, including the use of 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDl 0003-2670(94)00550-X