基于多层酶/纳米金固定甲胎蛋白免疫传感器的研究

Reagentless Amperometric Immunosensor for α-1-Fetoprotein Based on Multilayers of Horseradish Peroxidase/Nano Au

利用自组装技术和静电吸附作用,将甲胎蛋白抗体(anti-AFP)固定在多层辣根过氧化物酶/纳米金及L-半胱胺酸修饰的金电极表面,制备出用于检测甲胎蛋白抗原(AFP)的无试剂型免疫传感器.通过交流阻抗技术、循环伏安法和计时电流法考察了电极的电化学特性,并对该免疫传感器的作用机理及性能进行了详细的研究.用计时电流法测得AFP的线性范围为1.0～10.0和10～200ng?mL-1,检出限为0.5ng?mL-1.实验结果表明,该方法提高了抗体的固定量,增强了传感器的灵敏度和稳定性,且该传感器响应迅速、选择性好,血清中常见抗原不干扰测定.将其用于临床血清检验,与放射免疫测定法(RIA)的符合率为86.7%.

An immunosensor for direct amperometric determination of α-1-fetoprotein （AFP） was constructed by a-l-fetoprotein antibody （anti-AFP） immobilized on multilayers of horseradish peroxidase （HRP）/nano Au through electrostatic adsorption onto L-cysteine modified gold electrode surface. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The determination of AFP was established by chronoamperometry to record the direct electrochemistry response of HRP to reduction of H2O2. Under optimal conditions, the immunosensor was highly sensitive to AFP with a detection limit of 0.5 ng.mL 1 and the linear range was at two concentrations from 1 to 10 and from 10 to 200 ng. mL^-1 with 0.7 mmol.L^-1 H2O2 in analyte solution. The dependence of the number of HRP/nano Au layer, pH, incubation time, reproducibility and stability was studied. More than 86.7% of the results of the human serum samples obtained by this method were in agreement with those obtained by radioimmunoassay.