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目的:研制一种灵敏度高、电化学性能稳定的免疫传感器,构建准确检测阪崎肠杆菌(Enterobacter sakazakii,E.sakazakii)的电化学方法。方法:将硫堇(Thi)、辣根过氧化物酶标记的阪崎肠杆菌抗体(HRP-anti-E.sakazakii)依次自组装固定于多壁碳纳米管(MWCNT)/十二烷基苯磺酸钠(SDBS)修饰的四通道丝网印刷电极上,制得一次性免疫传感器。采用原子力显微镜(AFM)表征电极修饰和孵育抗原后的表面形态,用循环伏安法(CV)考察不同修饰电极的电化学特性,根据还原峰电流的变化对阪崎肠杆菌进行测定。结果:在优化的实验条件下,该方法检测阪崎肠杆菌的线性范围为102～108cfu/mL,检测限为5.7×101cfu/mL(S/N=3)。结论:该免疫传感器灵敏度很高,具有较好的特异性、重现性(RSD=6.3%)、稳定性(4℃无菌容器中放置15d后电流响应为初始值的93.24%)和准确性(与GB/T4789.40-2010符合率为96.67%),具有一定的应用潜力。
OBJECTIVE: To develop an electrochemical sensor with high sensitivity and electrochemical stability and construct an electrochemical method for accurate detection of Enterobacter sakazakii (E.sakazakii). Methods: HRP-anti-E.sakazakii was self-assembled and immobilized on multi-walled carbon nanotubes (MWCNTs) / dodecylbenzenes (Thi) and horseradish peroxidase Sulfonate (SDBS) modified four-channel screen printed electrodes, the disposable immunosensor. The surface morphology of the modified electrodes was characterized by atomic force microscopy (AFM). The electrochemical properties of the modified electrodes were investigated by cyclic voltammetry (CV). The Enterobacter sakazakii was determined by the reduction current. Results: Under the optimized experimental conditions, the linear range of this method for E. sakazakii detection was 102-108 cfu / mL with a detection limit of 5.7 × 101 cfu / mL (S / N = 3). Conclusion: The immunosensor has high sensitivity, good specificity, reproducibility (RSD = 6.3%), stability (93.24% of initial value after 15 days in 4 ℃ sterile container) and accuracy (With GB / T4789.40-2010 compliance rate of 96.67%), has some potential applications.