气性坏疽主要由梭状芽胞杆菌（Clostridium）等厌氧菌进入深部伤口，造成组织或器官感染、肢体坏死，是地震伤、战伤、工程创伤等的严重并发症之一；但目前临床缺乏对厌氧菌的快速、灵敏、高通量的检测手段，尤其是地震现场或野战条件下的检验。本项目拟设计：①利用环介导恒温扩增技术（LAMP）快速扩增厌氧菌DNA,生成大量的茎环状产物；设计一对特异的DNA探针（捕获探针和检测探针），通过茎环状产物，1个捕获探针最终可以连接4-8个电致化学发光体[邻菲罗啉（phen）/联吡啶（bpy）配合物]，有望解决常规厌氧菌检测方法灵敏度不高的难题；②利用Pd@Au纳米复合物改进传感器，结合阵列式丝网印刷电极(array SPE)，可以实现对多种厌氧菌的高通量检测。因此，该DNA传感器的构建有望实现对气性坏疽厌氧菌的高灵敏度、高通量检测；还可为其他类似微生物的检测提供可选的解决方案。

Gas gangrene is caused mainly by the Clostridium and other anaerobic bacteria got into the deep wound, led to the infection of deep tissue and organ. It is one of the serious complications in the earthquake, war，and engineering damage. But there is lack of rapid, sensitive, and high-throughput test method for the anaerobic bacteria in clinical today，especially in the piont of care test in the earthquake and war field conditions. The project of the study: ① Using Loop-mediated isothermal amplification (LAMP) to amplify the DNA of the bacterial rapidly under constant temperature condition, generating a large number of multiple stem-loop structures.Then a pair of specific DNA probes (a capture probe and a detect probe) are designed. Through the multiple stem-loop structures, one capture probe could connect 4-8 electrogenerated chemiluminescences [Ru(phen)3 2+/Ru(bpy)3 2+] eventually, thus greatly improve the sensitivity of DNA detection.It is expected to solve the problem of low sensitivity in the anaerobic bacteria detection by conventional methods. ② At the same time, using the Pd@Au nanoparticles composite to improve the biological compatibility of the sensor, combining with the array of screen printing electrode (array SPE ), we can achieve the high throughput detection of the anaerobic bacteria. Therefore, the construction of the DNA sensor is expected to realize the high sensitivity and high throughput detection of the anaerobic bacteria in gas gangrene. It can also provide a alternative solution for others similar microbial detection.

气性坏疽是战伤、地震伤的严重并发症之一，引起气性坏疽的病原菌主要有产气荚膜梭菌、破伤风梭菌、诺维梭菌等，准确、快速、简便地检测病原菌对气性坏疽的早期诊断和及时治疗具有十分重要的指导价值。本课题计划利用环介导等温扩增技术(LAMP)快速扩增病原菌DNA，结合电致化学发光技术(ECL)高灵敏度检测，构建丝网印刷电极(SPE)传感器，实现对气性坏疽多种病原菌的高通量检测。1）首先，完成了LAMP恒温扩增与检测技术的建立与优化。通过在线软件primerexplorer进行引物设计，利用交流阻抗谱法等对传感器包被效果进行表征、检测、LAMP反应条件优化。在LAMP恒温扩增技术的基础上，进一步探索DNA滚环扩增（RCA）技术，并通过Hemin结合RCA产物，实现了病原菌DNA的在传感器表面的高效扩增。2）其次，完成了纳米复合传感器的研制。构建了传感器表面的双催化信号放大系统，通过优化流程的电化学检测、电镜检测，对构建传感器进行表征及性能评价，调整修饰条件、保持温度、稳定剂和检测条件参数，进一步提高了传感器的灵敏度、特异性、重复性和稳定性。3）然后，利用石墨烯、纳米金等纳米材料在SPE上进行分层自组装，然后在SPE电极上组装ECL复合物，借助GDH高效快速催化产生ECL的共反应试剂，实现对DNA的高灵敏快速检测。优化了SPE传感器基质包被条件、纳米微粒的种类和修饰方式、LAMP扩增、DNA分子识别、电致化学发光检测等条件参数。整体而言，在传感器上实现了LAMP或RCA等恒温扩增、目标DNA捕获与信号放大、CV、DPV和ECL高灵敏度检测。完成了对气性坏疽主要病原菌检测的初步性能评价，实现了气性坏疽主要病原菌的高灵敏、特异性、现场快速检测。另外，课题改进的纳米材料修饰、ECL双催化信号放大等方法也可以对其他传感器构建提供解决策略。