基于通键能量转移的近红外双光子荧光染料平台构建及其生物应用研究

Two-photon (TP) dyes have become a preferred choice in the recent years in the biology-related field of fluorescent probe and imaging techniques, due to their ability to capture two low-energy near infrared photons, deep tissue penetration, their high spatial resolution, and excellent photostability. However, most of the commercially available TP dyes shows low emission wavelength (﹤600nm), which could not achieve ideal NIR-incoming-NIR-outgoing fluorescence features in the practical application. This project is mainly focus on the development of novel NIR TP dyes by using the strategy of through-bond energy transfer (TBET). The result will give some new ideas and experimental evidences for the development of TP fluorescent materials in the biological window. These fluorescent probes based on the new TP dyes are bound to have a major impact in the field of two-photon microscopy and disease diagnose, and display a promising application. The innovation of the article mainly presents in the following three aspects: (1)To address the above issues, a new method for the construction of NIR-emissive TP fluorophore was ideally developed with the TBET strategy; (2)In order to acquire a better biocompatibility, a fluorophore exhibiting high two-photon absorption cross-sections was chosen as the TP donor, and a fluorophore with emission wavelength in the red to NIR range was explored as the TBET acceptor; (3)Based on the platform, near-infrared fluorescent dyes with ratiometric character was developed and used to design fluorescent probes.

由于双光子激发染料可实现长波长激发、大的穿透深度、高的三维分辨成像等，已成为当前荧光探针和生物影像等领域首选染料。然而，目前大部分商业化的双光子染料存在发射波长较短（＜600nm）的问题，在实际应用中并未实现理想的“近红外输入——近红外输出”。本项目拟开发系列“基于通键能量转移的近红外双光子荧光染料平台”，并在此基础上构建系列近红外双光子荧光探针，促进该类染料在生物学方面的应用。研究结果有望为开发具有“生物光学窗口”的双光子荧光染料提供新思路和实验依据，所构建的荧光探针分子将在双光子显微成像、疾病诊断等方面具有切实应用价值。创新点主要体现在三方面：（1）创新性地提出“利用通键能量转移实现双光子激发的近红外荧光发射”；（2）选择双光子吸收截面大的染料作为能量供体，荧光量子产率高的近红外染料作为能量接受体，优化光物理性质，满足生物学研究需求；（3）构建了具有比率性质的近红外双光子荧光染料平台。

双光子荧光染料因采用远红外/近红外光激发，在荧光技术领域呈现出更多优势，更适合作为影像工具应用于生物学研究、药物靶向、疾病早期诊断等领域。然而，目前商业化的双光子荧光染料当前只是解决了荧光技术应用中的激发问题，并没有完全实现理想的“近红外输入——近红外输出”。因此，如何有效地解决双光子荧光染料发射波长太短这一科学问题成为该领域研究的重点。在经典的荧光机制中，解决这一问题的主要方法是利用荧光共振能量转移机制在一定程度上得以解决。其中通键（through bond）荧光共振能量转移机制（TBET），也称跨键能量转移机制，因不受限于供体发射光谱与接受体吸收光谱必须重叠的要求，在实践应用中具有更加明显的优势和可操作性。然而，当前“基于通键能量转移的近红外双光子荧光体系”报道实例较少，理论不完善。究其原因是受双光子染料分子结构不易修饰、近红外染料功能化位点少、桥梁基团单一、分离纯化等因素，使得合成制备过程复杂。针对以上问题，本项目合理地选择双光子能量供体（芴、1，8-萘酰亚胺）、桥连基团以及合适的近红外能量接受体（Si-罗丹明、萘荧光素），采用有机合成手段将它们组装，成功合成了系列基于通键能量转移的近红外双光子荧光染料平台，初步探讨了该类染料的光物理性质，并通过理论计算的方法证实其可以实现双光子激发的通键能量转移。该工作为基于通键能量转移的近红外双光子荧光平台构建提供合成基础和新的生物成像材料。