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.
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 (khet) 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-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.
Pemberton
R M
RM
Faculty of Applied Sciences, University of the West of England, Bristol, UK.
Hart
J P
JP
Stoddard
P
P
Foulkes
J A
JA
D000469:Alkaline Phosphatase; D000814:Aniline Compounds; D000818:Animals; D015374:Biosensing Techniques; D002417:Cattle; D004563:Electrochemistry; D005260:Female; D008892:Milk / Q000737:chemistry*; D009281:Naphthalenes; D009943:Organophosphorus Compounds; D011374:Progesterone / Q000032:analysis*; D013379:Substrate Specificity
D000814:Aniline Compounds; D009281:Naphthalenes; D009943:Organophosphorus Compounds; C014694:naphthyl phosphate; D011374:Progesterone; C068798:4-aminophenylphosphate; D000469:Alkaline Phosphatase