基于DNA三维纳米探针的微尺度电化学microRNAs检测新方法

.The quantitative detection of microRNAs provides great insight into understanding of pathogenic mechanism, tumorigenesis and the diagnostics of tumor. This project combines functional three-dimensional DNA nanostructured probe and microfluidic chip to assemble electrochemical sensing array and construct an integrated microdevice for highly sensitive and multiplexed detection of miRNAs. A one-step method will be taken to rapidly assemble 3-D DNA nanoprobes with specific recognition, uniform orientation and minimal steric effect, and the as-prepared nanoprobes can immobilize on arrayed electrode surface as capture probes. Reagent plugs including targets, signal probes and washing solutions that segmented by air spacers can be driven along microscale sensing surface with enhanced hybridization efficiency of nucleic acids, and thus a high-performance, rapid and sensitive electrochemical detection of miRNA can be realized. Moreover, it is really important to investigate the related scientific issues such as the directed conjugation and oriented assembly of biomolecular probes at micro- nanoscale surfaces, and the motion of nucleic acids in channel with herringbone microstructures. The distinguish feature of this project is that the limitations, including fall-down of single nucleic acid strands, weak binding ability and long diffusion distance at sensing surface, can be effectively addressed by combining the excellent biorecognition of 3D DNA nanostructured probes and the controllable fluids manipulation by microfluidic technology. The successful project implementation is helpful to advance the sensitive and rapid detection of miRNAs associated with breast cancer.

微小RNA(MiRNA)的定量检测对深入理解肿瘤的发生发展和诊断等具有重要意义。本项目拟以DNA纳米结构探针的设计和组装为基础，结合微流控芯片技术，构建阵列电化学传感界面，并藉此搭建功能集成的微型分析装置，从而实现乳腺癌相关的多种miRNA的高灵敏检测。本项目将快速组装具有特异识别能力、界面朝向均一、空间位阻小的3-D DNA纳米探针并固定在相应探针捕获区域。通过微通道引入被空气间隔开的靶标、信号探针和洗脱溶液等区带，对微流体进行有效操控，增强核酸杂交效率，以进行高效、快速、灵敏的电化学检测。并对其中所涉及的生物探针在微纳米界面上的定向偶联、有序组装和核酸分子在微通道中的运动特性等科学问题进行深入研究。本项目研究特色是：结合DNA纳米探针优异的分子识别能力和微流控独特的样品操控特性，解决了传统单链探针易倒伏，识别效率低和界面分子扩散距离过长的局限性。该研究有望对乳腺癌早期检测产生积极影响。

标志物的准确定量分析对疾病的早期诊断意义重大。通过本项目的研究，我们成功合成出不同靶标响应的3-D DNA纳米结构探针。在此基础上，结合液滴微阵列技术构建了可寻址的毛细管微阵列，有序组装DNA纳米结构探针，实现核酸目标物的高效捕获。研制出气泡介导的一步式多元检测微流控电化学传感器用于肿瘤标志物的高灵敏传感分析，并用于前列腺癌病人的血清样本分析。设计的miR21特异响应性DNA纳米结构探针实现了皮摩尔水平miR21的电化学检测。在深入研究传感界面核酸动态结构转换的基础上，利用电极表面的核酸动态组装原理设计了部分杂交的高响应性“开口式”颈环结构探针。该设计可彻底消除因结构转换后受限而无法移除的探针分子所引起的高背景噪音，从而大幅度提高检测的灵敏度（0.18 fM）。部分成果有望进一步用于临床疾病的早期检测研究。