等离子体共振诱导双光子荧光纳米探针对细胞内pH值波动的实时影像分析

The pH value in organelle is a critical marker of cell physiology and pathology. However, the current pH probes exhibit low sensitivity and poor photo-stability, limiting their applications in real-time monitoring of the small pH fluctuation in the organelles. Based on the previous work about two-photon fluorescence of metal nanoparticles, this project aims to develop a pH-dependent plasma resonance-induced two-photon fluorescence enhancement of metal nanoparticle probe that achieves simultaneous and dynamic tracing of pH fluctuations at subcellular level by reversible aggregation/disaggregation process of noble metal nanoparticles. First, to explore the functionalization of pH-sensitive ligands on the surface of metal nanoparticles, and to investigate the assembling methods and principles of targeting molecules to organelles, plasma resonance-induced two-photon fluorescence enhancement of nanoparticle probe was constructed. Second, to explore the organelles localization function and the relationship between the spectral response of the nanoparticle probe and the pH change, pH-responsive aggregation/disaggregation principle of metal nanoparticles was developed. Third, the effect of pH value on the aggregation effect of nanoparticle probe and the two-photon fluorescence enhancement, was illustrated in different organelles. The in situ imaging method of pH changes in living cells organelles was established to realize the simultaneous and real-time image analysis of pH fluctuation in different organelles. The expected results of this project will provide new ideas and technical guidance for developing new methods in monitoring carcinomatous lesions at subcellular level.

细胞器内pH值是决定细胞生理病理的重要标志。然而现有的pH探针由于敏感性低、光稳定性差，难以实现对细胞器内pH值微小波动的实时监测。在前期金属纳米粒子双光子荧光研究基础上，本项目拟发展一种pH依赖的等离子体共振诱导双光子荧光纳米探针，通过可逆的聚集-解聚集实现细胞水平pH值波动的动态示踪。具体研究内容如下：（1）探索金属纳米粒子表面pH敏感配体功能化、细胞器靶向分子的组装方法和原理，构建等离子体共振诱导双光子荧光增强型纳米探针。（2）探索纳米探针的细胞器定位功能及光谱响应与pH变化关系，发展pH响应的纳米探针聚集-解聚集原理。（3）阐明不同细胞器内pH值对探针聚集效应及双光子荧光增强的影响，建立活细胞内细胞器pH变化的原位影像学方法，实现对不同细胞器pH值波动的同步、实时影像分析。本项目的预期成果将为在亚细胞水平上发展细胞病变癌变的监测方法提供新的思路和技术手段。

细胞内pH值是决定细胞生理病理的重要标志。本项目利用激光共聚焦显微镜，进行了一系列细胞水平的荧光实时原位监测研究。主要包括（1）设计了基于pH依赖的聚集诱导双光子荧光增强型纳米探针，通过调节纳米探针表面11-巯基十一烷酸的用量来调控其酸度系数，成功实现了对肿瘤细胞偏弱酸性pH的影像分析。（2）通过构筑金棒和水溶性聚合物组成的核壳纳探针，系统研究距离对金纳米棒增强共轭聚合物的单光子和双光子激发荧光增强趋势，实现了对肿瘤细胞实时成像及同步光动力治疗。（3）构建基于荧光能量共振转移的核苷酸探针，实现对正常细胞和肿瘤细胞内端粒酶表达水平的准确区分。（4）发展基于生物分子特异性识别反应的双色比率荧光纳米探针，实现对生物分子的实时同步检测及可视化分析。这些内容为肿瘤细胞可视化治疗提供了新的思路和技术手段。在此基础上，我们将继续针对细胞水平或生物活体水平的生物分子进行荧光实时原位监测，将纳米材料与有机探针结合，进行精准成像和治疗方面的研究。