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目的:为增强响应信号,改善生物相容性,研制可以快速检测福氏志贺氏菌(Shigella flexneri,S.flexneri)的电化学免疫传感器。方法:将辣根过氧化物酶标记福氏志贺氏菌抗体(HRP-anti-S.flexneri)吸附在多壁碳纳米管(MWCNT)/海藻酸钠(SA)复合物修饰的四通道丝网印刷电极表面,制得一次性酶免疫传感器。采用原子力显微镜(AFM)表征免疫电极和孵育抗原后的电极表面形态以及循环伏安法(CV)考察不同修饰电极的电化学特性;采用一步法检测福氏志贺氏菌,CV监测酶促反应;根据免疫反应前、后还原峰峰电流的降低值来检测样品中的福氏志贺氏菌。结果:在优化的试验条件下,酶免疫传感器与福氏志贺氏菌浓度的对数在104~1011cfu/mL范围保持良好的线性关系,检出限为3.1×103cfu/mL(S/N=3)。结论:该酶免疫传感器放大了电化学反应信号,保持了生物物质的活性,具有较好的特异性、重现性(RSD=7.8%)、稳定性(4℃放置两周后电流响应为初始值的95.16%)和准确性(与GB/T 4789.5-2003符合率为97.5%),有望用于福氏志贺氏菌的快速筛检。
OBJECTIVE: To improve the biocompatibility of response signals and to develop an electrochemical immunosensor capable of rapid detection of Shigella flexneri (S. flexneri). Methods: HRP-anti-S.flexneri horseradish peroxidase (HRP-anti-S.flexneri) was adsorbed on four-channel silk modified with multi-walled carbon nanotubes (MWCNTs) / sodium alginate Screen printing electrode surface, obtained a one-time enzyme immune sensor. The morphology of the electrode after immunization and incubation of antigen was characterized by atomic force microscopy (AFM) and cyclic voltammetry (CV) were used to investigate the electrochemical characteristics of different modified electrodes. One-step method was used to detect Shigella flexneri. CV monitoring of enzymatic reaction ; Based on the immune response before and after reduction peak current peak reduction to detect Shigella flexneri in the sample. Results: Under the optimum conditions, the logarithm of the concentration of enzyme-immunosensor and Shigella flexneri maintained a good linearity in the range of 104-1011 cfu / mL with the detection limit of 3.1 × 103 cfu / mL (S / N = 3). Conclusion: The enzyme immunosensor amplifies the signal of electrochemical reaction and maintains the biological activity. It has good specificity and reproducibility (RSD = 7.8%) and stability (the initial response after two weeks at 4 ℃ Value of 95.16%) and accuracy (with GB / T 4789.5-2003 compliance rate of 97.5%), is expected to be used for rapid screening of Shigella flexneri.