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为改善免疫传感器的生物相容性和反应信号强度,采用恒电位沉积法将HAuCl4直接还原成纳米金,并修饰于四通道丝网印刷碳电极(4-SPCE)表面。以质量浓度为0.05g/L和HAuCl4和电沉积时间30s作为电沉积纳米金修饰4-SPCE的制备条件。利用静电吸附作用将辣根过氧化物酶标记副溶血性弧菌抗体(HRP-anti-VP)固定,制备副溶血性弧菌酶免疫电极。通过循环伏安法表征免疫电极和监测酶促反应,根据免疫反应前、后还原峰电流下降的比例(DP)来实现对VP的检测。在优化的免疫反应条件及电化学检测条件下,免疫电极线性检测范围为104～109cfu/mL,其线性回归方程为:DP=7.7lgC-12.63,线性相关系数为0.9973(n=6),检测限为8.1×104cfu/mL(S/N=3)。该免疫电极具有较好的特异性、重现性(RSD<6%)、稳定性(1周后电流响应为初始值的90%)和准确性(与GB/T4789.7-2003符合率93.3%)。所研制的免疫传感器用于快速筛检VP效果良好。
In order to improve the biocompatibility and signal intensity of immunosensor, HAuCl4 was directly reduced to nanogold by potentiostatic deposition and was modified on the surface of 4-channel screen-printed carbon electrode (4-SPCE). The preparation conditions of 4-SPCE electrodeposited gold nanoparticles were as follows: 0.05g / L mass concentration of HAuCl4 and 30s of electrodepositing time. The horseradish peroxidase-labeled Vibrio parahaemolyticus antibody (HRP-anti-VP) was immobilized by electrostatic adsorption to prepare a Vibrio parahaemolyticus immune electrode. The cyclic voltammetry was used to characterize the immunodelectrode and to monitor the enzymatic reaction. The detection of VP was based on the ratio of the reduction peak current before and after the immunoreaction (DP). Under the optimal conditions of immunoreaction and electrochemical detection, the linear range of the immunosensor was 104-109 cfu / mL. The linear regression equation was: DP = 7.7lgC-12.63, linear correlation coefficient was 0.9973 (n = 6) The limit was 8.1 × 10 4 cfu / mL (S / N = 3). The immune electrode has better specificity, reproducibility (RSD <6%), stability (current response after 90% of initial value after 1 week) and accuracy (with GB / T4789.7-2003 coincidence rate of 93.3 %). The developed immunosensor for rapid screening of VP works well.