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 (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) 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 E(2) conjugate. Calibration plots for E(2) 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 E(2) 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 E(2) 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.
Centre for Analytical, Materials and Sensors Science, University of the West of England, Bristol BS16 1QY, UK.
R M
RM
Pemberton
T T
TT
Mottram
J P
JP
Hart
D000911:Antibodies, Monoclonal / Q000737:chemistry; D019921:Biotinylation; D002138:Calibration; D002244:Carbon / Q000737:chemistry*; D003124:Colorimetry; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000097:blood*; D006801:Humans; D007118:Immunoassay; D007074:Immunoglobulin G / Q000737:chemistry; D008956:Models, Chemical; D009281:Naphthalenes / Q000737:chemistry; D009284:Naphthols / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009281:Naphthalenes; D009284:Naphthols; D009943:Organophosphorus Compounds; C029350:1-naphthol; C014694:naphthyl phosphate; D004958:Estradiol; D002244:Carbon