基于上/下转换发光的新型比率荧光温度探针

温度的可视化实时监测，一直都是科学研究的重点方向。荧光传感是一种具有高灵敏度、快速响应、可视化等优点的半侵入式测温方法，在生物医药等领域已被广泛应用。然而，传统荧光探针容易受到外界条件波动的影响而产生误差。为解决这一问题，可以采用两组荧光检测信号构建比率型荧光探针，通过两组信号的相互校准提高检测的准确性。传统的比率荧光温度探针大多基于下转换荧光发射，这类探针通常由短波长光激发，对生物组织穿透性差且有一定伤害，还会受到生物组织自发荧光的干扰。频率上转换是由长波长激发，短波长发射的一种光致发光现象，由其构建的荧光探针可以克服传统下转换荧光探针的上述缺点。而基于三线态-三线态湮灭(TTA)机理的频率上转换发光体系，由光敏剂和湮灭剂的双分子体系共同构成，因而自身就同时具有上/下转换的发光特性，满足了构建比率型荧光探针的条件。然而目前，基于TTA上转换体系的比率型荧光温度探针还鲜见报导，已报导的工作中仍需要另外添加参比探针。仅通过TTA双分子体系构建的上/下转换比率型荧光温度探针仍然是一大挑战。本文通过将传统的TTA上转换体系(PdOEP/DPA)负载于由温敏型两亲性聚合物Pluronic-F127组装形成的胶束中，形成上转换纳米胶束温度探针。随着温度的升高，聚合物亲水链段水溶性下降，向胶束核心位置收缩，导致负载上转换分子的胶束内部空间体积减小，TTA分子间碰撞概率增大，上转换效率提高，上转换发光的强度也随之提高；与此同时，光敏剂的下转换磷光发射也会发生小幅度的下降。由此上/下转换两组荧光信号构成的比率荧光，可成功实现25~60 ℃范围内对温度的线性检测，并可通过肉眼观察到体系发光由紫红色向蓝紫色的转变，检测结果的重复性良好。TTA上转换分子通过被温敏聚合物胶束的包覆，既解决了在实际应用中探针水溶性差，以及上转换发光易被氧气淬灭的问题，还为上转换体系提供了温敏性质，实现了上转换发光对温度的精确响应。这种基于上转换纳米胶束的比率型荧光温度探针不仅制备方法简单，具有良好的生物相容性，且检测灵敏度高，可以人眼识别，无需外加参比，对生物体内温度在线监测的实现具有重要意义。

Ratiometric Fluorescent Temperature Probe Based on Up/Down-Conversion Luminescence

The visual and real-time monitoring of temperature has always been an attractive research direction. Fluorescent sensing is a semi-invasive temperature detecting method with the advantages of high-sensitivity, rapid-response and real-time visualization, which has been widely applied in biomedicine. However, conventional fluorescent detecting results can be easily effected by the fluctuation of external conditions which can cause deviation. Therefore, ratiometric fluorescent probes have been developed to solve this problem, because the two fluorescent signals can achieve intercalibration, improving the accuracy of the method. Traditional ratiometric fluorescent temperature probes are always based on down-conversion emission (fluorescence). This type of probes requires excitation at short wavelength like ultraviolet, which has poor penetrability and potential damage to biological tissues. Besides, the auto fluorescence from tissues become strong interference to the probes. Frequency upconversion is a photoluminescence phenomenon excited at long wavelength and emitting at short wavelength. Fluorescent probes based on upconversion can overcome the drawbacks of conventional ones. Triplet-triplet annihilation (TTA) upconversion system requires two kinds of molecules, sensitizer and emitter, and has both up/down-conversion itself, which perfectly meets the requirement of ratiomatric fluorescent probes. However, ratiomatric fluorescent temperature probes based on TTA upconversion are barely reported, and even in the reported work, additional reference probe is still needed. Ratiomatric fluorescent temperature probes only based on the up/down-conversion luminescence of TTA system itself is still a great challenge. Herein, a traditional TTA system (PdOEP/DPA) is encapsulated into micelles assembled by amphipathic polymer, Pluronic-F127, yielding a TTA upconversion nanoscale micelle temperature probe. As the temperature rises, the hydrophilicity of PEO segment in Pluronic-F127 decreased, yielding the micelles shrink inward and become smaller. The confined space inside the micelles results in greater collision probability of the TTA molecules, causing higher TTA upconversion efficiency and intensity. Meanwhile, the phosphorescence intensity of sensitizer slightly declines. The ratiometric fluorescence composed of up/down-conversion fluorescence signals of the TTA system can achieve linear detection of temperature from 25 to 60 ℃, which can be observed by naked eye due to the color change of the emitting light from magenta to violet. The detecting results also have good repeatability. Encapsulated by thermo-sensitive polymer, the TTA system can be applied both in aqueous solution and in air atmosphere, solving the problems of poor water solubility and quenching by oxygen. Besides, the thermos-sensitive polymer brings the TTA system remarkable temperature response capability. This novel type of ratiometric fluorescent probe based on TTA upconversion micelles shows advantages of simple preparation, great biocompatibility, high sensitivity and human eye recognition. No extra reference probe is needed. This method will open an efficient avenue for vivo temperature detection.