Pemberton Roy M, Hart John P
Faculty of Health and Life Sciences, University of the West of England, Bristol, UK.
Methods Mol Biol. 2009;504:85-98. doi: 10.1007/978-1-60327-569-9_6.
The method of fabrication of a prototype electrochemical immunosensor for estradiol (E2) is described. Methodologies are also given for colorimetric assays, which can be used to verify and optimize reagent performance, prior to their use in the electrochemical immunoassay: these include an E2 ELISA and a colorimetric assay performed on the immunosensor surface. The electrochemical immunosensor system uses screen-printed carbon electrodes (SPCEs) upon which antibody against E2 is immobilized. Antibodies (rabbit anti-mouse IgG, then monoclonal mouse anti-E2) are immobilized by passive adsorption onto the working electrode surface. A competitive immunoassay is then performed using an alkaline-phosphatase-labeled E2 conjugate. Electrochemical measurements are performed using differential pulse voltammetry (DPV) to detect the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current vs. E2 concentration shows a measurable range of 25-500 pg/mL with a detection limit of 50 pg/mL. The immunosensor can be applied to the determination of E2 in spiked serum, following an extraction step with diethyl ether.
描述了一种用于雌二醇（E2）的原型电化学免疫传感器的制备方法。还给出了比色测定法，可用于在将试剂用于电化学免疫测定之前验证和优化其性能：这些方法包括E2酶联免疫吸附测定（ELISA）和在免疫传感器表面进行的比色测定。电化学免疫传感器系统使用丝网印刷碳电极（SPCE），在其上固定有抗E2抗体。抗体（兔抗小鼠IgG，然后是小鼠抗E2单克隆抗体）通过被动吸附固定在工作电极表面。然后使用碱性磷酸酶标记的E2缀合物进行竞争性免疫测定。使用差分脉冲伏安法（DPV）进行电化学测量，以检测从1-萘基磷酸酯产生的1-萘酚。DPV峰值电流与E2浓度的校准曲线显示可测量范围为25-500 pg/mL，检测限为50 pg/mL。在用乙醚萃取步骤之后，该免疫传感器可用于测定加标的血清中的E2。
