Novel substrates for use in an amperometric 3-electrode system are described for the determination of alkaline phosphatase (EC.3.1.3.1), the enzyme label most commonly used in electrochemical immunoassays. Previous problems encountered with a variety of substrates have led to passivation of the working electrode at low product concentrations. Our group has synthesised a number of novel substrates in an attempt to elucidate this fouling problem. These substrates were synthesised with various electron withdrawing groups at the para position as these groups seem to stabilise the reaction product in an alkaline medium [I. Rosen, J. Rishpon, J. Electroanal. Chem. 258 (1989) 27-39] and limit fouling of the working electrode [Y. Xu, H.B. Halsall, W.R. Heineman, J. Pharm. Biomed. Anal. 7 (12) (1989) 1301-1311]. These substrates were p-cyanophenylphosphate, p-methoxyphenylphosphate and p-formylphenylphosphate. Preventing the formation of phenolic radicals, which polymerise and adhere to the working electrode, is the primary goal when designing the synthesis of these substrates. Of these, p-cyanophenylphosphate showed the greatest stability of product, significant lowering of the fouling of the working electrode and comparable Michaelis-Menten kinetics. Four aminoethanol buffers were compared with respect to their abilities to stabilise the products and to promote enzyme activity. 0.1 M Tris(hydroxymethyl)aminomethane buffer, at pH 9.0, was found to be the optimum buffer system.
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