近红外荧光开关与双光子激发荧光探针的设计、合成与生物探测研究

Based on the existing photophysical platforms and the applicant's experiences in the design and construction of low-dimensional photoactive materials and investigation on their applications in biological detections, probes with photoswitchable near-infrared (NIR) fluorescence and with two-photon excited NIR fluorescence will be fabricated in this project. By designing and sythesizing NIR-emitting organic fluorophores, which function as energy acceptor, and bistable photochromic molecular switches, which function as photo-responsive components and energy donors, fluorescence resonance energy transfer (FRET) based nanoprobes possessing photoswitchable fluorescence emission in the optical window region will be fabricated.This new type of photoswitches with photoswitchable NIR fluorescence is expected to enhance the biological tissue and cell penetration ability of fluorescence signals, which will contribute to improved signal-to-noise ratio in bioimaging. Organic fluorophores possessing two-photon excitation features in the NIR optical window region will be synthesized and used as low-energy photon harvesting components and energy donor. By combining such energy donor with energy acceptor possessing NIR fluorescence, probes with low-energy two-photon excitation and NIR fluorescence emission in optical region window can be constructed based on a transition energy level match between the donor and acceptor. Such type of probes with excitation and emission spectra both in the NIR optical region, which has the lowest hemoglobin, melanin and water absorbance, will enable noninvasive imaging of biological tissues and cells and better imaging performance owing to excellent penetration ability of NIR fluorescence. Properties of the target probes such as structural stability, photostability, and suitability for cell uptake are definitely major concerns for biological applications and will be taken within the scope of this work. Rational optimization of the protective shell components of the core-shell nanoprobes, e.g. the oxygen permeability and biocompatibility, will be applied to improve the stability and facile cell uptake of the nanoprobes. Additionally,preliminary biological experiments will be carried out to test the practicality of the as-prepared probes for the future optimization.

本项目拟基于申请人所在课题组现有的光物理实验平台装置以及申请人在低维光学活性材料的设计、构建以及生物探测方面的研究经历，设计合成具有近红外荧光开关特性的荧光开关和双光子激发、近红外荧光发射的纳米探针；探索这些材料在纳米尺度细胞荧光影像、细胞内特定目标物传感和示踪分析方面的应用。通过设计合成具有红外荧光发射特性的有机荧光团作为能量受体、具有双稳特性的分子开关作为光响应组份和能量给体来构建基于荧光共振能量转移机制的荧光开关，将现有的荧光“开-关”波长扩展至“光学窗口”区域，从而增强荧光信号对生物组织、细胞等样品的穿透能力。通过合成优化在“光学窗口”区域有双光子激发荧光发射的荧光团作为低能量光子捕获组份和能量给体、具有红外荧光发射特性的荧光团作为能量受体；基于二者之间在跃迁能级上的匹配来构建低能量近红外双光子激发、在近红外区域发射荧光的纳米探针，从而克服高能光子激发对活细胞样品的侵害和目标荧光信号的穿透性问题。

根据研究计划，我们在近红外荧光开关及双光子激发荧光探针的设计构建及其应用方面开展了一系列探索性研究，取得了一系列阶段性研究成果。.1)设计构建了可用近红外光 （NIR）通过双光子吸收机制激发、发射NIR荧光的“NIR input-NIR outgoing”复合荧光探针；其荧光成像穿透深度可高达1200m、对深置目标及一定厚度的生物样品进行成像具有实用意义。.2)构建了斯托克斯位移超过200nm的光致NIR荧光开关探针；基于超常的斯托克斯位移、NIR荧光信号、及动态比照荧光开关三重机制的共同作用，降低背景噪音的干扰、将细胞内荧光成像的灵敏度提高两个数量级。.3)设计合成了一种可用近红外光(NIR)触发的光动力学治疗的仿生纳米载体；这种材料展现出了对小白鼠肿瘤极为有效的光动力学治疗效果、对于肿瘤的光动力学治疗具有重要意义。.4)设计构建了一种基于石墨烯碳量子点单元、能基于NIR荧光发射特性变化来程序化监控抗肿瘤药物递送、释放和响应的复合体系，实现了对抗肿瘤药物紫杉醇释放的NIR荧光成像监测。.5)基于单一发色团的双色荧光开关探针发展了“反应关联双光转换荧光成像”方法，目标信号因“反相关联”而被放大、假阳信号则因相位关联性的缺失而被滤除，将细胞内荧光成像的信噪比提高了两个数量级、大大提高探测的灵敏度。.6)设计合成了具有仿生特性的集成型“NIR input-NIR outgoing”复合荧光探针; 其在荷瘤小鼠活体肿瘤荧光成像方面的应用已得到验证；.7)设计构建了新型的比率计型的NIR钯荧光探针，该探针的荧光信号峰位在717 nm，能够在约3分钟时间内对浓度低至5.1ppb的钯作出响应、且具有识别选择性。