A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzymesubstrates for use with a screen-printed amperometric immunosensor for progesterone in cows' milk

4-Aminophenyl phosphate (4-APP) and 1-naphthyl phosphate (1-NP) were compar ed as enzyme substrates for an amperometric milk progesterone biosensor uti lising progesterone-conjugated alkaline phosphatase in a competitive immuno assay format. Cyclic voltammetry of the corresponding hydrolysis products, 4-aminophenol and 1-naphthol, at the surface of screen-printed carbon base transducers, uncoated or coated with anti-progesterone monoclonal antibody (mAb) showed well-defined anodic responses for both species, with the more sensitive being 4-aminophenol. Scan rate studies produced evidence that sur face mAb could impede the diffusion of 4-aminophenol, but not 1-naphthol, t oward the electrode surface. This was supported by computer simulation for the electrochemical rate constant (k(het)) using 4-aminophenol, which gave Values at uncoated and mAb-coated electrodes of 6.5 x 10(-4) and 3.0 x 10(- 4) cm s(-1), respectively. The applied potential for oxidation of 4-aminoph enol was 230 mV lower than for 1-naphthol. Nevertheless, by operating below +400 mV versus a saturated calomel reference electrode, it was possible to obtain a chronoamperometric signal for l-naphthol in the absence of electr ochemical interference from milk. Using mAb-coated SPCEs, calibration curve s were obtained for progesterone in oestrus whole cow's milk spiked with st andard concentrations over the range 0-50 ng/ml, using either 4-APP or 1NP as enzyme substrate. Precision values for triplicate sensors were 5.3-18.3% for 4-APP and 4.1-12.4% for 1-NP. An assay of real whole milk samples from different cows at various stages of the oestrus cycle produced correlation s against a commercial EIA of r = 0.840 and 0.946 for 4-APP and 1-NP, respe ctively. 1-NP possesses the advantages over 4-APP of being inexpensive, eas y to obtain and soluble (1-naphthol cf. 4-aminophenol) at high pH. From the se observations, it is concluded that 1-NP is the preferred substrate for u se with our proposed milk progesterone biosensor. (C) 1999 Elsevier Science S.A. All rights reserved.