基于液滴微流控技术的单个外泌体内miRNA数字化检测新方法及其响应机制研究

The detection of miRNA in exosome is of great significance in the early and rapid diagnosis of diseases. However, most of the existing detection techniques require extraction of total RNA in exosome before analysis, which have the disadvantages of cumbersome operation, time consuming and large sample volume. Simultaneously, the low-abundance disease-specific miRNA detection is susceptible to be interfered from other nucleic acids background signals in exosome. Therefore, it is urgent to establish a new convenient, sensitive and specific method for miRNA detection in single exosome to avoid background signals interference. In our previous work, we have realized the digital detection of proteins on single exosome membrane by the droplet microfluidic technology, and we have also constructed a catalytic hairpin assembly (CHA) technique to detect miRNA. Based on this, we propose to perform single encapsulation, recognition, localization and lysis of exosome on the microdroplet chip, so that the target miRNA released from the individual exosome can catalyze the CHA reaction in the microdroplet to amplify the detection signal, establishing the digital detection system of miRNA in single exosome, and followed by elucidating the reaction mechanism, optimizing the analysis conditions, evaluating the detection performance. Finally, we verify its clinical diagnostic performance by detecting the miRNA in the exosome of the plasma samples from breast cancer patients, aiming to provide experimental basis and theoretical basis for the construction of a new highly sensitive, rapid and simple digital detection technology of miRNA in single exosome for clinical diseases detection.

外泌体内miRNA的检测在疾病早期快速诊断中具有重要意义。然而，现有技术大多需要在分析前对外泌体中总RNA进行提取，存在操作繁琐、费时、需样量大等缺点，并且对于低丰度疾病特异miRNA的检测易受其他核酸背景信号的干扰，因此急需建立一个便捷、灵敏、特异、避免背景信号干扰的单个外泌体内miRNA检测新方法。前期，我们利用液滴微流控技术实现了单个外泌体膜蛋白的数字化检测，同时也构建了DNA催化发夹自组装技术（CHA）检测miRNA的方法。据此，我们提出在微液滴芯片上进行外泌体的单个包裹、识别、定位与裂解，使裂解释放的靶miRNA在微液滴中催化CHA反应，建立单个外泌体内miRNA的数字化检测体系，并阐明反应机理、优化分析条件、评价检测性能，最后通过检测乳腺癌病人血浆外泌体内miRNA验证其临床诊断性能。旨在为构建一种高敏、快速、简便的单个外泌体内miRNA数字化检测新技术提供实验基础与理论依据。

恶性肿瘤是严重危害人类健康的重大疾病，早期发现并及时治疗是有效阻止肿瘤进展、改善患者预后的关键，也是临床亟待解决的难题。细胞外囊泡（EVs）是一类由细胞向细胞外环境释放的粒径不均一的具有脂质双分子层膜的囊泡，研究发现肿瘤释放的EVs可以作为新型循环标志物，有望以无创的检测方式助力于肿瘤的精准诊疗，具有极大的临床应用前景。近年来，肿瘤EVs检测技术不断更新迭代，逐步从传统的纳米粒子跟踪分析测定、酶联免疫吸附测定、PCR等，转向基于光学、电学（电化学和电动力学）和小型微流控等多学科交叉的检测技术平台。本项目在基金委的资助下围绕液体活检EVs核酸标志物的检测开展了相应的工作。与香港科技大学姚舒怀教授课题组合作针对sEV携带的mRNA构建了基于液滴数字PCR的4重检测体系，实现了乳腺癌患者血浆标本来源sEV携带的GAPDH/PGR/ ESR1/ERBB2 mRNA的同时检测，以GAPDH mRNA作为内参筛选出了sEV携带的PGR和ERBB2 mRNA具有作为乳腺癌早期诊断潜在标志物的潜力，并联合机器学习算法的新策略，通过分析sEV来源的mRNA，构建了3种乳腺癌诊断模型。结果显示，相对于传统的统计分析，显著提高了乳腺癌的诊断效能。此外，针对EV所携带的蛋白标志物检测新技术也开展了一些共组，包括基于DNA纳米自组装策略建立了检测特异蛋白阳性EV亚群的癌症早期诊断新技术；其次，在与香港科技大学唐本忠院士组的学术交流中发现一种有望用于DNA 循环自组装放大系统的聚集诱导发光材料（Aggregation-induced Emission, AIE）材料，并开发了一种靶向EV特异蛋白的适体传感器，该传感器从一步反应体系混合到信号检测只需30min。