电极表面锁式探针滚环信号放大及其荧光、电化学、ECL多模式检测方法

Rolling Circle Amplification (RCA) is one kind of isothermal nucleic acid amplification techniques, which avoids the intense temperature change during the polymerase chain reaction (PCR), and the reaction time is shortened. Compared with PCR, RCA exhibits not only much convenience both in the actual operation and equipment requirements, but also good application prospect in rapid diagnosis and clinical application. While, there are still some shortcomings in RCA like false positives, background interference, lack of ideal quality control method, difficult to realize high throughput and multi-target detection simultaneously etc. The existing methods cannot solve these problems. Hence, in this proposal, DNA primers are firstly connected with the graphite carbon materials like graphene oxide (GO) and chemical reduced graphene (CCG) coated on the working electrode (glassy carbon, ITO) surface, then a new kind of padlock RCA probe are designed on the working electrode surface. After RCA, the electrode can be picked up from the system, and the false positive of RCA and background interference can be eliminated through simply rinsing. The fluorescent probe sequence adsorbed on the surface of the GO or CCG can be employed to check the RCA result through fluorescence, electrochemistry and ECL (electrochemiluminescence) detection, and a triple-mode RCA detecting method can be established with independent intellectual property rights. All these are helpful to expand the RCA structure design, the RCA result detection method and laying a foundation for the establishment of the RCA quality control standard. In order to check the applicability of the proposed method and the practical effect, a serious of virus threaten to human health such as the ebola virus, HIV, HBV are selected to develop RCA measuring technology, which can be employed for monitoring and clinic diagnosis of the related virus. To fulfill triple-mode detection of the three virus at the same time, three typical types of RCA probes like ebola, HIV, HBV are going to be modified on the same electrode surface, and the corresponding fluorescent probes sequences (labeled with Ru, Ir, Os, respectively) are loaded onto the corresponding material (GO, CCG) surface, then the detection can be realized through fluorescence, electrochemistry and ECL.

滚环扩增(RCA)是一种核酸等温扩增技术，避免了聚合酶链反应（PCR）剧烈的温度变化，反应时间更短，实际操作和仪器要求比PCR更为便捷，在临床和现场快速诊断中呈现良好前景。但目前RCA存在背景干扰、检测方法单一等缺陷，难以同时实现高特异、高灵敏度、高通量、多目标检测，现有方法都不能解决上述问题。本申请将引物DNA与修饰在电极表面的石墨材料连接，在工作电极表面构建锁式RCA探针，RCA后从体系中取出电极，简单冲洗即可得到电极表面的RCA产物，利用负载在材料上的荧光探针序列对其进行荧光、电化学、ECL检测，消除背景干扰，开发多模式RCA检测方法。进一步地，将埃博拉、HIV、乙肝病毒等锁式RCA探针共同接枝到同一工作电极表面，再将与其对应的荧光探针序列共同负载到同一探测材料表面，实现三种病毒的高通量、多目标、多模式检测。为埃博拉病毒、HIV、乙肝病毒的筛查、监控及临床高效、快速诊断创造条件。

滚环扩增(RCA)是一种核酸等温扩增技术，相比聚合酶链反应（PCR）反应时间更短，实际操作和仪器要求更为便捷，在临床和现场快速诊断中呈现良好前景。但目前RCA存在背景干扰、检测方法单一等缺陷，难以同时实现高特异、高灵敏度、高通量、多目标检测，现有方法都不能解决上述问题。因此本项目建立了RCA埃博拉病毒荧光可视化检测方法，同时开发了Hg2+、Cu2+等金属离子、肿瘤可视化探测、肿瘤靶向给药等ECL、荧光可视化检测技术。在此基础上，聚焦肿瘤治疗热点领域，对近年来相关研究做了详细综述，设计了系列荧光可视化靶向药物递送系统及诊疗一体化试剂，实现了荧光可视化光热/光动/化疗组合治疗肿瘤。相关研究为今后肿瘤组合治疗及其多功能给药平台的设计和应用奠定基础。