基于单颗粒暗场成像策略的体液microRNA灵敏检测

Endogenous microRNAs in blood circulation or other body fluids have good stability, and their expression levels are directly related to some diseases, which supposed to be a new non-invasive biomarker. However, due to the low content of microRNA in body fluids in the early stage of disease, a large number of sample are required with complex pretreatment, which cannot meet the needs of accurate diagnosis in early clinical treatment. Therefore, it is urgent to develop a direct and ultra-sensitive detection method to evaluate the performance of microRNA with low abundance..The dark-field imaging strategy based on the localized surface plasmon resonance of metal nanoparticles shows great prospect in recognition and detection of single molecule with the ability of against photobleaching, high sensitivity, fast response, and could realize the spatial-temporal resolution. In this project, the nucleic acid based signal amplification strategies are combined with the dark-field imaging techniques to build a novel sensing platform on the surface of single nanoparticle as a palsmon probe. After the recognition of specific microRNA, the dark-field scattering property of nanocage probes showed obvious color changes, and the reaction products could accelerate the autonomous motion of nanomotors, realizing the synchronous output of optical and motion signals for single nanoparticles under dark-field microscopy. This dark-field based optical analysis with static, dynamic, and dual-signal combined recognition strategies would finally apply to the ultra-sensitive detection of microRNAs in blood or other body fluids, providing technical support for the early diagnosis and monitoring, as well as the establishment of databases for major diseases.

血循环或其他体液中内源性microRNA具有较好的稳定性，且表达量与疾病状态直接相关，因此可作为一种新型无创伤性生物标志物。然而疾病早期体液microRNA含量较低，大量的样本需求和复杂的预处理操作无法满足临床早期精准诊疗的需求，因此需开发低丰度microRNA的直接超灵敏检测方法。.基于金属纳米颗粒局域表面等离子体共振效应的暗场光学监测方法具有无光漂白、灵敏度高、响应快、可时/空分辨的特点，广泛用于单分子识别和检测过程。本项目中我们将核酸信号放大技术与暗场成像方法结合，在单颗粒表面构建传感平台。利用目标识别引发纳米笼探针暗场光学信号变化，同时引起马达探针运动性能的改变，实现暗场下单个颗粒的光学信号和运动信号的同步输出。通过构建静态、动态和动静双信号结合的暗场成像光学分析方法，实现血循环或其他体液microRNA的直接灵敏检测，为重大疾病的早期诊断、监测和数据库的建立提供技术支撑。

MicroRNA作为重要的肿瘤标志物在疾病早期表达量较低，大量的样本需求和复杂的预处理操作无法满足临床早期精准诊疗的需求，因此需开发低丰度microRNA的直接超灵敏检测方法。本研究基于暗场光学监测方法的无光漂白、实时响应、可时/空分辨的特点，以及电致化学发光检测技术的灵敏度高、背景信号低、电化学参数可控等优点，构建了一系列暗场和电化学传感平台。我们将纳米材料和DNA组装及信号放大技术相结合，通过纳米材料合成及功能化修饰，构建光学和电化学信号探针；通过设计目标识别引发的链取代、杂交链式反应、催化发卡组装等DNA链组装反应，实现信号放大过程；对细胞等复杂样本中的microRNA进行灵敏检测。主要包括：（1）构建纳米笼颗粒探针，利用目标识别触发DNA链反应引起探针“灭灯”响应，通过暗场成像实时监测反应过程，实现对microRNA的灵敏时空监测和定量分析。（2）开发新型的核-卫星Au/Ag NCs等离子体纳米探针，利用目标链引发连续链取代反应，实现DNA辅助的荧光增强和暗场成像双模式检测microRNA。（3）构建3D步行器探针，通过walker-HCR-DNAzyme级联放大，实现细胞液中microRNA含量的精准测量。（4）通过构建具有内参比信号的双电位ECL比率传感策略，提升microRNA检测的抗干扰能力和检测结果的可靠性。（5）设计基于杂交链式反应的无标记均相传感策略，实现复杂样本环境中的microRNA检测。（6）开发基于热敏脂质体水凝胶体系的可控药物释放和联合肿瘤治疗平台，将水凝胶系统与化疗和光热治疗结合起来，实现良好的肿瘤治疗效果。本项目的研究成果为肿瘤标志物的检测和肿瘤治疗提供重要的策略。