基于核酸适配体的结核杆菌电化学传感器的研制与应用

M.tuberculosis is the most serious disease of single factor hazard to human health. The appearance of resistance strains and AIDS co-infection have made the TB become the biggest challenge of public health we have to face.Through the research process of " the researching and application of new M.tuberculosis detection technology based on piezoelectric sensor", We have found that the detection time of M. tuberculosis based on the cultivation method were too long to meet the demands of TB control and prevention because of its long generation cycle.Based on the research results of previous project, we plan to construct new M.tuberculosis detection technologies based on the sensitive response of electrochemical sensor to electric parameters and specific recognition of aptamer.M.tuberculosis will be rapid and specific detected by this new method.The constructed method will meet the demand of clinic test with characteristics of simple, rapid and specific, not affected by the cycle time of T. tuberculosis.It will overcome the disadvantages of method based on antigen-antibody reaciton, such as high false positive results and difficult to obtain monocolone antibody.The main contents are described as follows: ⑴ the aptamer screening for the detection of M. tuberculosis by the TECS-SELEX methods; ⑵ preparation of electrochemical probe based on aptamer; ⑶the estalishment of rapid detection of M. tuberculosis based on aptamer response;⑷Application and evaluation of new detection methods in the assay of actual sample analysis by statistical method.

结核病是单因素危害人类健康最严重的疾病。耐药菌株的出现和艾滋病合并感染使结核成为我们面临的最大公共卫生挑战。通过"结核分枝杆菌压电测试新技术研究和应用"课题的研究，发现以培养法为基础建立的分析检测方法，检测速度受结核分枝杆菌分裂周期长限制，无法再提高。本课题拟在前课题的基础上，构建基于适配体分子识别的结核分支杆菌电化学传感测试新技术。利用适配体对靶细胞的特异性识别和电化学传感器对电参数的灵敏响应，实现结核分枝杆菌的快速特异检测。所构建的传感方法既能克服培养法受细菌增代时间限制的缺点，又能克服抗原抗体法单克隆抗体难得，假阳性太高的缺点；具有简单、快速和特异性，满足临床检测需求。 具体内容有：⑴筛选用于检测的结核分枝杆菌适配体；⑵制备基于核酸适配体的电化学敏感探针；⑶建立基于核酸适配体响应的结核分枝杆菌电化学检测新方法⑷ 分析统计评价所建分析方法在临床应用的结果。

结核病是严重危害人类健康的慢性传染病，是全球关注的公共卫生问题和社会问题。快速准确的早期诊断是防治结核病的有效措施和控制结核病传播的关键步骤之一。针对目前结核杆菌检测方法存在检测速度、特异性和灵敏度不能满足检测需求这一实际问题，展开了基于核酸适配体的结核杆菌压电传感方法的研究。小组以结核分枝杆菌菌体为原材料进行正向筛选，以耻垢分枝杆菌、卡介苗、大肠杆菌、绿脓杆菌、金黄色葡萄球菌、肠炎沙门氏菌的混合物为靶标进行反向筛选，得到了新的与结核分枝杆菌具有高特异性和结合力的适配体（Apt），解决响应的特异性问题；研究了电极形状对传感器灵敏度的影响，解决传感器响应灵敏度问题；利用碳纳米管的优良导电性能，巧妙设计和解决了传感信号的响应问题；最终构建了构建了新型多通道单壁碳纳米管\结核分枝杆菌适配体压电传感器（SWCNTs/Apt/ IDE-MSPQC），用于结核分枝杆菌快速特异性检测。通过Au-S键将Apt与金叉指电极结合，再通过π-π作用，将单壁碳纳米管吸附在电极表面。当检测液中有结核分枝杆菌存在时，与Apt具有高亲合力和特异性的结核分枝杆菌，会与Apt发生特异性结合，从而将单壁碳纳米管从Apt表面置换出来，导致叉指电极表面电参数发生变化，从而引起压电传感器的频率响应。研究结果表明，新构建的传感器，检测结核分枝杆菌所需时间为1h，检测下限是100cfu/mL。该传感器不仅快速、灵敏，而且特异性强高，不仅能够区分分枝杆菌与其它杆菌，还能区分结核分枝杆菌、牛型分枝杆菌（卡介苗）、耻垢分枝杆菌。此法有望在结核病的早期临床诊断方面得到广泛的应用。