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

Abstract

4-Aminophenyl phosphate (4-APP) and 1-naphthyl phosphate (1-NP) were compared as enzyme substrates for an amperometric milk progesterone biosensor utilising progesterone-conjugated alkaline phosphatase in a competitive immunoassay 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 surface mAb could impede the diffusion of 4-aminophenol, but not 1-naphthol, toward 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 6.5×10 −4 and 3.0×10 −4 cm s −1, respectively. The applied potential for oxidation of 4-aminophenol 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 1-naphthol in the absence of electrochemical interference from milk. Using mAb-coated SPCEs, calibration curves were obtained for progesterone in oestrus whole cow’s milk spiked with standard 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 correlations against a commercial EIA of r=0.840 and 0.946 for 4-APP and 1-NP, respectively. 1-NP possesses the advantages over 4-APP of being inexpensive, easy to obtain and soluble (1-naphthol cf. 4-aminophenol) at high pH. From these observations, it is concluded that 1-NP is the preferred substrate for use with our proposed milk progesterone biosensor.