Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E2) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E2) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E2 conjugate. Calibration plots for E2 buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E2 concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E2 in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.