一种检测结核分枝杆菌抗原标志物的方法学研究——基于signal-on型电化学适体检测体系的构建及应用

Tuberculosis is a chronic infectious disease which seriously imperils health of human beings. However, the present methods for tuberculosis diagnosis have the defects of complicated time consuming, expensive equipment, low sensitivity and poor specificity. Signal-on electrochemical aptasensors have the advantages of simple operation, high sensitivity, low cost and good selectivity. However, these aptasensors have the requirements of target binding sites and number of binding aptamers, which limits its application. This project intends to take Mycobacterium tuberculosis (MTB) antigen as the research object and organically combine the signal conversion with biological amplification technology through the reasonable design of base sequence of aptamers and the corresponding probes. Then the general versatility of the aptasensor and target-cycling amplification will be realized. The enzyme labeled C60-polyaniline (C60-PANI) with excellent performance are prepared and used for the generation and multiplex amplification of the detection signal for the first time. And on this basis, high throughput detection of MTB antigens would be achieved combined with screen-printed electrode (SPE) array technology. Thereby, a new and versatile signal-on elctrochemical aptamer detection method with high sensitivity and specificity, good stability and rapid response, will be established for the combined detection a variety of MTB antigen markers (such as CFP10, ESAT6, Ag85, etc), which provides an innovative technology platform for the early diagnosis of tuberculosis.

结核病是严重危害人类健康的慢性传染病，但现有的结核诊断方法存在繁琐耗时、仪器昂贵、灵敏度和特异性低等缺陷。Signal-on型电化学适体传感器具有简便快速、成本低、灵敏度高、选择性好等优点，但对于目标分析物的结合位点和亲合适体数量均有要求，因而其应用受限。本项目拟以结核分枝杆菌（MTB）抗原标志物为研究对象，通过合理设计适体及相应探针序列而将信号转换与生物放大技术有机结合，提高传感器的适用性并实现目标物的循环放大；首次设计制备性能优异的酶标记C60-PANI复合物用于检测信号的产生及多重放大；采用SPE阵列技术实现MTB抗原的高通量检测；从而建立一种敏感特异、高效快速的signal-on型电化学适体检测新方法用于临床样本中多种MTB抗原标志物（如CFP10、ESAT6、Ag85等）的联合检测，为结核病的早期诊断提供创新性的技术平台。

结核病是由结核分枝杆菌（MTB）感染引起的慢性传染病，致死率高、危害大，但现有的结核诊断方法存在繁琐耗时、仪器昂贵、灵敏度和特异性低等缺陷。电化学适体传感器具有简便快速、成本低、灵敏度高、选择性好等优点，但对于目标分析物的结合位点和亲合适体数量均有要求，因而其应用受限。本项目以结核分枝杆菌（MTB）抗原标志物（MPT64、CFP10、ESAT6）和MTB插入序列6110的特异性DNA为研究对象，通过合理设计适体及相应探针序列而将信号转换与生物放大技术有机结合，提高传感器的适用性并实现目标物的循环放大；设计制备多种性能优异的复合纳米材料用于检测信号的产生及多重放大；临床样本检测，并与现有的临床测试方法进行对照，从而建立一种敏感特异、高效快速的MTB电化学适体检测新方法，为结核病的早期诊断提供创新性的技术平台。相关研究成果在Biomaterials（IF=10.273）、Biosensors and Bioelectronics（IF=9.518）、Sensors and Actuators B: Chemical（IF=6.393）等国际知名学术期刊发表SCI论文11篇，累计影响因子IF大于70。申请国家发明专利4项，其中1项已授权。