基于PBP2a特异性识别的电化学免疫传感器检测耐甲氧西林金黄色葡萄球菌

Electrochemical immunosensor based on PBP2a specific recognition for MRSA detection

目的构建一种高性能的电化学免疫传感器用于耐甲氧西林金黄色葡萄球菌(MRSA)检测。方法制备MWCNT-PDA-AuNPs纳米复合物修饰至金电极表面,将MRSA标志蛋白PBP2a的抗体连接于纳米复合物表面,用牛血清白蛋白(BSA)封闭非特异性位点,得到一种检测MRSA的电化学免疫传感器。通过对传感器构建过程关键因素的考察确定最优条件,考察其线性范围及检出限,最后对传感器性能进行评价。结果通过CV、EIS表征分析确定传感器的成功构建;明确最优构建条件为:MWCNT-PDA∶AuNPs混合比例为1∶2,材料干燥温度为37℃,抗体孵育时间为1 h,样本孵育时间为30 min;MRSA在20 CFU/ml~2.0×10^(7)CFU/ml范围内浓度对数值与DPV峰电流值呈良好的线性关系,回归方程为I(μA)=-6.72lgC+84.26(R^(2)=0.9878),检出限为1 CFU/ml;传感器不易被杂质蛋白干扰,其对低、中、高浓度MRSA重复测定结果RSD分别为4.18%、3.59%、4.38%(n=8),保存20 d内,传感器电信号不低于初始的92%;传感器对低、中、高浓度MRSA样本检测回收率与平板计数法基本一致。结论该电化学免疫传感器构建简便,检测迅速、准确,灵敏度高,具有良好的特异性、重复性和稳定性,可以作为MRSA临床检测的备选方法。

This study was designed to constructed a high performance electrochemical immunosensor for the detection of methicillin-resistant Staphylococcus aureus(MRSA).The nanocomposite of MWCNT-PDA-AuNPs was prepared and modified to the surface of gold electrode;the antibody of MRSA marker PBP2a was linked to the surface of nanocomposite electrode,and the non-specific site was blocked with bovine serum albumin acid(BSA),then the electrochemical immunosensor for detecting MRSA was developed.The optimal condition,linear range and detection limit of the electrochemical sensor were determined by investigating the key factors in the construction process,and the performance of the sensor was evaluated.The construction of the sensor was confirmed by the analysis of CV and EIS.The optimal construction conditions were determined as follows:the ratio of MWCNT-PDA∶AuNps was 1∶2;the drying temperature was 37°C;the incubation time of antibody was 1 h;and the incubation time of sample was 30 min.There was a good linear relationship between the logarithm of MRSA concentration and the peak current of DPV in the range of 20 CFU/ml-2.0×10^(7) CFU/ml,the regression equation was I(μA)=-6.72lgC+84.26(R^(2)=0.9878),and the detection limit was 1 CFU/ml.The sensor has strong anti-interference ability to the impurity protein,and the RSD of repeated determination results for low,medium and high concentration MRSA were 4.18%,3.59%and 4.38%,respectively(n=8).The sensor electrical signal is no less than 92%of the initial value within 20 days of storage.The recovery rates of low,medium and high concentration MRSA samples detected by the sensor were basically consistent with that of plate counting method.In conclusion,the constructed electrochemical immunosensor has the advantages of simple construction,rapid,accurate,high sensitivity,good specificity,good repeatability and stability,and can be used as a candidate method for clinical detection of MRSA.