基于链置换扩增的电化学适配体生物传感器检测食品中的赭曲霉毒素A

Detection of Ochratoxin A in Food by Electrochemical Aptamer Biosensor Based on Strand Displacement Amplification

为构建一种基于链置换扩增技术(SDA)和电化学适配体传感器技术检测食品中赭曲霉毒素A(OTA)的方法,根据OTA特异性适配体设计发卡结构,并在发卡结构的茎部设置SDA反应识别位点,进行SDA扩增。将扩增产物与修饰二茂铁(Fc)的电化学探针进行杂交,使电信号发生变化,从而建立电化学适配体传感器检测OTA的方法。通过对7组不同毒素的测定评价该方法的特异性,测定其灵敏度和检出限,并与赭曲霉毒素A酶联免疫分析方法(ELISA)国家标准(GB 5009.96-2016)进行对比试验。结果表明:最优条件下,电化学适配体传感器灵敏度线性范围为0.1 pg/mL~10 ng/mL,检出限(LOD)为0.05 pg/mL。当OTA存在时,检测结果为阳性,当OTA不存在时,检测为阴性,说明该方法特异性良好。在人工加标试验中,该电化学适配体传感器的加标回收率为96.60%~99.04%,ELISA(国家标准)的加标回收率为94.00%~98.50%,该方法的加标回收率优于国家标准。结论:该方法能够快速检测食品中的OTA,具有实用应用价值。

A method based on strand displacement amplification(SDA)and electrochemical aptamer sensor technology was constructed to detect ochratoxin A(OTA)in food.According to the OTA specific aptamers,the hairpin structure was designed,and the SDA reaction recognition sites were set in the stems of the hairpin structure.SDA amplification was carried out.The amplified products were hybridized with the electrochemical probe modified ferrocene(FC)to change the electrical signal,so as to establish an electrochemical aptamer sensor for detecting OTA.The specificity of the method was evaluated through the determination of seven groups of different toxins,and its sensitivity and detection limit were determined.The results were compared with the national standard of ochratoxin A enzyme linked immunosorbent assay(ELISA)(GB 5009.96-2016).The results showed that under the optimal conditions,the linear range of the sensitivity of the electrochemical aptamer sensor was 0.1 pg/mL-10 ng/mL,the detection limit(LOD)was 0.05 pg/mL.When OTA exists,the test result is positive,and when OTA does not exist,the test result is negative,indicating that the specificity of this method is good.In the manual spiked test,the spiked recovery of the electrochemical aptamer sensor is 96.60%-99.04%and that of ELISA(national standard)is 94.00%-98.50%.Therefore,the spiked recovery of this method is better than that of the national standard method.In conclusion,this method has the potential of rapid detection of OTA in food,provides a new detection idea for rapid detection of OTA in food,and has high practical value.