基于长程共振能量转移的生物医学成像分析基础研究

Förster resonance energy transfer (FRET) based fluorescence microscopic imaging (FMI) can be used to monitor the molecular motion trajectory of life activities, thus has attracted much attention, yet the techniques often suffer from photobleaching of the fluorescence organic molecular probes, limited number of energy-transfer donor-acceptor pairs, and the poor penetrating ability of visible light. This program focuses on the development of FMI for the biomedical analysis by introducing photoluminescent nanoprobes such as quantum dots and carbon dots, which have different composition, morphologies and dimensions, and excellent photoluminescent properties. By discussion of the long range resonance energy transfer (LrRET) mechanism of these photoluminescent nanoprobes composing of energy-transfer donor–acceptor pairs, much higher energy-transfer efficiency and longer distance than traditional FRET route, in which fluorescence organic molecular probes are employed, can be achieved. This program tries to develop new FMI techniques involving multple energy-transfer mechanisms, which could be adapted to the imaging analysis of multi-components, multi-color, and in the near IR regions, and might be applied to the visual analysis of virus-effected tissues and the real-time monitoring of the virus entrance of their host cells. The program tries to answer the basic problems, i.e. the spatiotemporal factors affecting the LrRET efficiency, and to elucidate the mechanism of LrRET, in the hope to have significant achievements in developing new FMI techniques with high contrast, high visualization capability, which could be applied to long-term, real-time monitoring. Implements of this program is far reaching for the development of new photoluminescent nanoprobes, breeding of new imaging techniques and the improvement of the biomedical researches.

基于Förster共振能量转移（FRET）的荧光显微成像（FMI）因可监测生命活动的分子运动轨迹已在生物医学中得到广泛关注，但存在有机荧光分子探针光漂白严重、能量转移供体-受体对有限和可见光穿透力差等问题。本项目拟引入不同材质、形貌和维度、具有优越发光性能的量子点、碳点等纳米探针开展FMI生物医学研究，探讨这些探针组成的能量转移供体–受体对所带来的比传统FRET效率更高、转移距离更远的长程共振能量转移（LrRET）机制，据此发展基于多种能量转移机制共存、且适于多成分、多色和近红外区的成像技术，并应用于病毒感染组织的可视化分析和病毒入侵宿主细胞的实时动态监控。项目以剖析影响LrRET效率的主要时空因素、明确LrRET原理为关键科学问题，以获得具有高对比和高可见度，且适于长时间实时动态监控的FMI技术为标志性成果。项目的实施对开发新型发光纳米探针、发展新成像技术和促进生物医学研究具有重要意义。

本项目的主体思路是通过引入不同材质、形貌和维度、具有优越发光性能的量子点、碳点等纳米探针开展荧光显微成像生物医学研究，探讨这些探针组成的能量转移供体–受体对所带来的比传统FRET效率更高、转移距离更远的长程共振能量转移（LrRET）机制，据此发展基于多种能量转移机制共存、且适于多成分、多色和近红外区的成像技术，并应用于病毒感染组织的可视化分析和病毒入侵宿主细胞的实时动态监控。按照预期计划，项目顺利完成了以下工作：.（1）利用有机染料分子作为能量转移供受体对，构建了Förster共振能量转移（FRET）体系，一方面应用于单核苷酸突变区分和肿瘤细胞中端粒酶成像指导下的肿瘤治疗，另一方面也极大地提高了显微成像分析性能，通过合理优化设计发展了高分辨、高可见度的显微成像技术，应用于肿瘤标志物检测、核酸分析及病毒入侵细胞的实时动态监测；.（2）为克服有机染料的光不稳定性，利用发光纳米颗粒作为能量转移供体，研究其参与的荧光共振能量转移（FRET）、纳米表面能量转移（NSET）、等离子体共振能量转移（PRET）、多重能量转移等多种转移机制的长程共振能量转移（Long range resonance energy transfer, LrRET）新体系；.（3）在多重能量转移研究中，探讨多重能量转移共存下长程共振能量转移效率与距离、发光材料特性的关系，对提高荧光显微成像性能具有十分重要的指导意义；.（4）基于多重能量转移建立了一系列多色生物成像分析方法，为多参数和高通量分析带来新进展；.（5）基于DNA光子线实现了长程共振能量转移，实现了近红外区多元显微成像分析。.项目的实施对开发新型发光纳米探针、发展新成像技术和促进生物医学研究具有重要意义。