DNA点击连接链式反应放大电化学传感的构建及超灵敏检测胶质瘤脑脊液IDH1突变的研究

Glioma is the most common intracerebral malignant tumor, in which IDH1 gene mutation often occurs in early stage and has become a key therapy target. Currently, repeatedly invasive operations in the brain required to obtain tumor tissue for mutation analysis are of great risk, while liquid biopsy-based method via cerebrospinal fluid has the significant advantages of little injury, representative integrity, easy sampling and fast speed, so that dynamic monitoring the progression of glioma could be easily achieved. However, the detection of low abundance IDH1 mutation in cerebrospinal fluid is still a great challenge. Herein, this project, on the basis of our previously proposed strategy “a dsDNA-assembly electrochemical biosensor developed by probe lengthening through ligase chain reaction”, provides a new ultrasensitive electrochemical biosensor amplified by DNA clickable ligation chain reaction via combining click chemistry and DNA-templated organic synthesis. Specifically, this work focuses on the study of a mutant-DNA promoted non-enzymatic exponential amplification system using synergistic effect between hybridization kinetics and spatial distance requirement, and also the study of the construction of electrochemical biosensing interface, both of which is aimed to solve the poor reproducibility caused by use of enzymes and the high sensitivity requirement of low-abundance mutation detection. Importantly, this project would realize the monitoring of IDH1 mutation in cerebrospinal fluid by means of electrochemical sensing, with low cost, fast speed, high stability and sensitivity, to help clinician to make better treatment plans, and provide important research basis and scientific guidance for the detection of extremely low abundance point mutation and clinical precision medicine.

胶质瘤是最常见的颅内恶性肿瘤，IDH1基因突变是胶质瘤早期发生及治疗关键靶点。目前，临床反复的脑内有创性操作来获取肿瘤组织进行突变分析具有极大的风险。基于脑脊液的液体活检方法具有损伤小、代表全、易取样、快速等优点，可以反复、动态地监测胶质瘤的变化，然而，脑脊液中极低丰度的IDH1突变检测是一项极具挑战性的工作。本项目在前期发展的一种基于连接酶链式反应的探针加长式dsDNA组装型电化学传感器基础上，结合点击化学和DNA模板有机合成技术，利用杂交动力学和点击官能团空间要求两者的协同作用，研究无酶催化的突变DNA指数扩增体系和电化学传感界面的构建，解决使用生物酶造成的重现性差以及低丰度突变检测的高灵敏度要求，实现以电化学传感的方式低成本、快速、稳定、高灵敏的监测脑脊液中IDH1突变，帮助胶质瘤患者制定更好的治疗方案，为极低丰度核酸突变检测和临床精准医疗提供重要的研究基础和科学指导。

胶质瘤是最常见的颅内恶性肿瘤，IDH1基因突变是胶质瘤早期发生及治疗关键靶点。目前，临床反复的脑内有创性操作来获取肿瘤组织进行突变分析具有极大的风险。基于脑脊液的液体活检方法具有损伤小、代表全、易取样、快速等优点，可以反复、动态地监测胶质瘤的变化，然而，脑脊液中极低丰度的IDH1突变检测是一项极具挑战性的工作。本项目首先构建了基于点击化学介导连接链式反应的DNA电化学传感平台，实现了核酸靶标的超灵敏检测。为了进一步提高方法学特异性，我们提出了基于嵌合体DNA探针辅助连接链式反应和基于“邻近双摇摆策略”的连接反应分析，构建了多种新型DNA电化学传感策略，为痕量点突变的检测提供了新思路，同时也为实现临床IDH1突变基因检测奠定扎实的研究基础。

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