【作者】 刘敏；

【导师】 谢国明；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2014， 硕士

【摘要】 目的本文将电化学生物传感器方法的高特异性和纳米金双标记的信号放大作用结合在一起，构建一种超灵敏检测耐甲氧西林金黄色葡萄球菌上mecA基因片段的电化学生物传感器,有望用于临床实际标本的早期快速检测。方法制备核酸和酶双标记纳米复合物，并分步探讨核酸、核酸和酶双标记的纳米金复合物等不同修饰步骤的电极的电化学行为。研究超灵敏检测金黄色葡萄球菌上mecA片段的电化学生物传感器方法，并对其检测条件进行优化，然后对该生物传感器进行方法学评价。首先在恒电势下，将裸玻碳电极浸入在氯金酸溶液中，电沉积纳米金颗粒。然后固定捕获探针，MCH封闭电极表面的非特异结合位点，加入目标探针与捕获探针形成“A=T”，“C=G”互补配对，最后加入双标记的检测探针,形成“三明治”夹心结构，在酶的催化作用下，产生电流信号。与其他同类方法相比，该方法明显减弱分别加入检测探针和酶繁琐的程度，大大缩减了反应时间，避免了实验中的相对误差，提高了整个实验的灵敏度。利用优化实验中探索出的最佳反应时间和酶的浓度，用差分脉冲伏安法对待测物所引起的电流信号变化进行检测，电极的不同修饰步骤用扫描电子显微镜（SEM）进行表征，电极的阻抗变化用电阻抗谱（EIS）进行表征。结果基于以上检测探针与酶同时标记纳米金的双标记结构，目标探针分别与捕获探针，检测探针形成“三明治”夹心结构，所构成的DNA生物传感器，在最优化实验条件下，检测信号与目标DNA的浓度在50-250pM内形成良好的线性关系，最低检测限达23pM。结论本文已成功应用检测探针与碱性磷酸酶同时标记纳米金的检测技术，与DNA电化学生物传感器相结合；实现了对金黄色葡萄球菌mecA基因片段的灵敏检测，检测时间控制在2小时以内。再对检测的最佳条件摸索后显示较宽的检测线和良好的重现性，对耐甲氧西林金黄色葡萄球菌的检测提供了较好的平台，该传感器具有很高的灵敏度和良好的特异性，为临床医生对疾病的及时诊断提供了重要线索。更多还原

【Abstract】 ObjectiveAn ultrasensitive and specific electrochemical biosensor for thedetection of the mecA gene, which is associated with methicillin resistantstrains, was successfully developed.Gold nanoparticles and alkaline phosphatase as the signalamplification, binding with electrochemical biosensors are used to theultrasensitive detection of the gene. This method is expected to be used todetect the actual bacteria in clinical specimens for clinical diagnosis.MethodsCapture probes were immobilized on the surface of a goldnanoparticles modified glassy carbon electrode. After hybridizationbetween with target probes and capture probes, dual labeled goldnanoparticles with alkaline phosphatase and detection probes were hybridized with target probes for sensitive detection of mecA genefragments. Dual labeled gold nanoparticles, which acted as ahigh-efficiency signal amplification strategy, were successfully prepared.The gold nanoparticles and dual labeled gold nanoparticles werecharacterized by transmission electron microscopy (TEM).ResultsDifferent concentrations of gene fragments were detected to inspectthe specificity and sensitivity of the proposed biosensor. This biosensordisplayed outstanding selectivity and a remarkable sensitivity LOD=23pM,S/N=3. Good linearity over the range of50pM to250pM wasdemonstrated. Moreover, the measurement process required less than2hours. This device was employed for the successful detection of mecAgene in methicillin resistant Staphylococcus aureus was confirmed byscanning electron microscopy (SEM), and electrochemical impedancespectroscopy (EIS). With the above amplification stratergy, the currentresponded to mecA gene linearly in the concentration range of50-250pM under optimal detection conditions with a detection limit of23pM.ConclusionA novel gene biosensor was developed for the electrochemicaldetection of the mecA gene, which was associated with methicillin resistantstrains. Dual marked gold nanoparticles with alkaline phosphatase and detection probe were successfully prepared and used for highly sensitivedetection of the mecA gene fragments. Under optimized detectionconditions, the proposed biosensor showed high sensitivity and a broadlinear range. More importantly, the whole detection time was performed in2h, which allows rapid identification of methicillin resistant strains andmay assist clinicians in making accurate decisions. Moreover, this proposedbiosensing strategy may be used to develop other biosensors for pathogenicbacterias and drug resistance analysis.更多还原