热激活延迟荧光分子探针的构建及其在生物荧光成像中的应用研究

Fluorescence imaging is a powerful and widely used method for complex biological environments owing to its high sensitivity and good spatial resolution. However, the autofluorescence of the tested samples and the light scattering in the instruments always reduce the sensitivity or give rise to false signals of this detection method. To overcome the disadvantages, time-resolved fluorescence microscopy is now emerging as a promising supplement or alternative to conventional fluorescence microscopy. A major challenge for time resolved imaging is to develop long lifetime probes with good biocompatibility and high specificity. Thermally activated delayed fluorescence (TADF) probes offer the advantages of long fluorescence lifetime and high fluorescence efficiency, which can eliminate the prompt fluorescence interference, improve signal-to-noise ratio, and avoid the high cost and high toxicity of rare metal probes.. The project aims to develop novel TADF probes based on naphthalene imide with improved biocompatibility and targeting abilities by introducing the hydrophilic side chain and targeting moieties. Several strategies will be applied in fluorescence lifetime imaging to minimize the influence of oxygen to the emission of TADF probes, including host–guest systems, self-assembling, and anaerobic bacteria imaging under deaeration conditions. These novel naphthalene imide-based TADF probes will have great potential as a time-resolved fluorescent sensing platform to overcome the drawbacks of luminescent complexes containing heavy metals.

荧光信号高特异性、高信噪比是生物荧光成像质量关键。在时间分辨成像中，长寿命荧光信号可方便的区分来自生物内源性物质自身荧光及光源杂散光等短寿命荧光信号背景干扰，因此利用长荧光寿命分子探针对生物体进行时间分辨成像可高效避免背景干扰。时间分辨成像的一个主要挑战是如何开发具有良好生物相容性、高特异性长荧光寿命分子探针。热激活延迟荧光(TADF)分子具有荧光寿命长、荧光效率高等优点，在生物成像中，可以消除短寿命荧光背景干扰，提高信噪比，并避免金属类探针成本高、毒性大等缺点。本项目拟基于萘酰亚胺母核构建新型TADF探针，通过在探针分子中引入亲水性侧链及细胞器、细菌靶向基团，增加探针分子生物相容性及靶向性；建立时间分辨成像方法并通过生物大分子包裹探针、两性探针分子自组装聚合及无氧状态下厌氧菌成像等策略，避免氧气对探针的影响，实现生物体时间分辨成像研究，为进一步组织、活体时间分辨成像研究奠定基础。

与荧光强度信号相比，荧光寿命信号值更稳定，且受探针浓度变化、测量方法、激发激光功率或体内外环境的影响最小。将荧光寿命这一时间维度引入生物成像，可监测生物活动的动态演变，具有重要的科学价值。热激活延迟荧光（TADF）分子具有荧光寿命长、荧光效率高等优点，然而其生物相容性差和易受氧气淬灭的特点限制了其生物成像应用。本项目首先通过引入亲水基团和靶向基团，发展了用细胞器内源性生物大分子包裹TADF分子策略，构建了一类新型TADF分子探针，增强了探针的生物相容性，避免了生物环境中氧气对探针三线态的淬灭。随后，通过两亲性TADF单体的自组装策略构建了长寿命的荧光纳米探针，这种两亲性的TADF单体可以自发地自组装成水溶性好的、生物相容性好的纳米探针。并且纳米颗粒表面的正电荷可以显著增强其细胞穿透能力。再次，通过将 TADF 发光团与具有细菌 16S rRNA 靶向能力的新霉素结合，发展了新型细菌靶向性长寿命荧光探针，为细菌核糖体16S rRNA 的特异性检测提供双模荧光分子工具。该项目利用新型TADF分子探针，成功实现了生物体时间分辨成像，为进一步生物时间分辨成像研究奠定了基础。