对流驱动下的三维DNA纳米结构探针芯片设计及其在胃癌循环miRNA的快速微阵列分析中的研究

Gastric cancer (GC) is one of the most common malignant tumors in China. Circulating microRNA (cmiRNA) is well-known as highly sensitive and non-invasive biomarkers for early diagnosis of GC. However, the overall amount of cmiRNA in blood was extremely low, therefore, the development of a rapid and sensitive analytical tool is highly desirable for early diagnosis of GC. This project build the regulation system for nonequilibrium state bio-interfaces, low cost fabrication of highly reactive DNA tetrahedron nanostructured probes microarray in microchannel of microfluidic chip. Introduction of dynamic hybridization system, convection-driven sample to hybridize with probes array in microchannel, greatly reduces the analysis time and improve detection efficiency. Objective to realize rapid determination of multiple microRNAs and establish a high throughput analysis approach for miRNA. Laser induced fluorescence detection technology and hybridization chain reaction (HCR) technology were used to enhance the fluorescent signal, and it improved signal-to-noise ratio. The project aims at achieving ultra-sensitive, high accuracy, easy and simple to handle and low cost rapid detection of cmiRNA in blood serum samples, so that ultimately realizes the non-invasive and efficient “real-time liquid biopsy” and realizes early diagnosis of gastric cancer to provide technical support.

胃癌是中国最常见的恶性肿瘤之一。循环miRNA（Circulating MicroRNA，cmiRNA）是肿瘤早期诊断重要的敏感性非侵入性肿瘤标志物，但其在血液中含量低，因此，发展快速灵敏的早期检测系统是当前胃癌研究中亟待解决的难题之一。本项目拟通过构建非平衡态生物界面调控系统，在微通道内固定高活性的四面体DNA结构探针，引入动态杂交系统，在对流驱动下建立非平衡态检测，实现样品的快速杂交反应，缩短样品检测时间和提高检测效率，建立高通量miRNA检测分析方法。同时利用激光诱导荧光检测技术和杂交链式反应（HCR）放大技术增强荧光检测信号，提高检测信噪比，并将其用于检测多个与胃癌相关的miRNA，发展一种用于早期胃癌诊断的高灵敏度、高准确度、操作简便且成本低廉的快速miRNA定量分析方法，从而实现对胃癌患者的微创、灵敏、特异、高效的“即时液体活检”，为实现其在胃癌早期诊断中的应用提供技术支持。

本项目瞄准“健康中国战略”，针对慢性病筛查和早期诊断这一社会需求问题。通过融合临床医学、化学测量学和仪器与技术等多种相关学科，解决胃癌早期症状不典型、常规筛查技术不普及等问题。借助“液体活检技术”，以早期胃癌相关的三种血液循环miRNA（miRNA-627，miRNA-629和miRNA-652）为研究对象，构建一种早期胃癌诊断的高灵敏度、高准确度、操作简便且成本低廉的快速miRNA定量分析方法和设备，为其他生物、化学分子的超灵敏高通量快速检测分析提供了一种可行的技术途径。本项目分别从基于激光诱导荧光的微流控液滴微阵列分析系统的搭建和优化；DNA四面体结构探针在微通道三维传感界面密度的调控；基于DNA纳米技术的信号扩增技术的开发和多样本同时检测等方面开展工作，进行项目的实施，已完成微流控液滴微阵列生成系统原型机的搭建，并与激光诱导荧光检测装置结合，验证了微通道三维探针微阵列的稳定性和可靠性，解决了多样本同时实时检测的问题，提高了诊断的准确性；通过设计多种尺寸四面体结构DNA探针，完成了对微通道三维传感界面活性的调控，同时通过与基于目标miRNA触发的HCR信号扩增方法相结合，显著提高了检测灵敏度，检出限达到pM级别，有效解决了循环miRNA在血液样本中丰度低难以检测的问题。