壳聚糖-SiO_2杂化膜酶免疫传感器的研制及其快速检测副溶血性弧菌的评价

目的 为改善已有免疫传感器存在的关键技术问题--生物敏感元件的固定化方法 及其生物活性的保持,研制壳聚糖(CS)-SiO_2杂化膜酶免疫传感器,并对研制的新型传感器进行评价.方法 采用溶胶-凝胶法制备CS-SiO_2杂化膜,将辣根过氧化物酶标记的副溶血性弧菌抗体(HRP-anti-VP)掺杂于该杂化膜中,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降低率(DP)来实现对副溶血性弧菌的检测.选择溶藻弧菌等7种菌进行特异度实验.结果 通过研究3种膜的红外光谱得出,CS-SiO_2杂化复合膜制备成功,HRP-anti-VP被固定于杂化膜中.在优化的免疫反应条件及电化学检测条件下,在10~4～10~9 cfu/ml浓度范围内,免疫电极免疫反应前后还原峰电流下降的DP与副溶血件弧菌浓度C的对数呈线性关系,线性回归方程为DP=6.5 lgC-3.319,线性相关系数0.9958,检出限为6.9×10~3cfu/ml[信/噪比(S/N)=3].该免疫电极具有较好的特异度、重现性[相对标准偏差(RSD)<6%]、稳定性(1周后电流响应为初始值的95%)和准确性(与GB/T4789.7-2003的符合率为96.7%).结论研制出的CS-SiO_2杂化膜酶免疫传感器用于快速筛检副溶血性弧菌效果良好.

Fabrication and evaluation of the enzyme immunosensor for rapid detection of Vibrio parahaemolyticus based on chitosan-SiO_2 hybrid membrane

ObjectiveTo improve the key technology of immunesensors in immobilizing biosensitive element and keeping its hioactivity, an enzyme immunosensor based on chitosan-SiO_2 ( CS-Sio_2 )hybrid membrane was fabricated. To estimate the new immunosensor Vibrio parahaemolyticus which was the main pathogens of aquatic products. MethodsA CS-SiO_2 hybrid membrane was prepared using sol-gel method. The enzyme immunosensor was fabricated by coating the membrane and horeseradish peroxidase labeled Vibrio parahaemolyticus antibody (HRP-anti-VP) on the surface of four-channel screen-printed carbon electrode. The immunosensor was characterized by cyclic voltammetry. Vibrio parahaemolyticus could be detected according to the decrease percentage (DP) of peak current before and after immune response,while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction. Seven kinds of bacteria, like Vibrio alginolyticus, were selected for specific experiments. Results By studying the infrared spectrum of three kinds of films, the CS-SiO_2 hybrid membrane was prepared and HRP-anti-VP was fixed in the hybrid membrane. Under the optimum conditions of immunoreactian and electrochemical detection,the DP of peak current before and after immune response showed a linear relation with lgC in the range of 10~4 - 10~9 cfu/ml,while the linear regression equation was: DP =6. 5 lgC -3. 319,the correlation coefficient was 0. 9958 and the detection limit was 6. 9 × 10~3 cfu/ml ( S/N = 3 ). The immunosensor possessed acceptable specificity, reproducibility ( RSD < 6% ), stability ( the amperometric response was 95% of the initial response after a week) and accuracy (96. 7% of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789. 7-2003 ). ConclusionThe enzyme immunosensor based on CS-SiO_2 hybrid membrane gave a good performance in rapid detection of Vibrio parahaemolyticus.