基于致病菌aptamer/分体式DNAzyme双功能核酸的新检测体系研究及应用

Development of sensitive detection methods is of great significance for preventing and controlling public health security caused by pathogenic bacteria, which pose a serious threat to human health. Several integrated DNAzyme-based probes have been developed for the detection of pathogenic bacteria, depending on the principle that combining with targets from pathogenic bacteria can causes catalytic cutting reaction. However, very small amount of bacteria produce a little targets so that free DNAzyme cannot combine with targets rapidly and efficiently. Limited number of targets just active limited amount of DNAzyme for catalytic cutting reaction. Therefore, integrated DNAzyme-based detection methods lead to difficulties in increasing sensitivity. To developing a novel detection system for targeted and high efficient detection, this project will combine pathogenic bacteria-specific aptamer and the same pathogenic bacteria-specific DNAzyme. Moreover, designing a cycle amplification strategy based on catalytic cutting reaction can be achieved by splited DNAzyme (including enzyme strand and substrate stand), resulting in improving utilization of targets from pathogenic bacteria. On this basis, simple, rapid, high sensitive and selective detection methods will be developed. Then, combining with glucometer to develop a simple detection method for daily monitoring, this method could be readily converted into a mature product. Furthermore, integrating porous nanomaterials for bactericide loading with functional nucleic acids as smart switches to control the drug release, this project will carry out the preliminary study of the integration of targeted detection and sterilization.

致病菌极易威胁人类健康，发展高灵敏检测方法对防控致病菌引起的公共卫生安全问题有重要意义。目前已有部分基于一体式脱氧核酶（DNAzyme）发展的致病菌检测方法，主要原理是结合目标致病菌分泌出的靶标物引起催化切割。但低浓度的目标致病菌导致靶标物浓度也低，此时游离的DNAzyme无法快速有效地结合靶标物，且有限的靶标物能激发的切割反应也有限，导致灵敏度大大受限。本项目拟结合同一目标致病菌的核酸适配体（aptamer）和DNAzyme，建立一种双功能核酸新检测体系，实现定向高效检测。同时，设计分体式DNAzyme（酶链和底物链），采用酶切循环放大策略，提高靶标物利用率。基于此，本项目将发展快速简单、高灵敏、强特异性的致病菌检测探针，并结合血糖仪开发便携检测方法，进行产品转化。后期将结合多孔纳米材料载药，设计功能核酸为智能开关，实现靶向检测同时给药杀菌，初步开展致病菌查杀一体化研究。

致病菌感染可引发多种疾病，该种公共安全问题对人类健康、社会经济和生态环境等方面造成极大影响。目前致病菌的各类检测方法仍旧存在灵敏度低、选择性差、耗时耗力或信号不准等问题。因此，发展简单快速、高灵敏及强特异性的致病菌现场检测技术具有重要的研究意义。本项目结合了功能核酸的识别功能、DNA自组装特性和纳米材料的光学特性、催化特性，建立了多种性能优良的功能核酸纳米传感体系用于对致病菌的超灵敏、可视化、实时检测。① 本研究基于特殊DNA序列的模板及识别作用，设计发夹型DNA-CuCNs荧光纳米探针用于沙眼梭菌的靶向快速检测。该方法创新性地将合成与检测合成一步，具有成本低、操作简单、室温5 min即可完成检测的优点。② 本研究基于触发式DNA自组装实现杂交链式反应（HCR）策略，构建了功能核酸荧光传感体系用于大肠埃希菌的检测。本方法结合HCR反应和其产物增强SYBR GREEN I荧光作用，实现了荧光的双重放大，检测灵敏度大大提高。③ 本研究采用aptamer和凝集素作为双识别分子，结合磁分离及诱导聚集变色策略，建立了一种具有高效浓缩富集和高特异性的可视化新方法用于鼠伤寒沙门氏菌的检测。通过优化磁珠修饰aptamer浓度和其复合物浓度，该磁性捕获探针对目标菌的捕获率可达81.48%。本方法可通过肉眼进行半定量可视化检测，也可通过仪器分析进行定量检测。④ 本研究在前期磁分离富集和DNA自组装基础上，进一步设计了一种比色-荧光双信号检测体系。与目前已报道的方法相比，本方法表现出了宽检测范围的同时具备更高的灵敏度，其荧光法和比色法分析检出限分别可达1 CFU/mL和3 CFU/mL。本检测体系可通过比色法进行简单、便携的可视化检测需要，还可通过荧光法实现更高的灵敏度及准确度检测，双信号输出相辅相成使得检测结果更加准确可靠。本项目的完成，将拓宽核酸-纳米材料的设计策略，便于开发致病菌高灵敏、简便的新检测方法，为食品安全分析、细菌感染的早期诊断提供一定的技术支撑。并且为细菌感染的诊疗一体化提供新思路、理论依据和技术支持。