DNA微流控芯片的微纳循环肿瘤标志物跨尺度精准分离与单标志物水平检测

Liquid biopsy based on the detection of circulating tumor biomarkers, such as circulating tumor cells, tumor extracellular vesicle, can be used for early diagnosis of cancer, classification and staged diagnosis and companion diagnosis, showing significant value in fundamental research and clinical application. Combined detection of multiple markers at the single marker level can effectively improve detection sensitivity and specificity, and comprehensively cover tumor heterogeneity. This project aims at developing a DNA system-based integrated, without external field manipulation of microfluidic device, targeting at the difficulty of cross-scale separation of circulating tumor markers and detection of single markers. Numerical simulations and experimental methods will be used to systematically investigate separation mechanism of micro/nano-particles under the interface of DNA viscoelastic-Newtonian fluid in microfluidic devices, realize cross-scale, label-free separation and enrichment of CTC and EV in whole blood. Detect single CTC gene mutations and cancer-associated membrane proteins of single CTC and EV in isolated and enriched samples using aptamer recognition and digital PCR technology. Analysis of the correlation between the rich molecular information of heterogeneous CTC and EV and clinical test results through machine learning algorithm. The successful implementation of the project will effectively promote the development of new tumor liquid biopsy for multi-marker joint detection and its clinical application in various stages of cancer early detection.

针对外周血中的循环肿瘤标志物（循环肿瘤细胞、肿瘤细胞外囊泡等）检测的液体活检技术，可用于癌症早期诊断、分型分期及伴随诊断，兼具重要的科学研究及临床应用价值。在单标志物层面进行多种标志物的联合检测，能够有效提高检测灵敏度与特异性，并全面覆盖肿瘤异质性。针对循环肿瘤标志物跨尺度分离难与单标志物检测难的挑战，本项目拟设计开发基于DNA体系的集成化、无需外场操控微流控器件：采用数值仿真和实验手段系统研究微纳颗粒在微流控器件中DNA黏弹性-牛顿流体界面中分离机理，实现全血中CTC与EV的跨尺度、无标记分离、富集；利用核酸适体识别与数字PCR技术，对分离富集样本进行单CTC基因突变检测与单CTC、EV的癌症相关膜蛋白检测；通过机器学习算法分析异质性CTC、EV中丰富的分子信息与临床检测结果的相关性。项目的成功实施将有效推进多标志物联合检测的新型肿瘤液体活检的开发，以及其在癌症早期检测等各阶段的临床应用

针对肿瘤发生发展过程中释放到外周血的细胞外囊泡（EVs）和循环肿瘤细胞（CTCs）的分子检测，对肿瘤的早期精准诊断、分型及预后监控具有重要意义。本项目发展了系列微流控分离检测新工具，力求解决复杂生物样本中CTC、EV分离效率低、检测灵敏度低等问题，取得如下创新性成果：.（1）设计开发微流控无标记分离系统，无需复杂前处理与复杂外围设备，实现了全血样本中EV与CTC的直接、快速、高效分离富集：EV的分离效率> 70 %，优于当前金标准超速离心法；对低丰度CTC的富集倍数>700，分离后CTC的免疫荧光分析与单细胞计数与HER2分析可用于乳腺癌诊断。.（2）结合DNA纳米探针与基于热泳的EV定向富集，发展了EV检测新方法，实现了临床血浆血清样本中EV蛋白、核酸靶标的原位、高灵敏检测：对EV蛋白的灵敏度比传统ELISA高三个数量级，对EV miRNA检出限为0.36 fM、EV mRNA检出限低至14 aM与qRT-PCR相当。结合DNA分子运算与微流控热泳，实现了单EV表面多蛋白共表达检测。微流控热泳检测灵敏度高、样本消耗量小、原位定量测量、有效排除基质干扰，为基于EV的肿瘤液体活检提供了有力工具。.（3）在此基础上，利用机器学习基于EV分子表达图谱构建EV分子特征，用于区分转移乳腺癌（MBC）患者、非转移性乳腺癌患者、年龄配对的健康对照者，准确率高达91.1 %；监测MBC的治疗效果，区分治疗后疾病进展（PD）与稳定/部分缓解（SD/PR）的准确率为88.5 %；并可作为MBC患者的无进展生存期（PFS）的独立预后因素。

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