基于纳米金固定半抗原的电化学免疫传感器灵敏检测克伦特罗

摘要

研制了一种基于纳米金固定半抗原的间接竞争电化学免疫传感器,可灵敏检测克伦特罗.在金电极表面组装1,6-己二硫醇单分子膜,通过Au-S共价作用连接纳米金颗粒,通过吸附作用固定克伦特罗牛血清白蛋白偶联物.样品中的待测组分与固定化的克伦特罗偶联物竞争结合单克隆抗体,碱性磷酸酯酶标记的二抗选择性地与电极表面捕获的一抗反应,进而催化底物1-萘酚磷酸酯水解生成1-萘酚,在电极表面氧化产生电信号.在优化的实验条件下,克伦特罗浓度在0.1～1000 μg/L范围内与电流强度线性相关,线性方程为I(A)-8.79× 10-7-2.66× 10-7logC (μg/L),相关系数0.9960,检出限达20 ng/L.同时测定了猪肉及猪肝样品中克伦特罗含量,相对标准偏差平均值为7.0％,加标回收率在89.1％～105.6％之间,与传统的间接竞争酶联免疫吸附法对照,结果无显著性差异.%An indirect competitive electrochemical immunosensor based on gold colloid-mediated hapten assembly was developed for the determination of clenbuterol (CLB), a β-agonist drug that has been proved to be very toxic to human beings. The sensing substrate was prepared using a gold electrode modified with a self-assembled monolayer of 1, 6-hexanedithiol that mediated the assembly of a gold colloid layer, which adsorbed clenbuterol and bovine serum albumin conjugates onto the electrode surface. After competition for the limited anti-CLB mouse monoclonal antibody between immobilized hapten and CLB analyte in sample solution, alkaline phosphatase (ALP)-labeled horse anti-mouse IgG antibody reacted selectively with the primary antibodies captured on the electrode surface. Electrochemical response was produced by the oxidation of enzymatic product of 1-naphthy1 phosphate. Under the optimal conditions, the ALP-mediated redox signals were inversely correlated to the concentration of the CLB analyte in the range of 0. 1μg/L to 1000μg/L. The regression equation was I(A) =8. 79 × 10-7 -2. 66×10-7 logC (μg/L) with a correlation coefficient of 0. 9960.The detection limit was estimated to be 20 pg/mL. Furthermore, the proposed electrochemical immunosensor was used to detect the content of clenbuterol in pork- tissues with an average relative standard of 7. 0%, and the recoveries were in the range from 89. 1% to 105. 6%. The statistical results for the same concentration level obtained by the proposed method and traditional indirect competitive ELISA were comparable, proving the feasibility of the electrochemical immunosensor for accurate determination of CLB in real samples.