基于DNA电化学生物传感技术的甘草提取液中大肠杆菌快检方法研究

Application of DNA-based electrochemical biosensor in rapid detection of Escherichia coli exist in licorice decoction

该文建立了一种基于DNA电化学生物传感技术的甘草提取液中大肠杆菌测试新方法。该研究首先将硫醇基修饰的捕获探针固定在金电极上，之后核酸适配体与捕获探针杂交。当甘草提取液中存在大肠杆菌时，它与适配体结合后，信号探针又与捕获探针结合并在底物中产生电化学信号，运用差分脉冲伏安法对甘草提取液中的大肠杆菌进行测试。实验结果显示，该测定方法线性范围为2．7×10^2-2．7×10^8CFU·mL^-1，最低检出限为50CFU·mL^-1，且具有较高的特异性和较好的重现性。在实际样品甘草浸膏的测试中，该方法的测定结果与周标法的计数结果呈现较好的一致性。以上结果显示DNA电化学生物传感技术可以用于甘草提取液中大肠杆菌测试，为中药制品的快速菌检提供了新的方法。

A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electro- chemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochenfical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7 × 10^2 to 2. 7 ×l0s CFU.mL-1 displayed a linear relationship with a detection limit of 50 CFU .mL-1. The relative standard deviation of 3 successive scans was 2. 5% ,2. 1% ,4. 6% for 2 × 10^2 ,2 × 10^4 ,2 × 10^6 CFU·mL^-1 E. coli, respectively. The proposed pro- cedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine.