基于毛细管液滴多重不对称MSP/阵列DNA纳米探针的微流控电化学肿瘤多基因甲基化传感新方法

As an early event, multigene promoter hypermethylation is closely associated with tumor occurrence, progression, metastasis and recurrence. In this project, we make a deteiled research about interfacial assembly of DNA nanostructure probe and solution manipulation, droplet multiple asymmetric MSP (MA-MSP) and molecular recognition in microfluidic channel. And an intelligent electrochemical microfluidic array platform is proposed for the biosensing of multigene methylation. By employing the solution delivery system, the water-in-oil amplification reaction mixture and several air-segmented buffer are simply formed in the teflon capillary tube, respectively. Lots of different single-strand (ss) DNA could be generated at the same time by the fast and highly efficient amplification of target genes in the heating zone of the tube. The separation in space between the amplification process and subsequent detection process effectively prevents their mutual interference as well as the complicated design of chip. The MA-MSP mixture containing ssDNA products will be easily separated from the oil and mix with the hybridization buffer during the controllable and low-loss solution delivery. Subsequently, the mixed buffer and latter signal molecule (poly-HRP) is successively delivered into the DNA nanoprobe-functionalized chip, resulting in the efficient capture of ssDNA followed by poly-HRP. After that, a high HRP-catalyzed amplified electrochemical signals is obtained, which will contribute to the decrease of PCR cycle and false positive. Eventually, the simultaneous, fast, accurate and highly sensitive detection of multigene methylation in trace samples could be achieved with the accomplishment of the project, which would provide a new ideas and technology platform for the early diagnosis of cancer.

多基因甲基化是与肿瘤发生、发展、转移密切相关的早期事件。本项目围绕DNA纳米探针的界面组装及微尺度下的溶液操控、液滴多重不对称MSP(MA-MSP)、分子识别等问题进行研究，以肺癌为研究对象，拟构建新型智能化阵列微流控电化学多基因甲基化传感平台。利用溶液传递系统，在聚四氟乙烯毛细进样管内，简单形成"油包扩增反应液"液滴及空气间隔的溶液区带。通过循环加热在管内实现目标基因的同时、快速、高效MA-MSP，产生大量单链DNA。扩增反应与随后检测的空间分离可有效避免相互影响及芯片设计的复杂性。操纵溶液进行可控、低损移动，扩增产物溶液与油相分离后同杂交缓冲液融合，并与后续溶液依次流入阵列DNA纳米探针的微流控芯片，实现单链产物和信号探针高效捕获、酶催化放大的高灵敏电化学检测，以期减少扩增循环数，降低假阳性，最终实现微量提取样本中多基因甲基化的同时、快速、高灵敏及准确检测，为肿瘤早期诊断提供新方法。

基因甲基化是与肿瘤发生、发展、转移密切相关的早期事件。本项目围绕DNA纳米探针的界面组装及分子识别、微尺度下溶液操控与非对称甲基化特异性PCR等问题进行研究。以肺癌为研究对象，设计具有连续识别模块的系列核酸定向扩增策略，消除体系非特异性扩增的干扰，提升甲基化DNA分辨率达到0.1%；设计DNA纳米结构探针，调控纳米探针在界面的定向偶联及有序组装，同时整合非对称甲基化特异性PCR进行电化学催化扩增，级联放大检测信号，灵敏度高达单拷贝，已成功用于微量（200μL）肺癌患者血浆样本中的甲基化DNA分析，显著优于传统PCR；利用溶液传递系统，在聚四氟乙烯毛细进样管内，简单形成“扩增反应液”液滴及空气间隔的溶液区带。通过循环加热在管内实现目标基因快速、高效的扩增，产生大量扩增产物；采用电子束蒸发制备表面均一的阵列金电极，设计并制作可以匹配阵列金电极结构的PDMS微通道，利用注射泵和阵列电极接口装置组合一体化的电化学传感平台，经DNA纳米探针功能化修饰传感界面，可实现单链产物和信号探针高效捕获、酶催化放大的高灵敏电化学检测。本项目在研期间共培养博士研究生4人，硕士研究生5人，相关研究成果已发表SCI论文21篇，申请国家发明专利1项。本项目的完成将为微量提取样本中多基因甲基化的同时、快速、高灵敏及准确检测提供新方法，为肿瘤的早期诊断、预后评估、治疗监测和复发检测提供新思路。

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