细胞膜成像探针的构建及对病毒-细胞膜结合过程的实时监测

It is of great importance to develop techniques for real-time monitoring the process of viral attachment to cell membrane, which is significant for the study of viral infections and the early diagnosis or treatment. However, most of the traditional assays involve the labelling of viruses, which needs complicated design and operation process. Furthermore, the labelling process may reduce the viral activity and affect the inherent infection process of viruses. Imaging plasma membrane has been proved to be powerful tool to monitor what cells sense and how they respond in real time to surrounding signals, thus it is possible to monitor the viral attachment events by fabricating imaging probes on plasma membrane. Therefore, by taking advantage of the high brightness of quantum-dots (QDs) and carbon dots (CDs), and the unique localized surface plasmon resonance (LSPR) properties of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs), this research is to engineer the above nanoparticles as imaging probes on cell surface. By controlling the parameters, such as nanoparticles size, surface chemistry and ligand density, the imaging probes with good biocompatibility, strong stability and high sensitivity could be anchored on cell membrane through affinity labeling, covalent coupling or hydrophobic interaction. Subsequently, the alteration of cell surface microenvironment such as receptor clustering, the change of molecular interactions and lipid raft structure resulting from viral attachment to cell membranes can be real-time monitored by fluorescence imaging or dark-field light scattering imaging. This project is aiming to establishing a set of sensitive assays for quantitative analyzing the amount of viruses adsorbed on cell surface and understanding the molecular events associated with process of viral attachment to cell membranes. Overall, we propose a novel strategy to real-time monitor how cells respond during the process of viral attachment through a membrane-anchored imaging probe. In this way, the problems of viruses labelling can be successfully avoided. This research is significant for investigating the mechanism of viral infections at molecular level, which can provide valuable information for the early diagnosis and treatment.

建立实时监测病毒-细胞膜结合过程的方法有利于研究病毒侵染机制及早期诊疗。传统方法往往需要对病毒进行标记，操作繁琐，并影响病毒感染力及侵染途径。细胞膜成像技术是探测细胞表面微环境变化的有力工具，为研究病毒侵染提供了良好的技术支持。本项目拟选择具有高发光亮度的量子点、碳点，和具有独特局域表面等离子体共振性质的金、银纳米颗粒，通过调节颗粒尺寸、表面化学及配体密度等参数，以亲和作用、共价偶联或疏水作用等方式构建低毒、稳定、灵敏的细胞膜成像探针；利用荧光成像和暗场光散射成像技术实时监测病毒-细胞膜结合过程触发的细胞表面受体聚集、分子间作用或脂筏结构改变等微环境的变化，建立病毒吸附量与信号变化之间的定量关系；在此基础上探讨与病毒-细胞膜结合过程相关的分子事件。本项目创新性地利用细胞膜成像探针点亮病毒“登陆”细胞膜过程，避免了传统的病毒标记问题，从而在分子水平上获得病毒侵染机制，对早期诊疗具有重要意义。

本项目以探究病毒-细胞膜结合过程相关的分子事件为目标，研究思路是通过构建生物相容性好、稳定性强及灵敏度高的细胞膜成像探针，发展荧光成像和暗场光散射成像技术；以实时监测病毒-细胞膜结合过程触发的细胞表面受体聚集、分子间作用或脂筏结构改变等微环境的变化贯穿整个研究过程；建立病毒吸附量与信号变化之间的定量关系，为在分子水平上研究病毒侵染机制及早期诊疗提供依据。按照预期计划，项目顺利完成了以下工作：.① 以乙二醇壳聚糖为骨架，侧链接枝胆固醇和核酸适配体，构建了生物相容性好、稳定性强及灵敏度高的细胞膜成像探针，基于“多足”锚定策略大大提高了探针与细胞的亲和力，克服了传统探针容易被內吞而无法长时间成像的问题。.② 利用量子点（QDs）和核酸适配体构建了可逆的细胞表面aptasensor，以细胞膜染料为参比信号，提出了基于细胞表面荧光比率法实时监测呼吸道合胞病毒侵染宿主细胞过程的方法，并建立了病毒吸附量与荧光信号变化之间的定量关系。.③ 以荧光碳点（CDs）为发光探针，创新性地构建了荧光共振能量转移软纳米球，克服了传统的碳点表面修饰困难的问题，实现了生物标志物的简单、快速、灵敏分析以及细胞内RNA长时间成像。.④ 以CDs、QDs等发光纳米材料为能量供体，以具有良好猝灭能力的金纳米颗粒、氧化石墨烯及BHQ等作为能量受体，构建了一系列双重能量转移荧光纳米探针，有效提高了能量转移效率及探针在复杂生物样本中的抗干扰能力和准确度，解决了传统荧光成像背景高、灵敏度低以及GO探针非特异性解吸附的问题，实现了病毒侵染、细胞凋亡过程相关生物标志物及其它疾病标志物的低背景、高准确度和高选择性成像。.本项目创新性地利用细胞膜成像探针点亮病毒“登陆”细胞膜过程，避免了传统的病毒标记问题，并建立了一系列简单、快速、灵敏的生物标志物含量测定方法，对早期诊疗具有重要意义。