基于特定结构蛋白质高选择性荧光探针的设计合成及应用研究

Proteins play important functional roles in a variety of physiological systems. Selective and sensitive detection of proteins is critical for diagnosis and therapy of diseases. Fluorometric technique has attracted much attention in proteomics research due to its virtues of real-time, sensitivity, selectivity, non-invasion and fluorescent imaging. In this project, we are going to design and synthesize fluorescent probes with high sensitivity, selectivity and relative long wavelengths for thiol-proteins, glycoproteins and (strept)avidin. Based on the structural property of having hydrophobic cavities with proteins, the fluorescent probes are constructed by integrating polarity-sensitive fluorophores (coumarin, BODIPY, rhodamine) and the corresponding receptors (2,4-dinitrobenzenesulfonyl, benzoboric acid, biotin) for the target proteins. Fluorescent nanomaterials, by incorporating fluorescent probes into nano-partials, will be developed for enriching, sorting and sensing the proteins with low abundances in biosamples. This work may inspire the design of fluorescent probes for sensitive and selective detection of proteins with special structures and functions. The relationship between protein expression and diseases will be explored by fluorescent imaging of different tumor and normal cells. The results of this project are expected to provide useful information for studying the functions of proteins and for disease diagnosis.

荧光检测技术因其实时、灵敏等优点在生物成像、高灵敏特异性蛋白质检测等领域至关重要。本项目以香豆素、氟硼吡硌、罗丹明等为荧光团；2,4-二硝基苯磺酸基、苯硼酸以及生物素为受体，针对巯基蛋白、糖蛋白和亲和素设计、合成高选择性、高灵敏度、长发射波长的荧光探针。利用荧光团对溶剂极性敏感的特性，以蛋白质的输水空腔作为辅助作用位点，使探针分子与蛋白质结合后产生显著的荧光增强，实现特定蛋白质的高选择性检测和精确定量。将荧光探针键合到纳米SiO2表面，制备针对生物样品中特殊结构低丰度蛋白质的荧光纳米分离介质，不仅能够对实际试样中目标蛋白分离、富集，还能通过蛋白质在纳米颗粒表面聚集产生的局部浓度效应实现对其高灵敏度荧光检测。发展的方法可以为特殊结构蛋白质的高选择性、高灵敏度荧光检测提供指导。通过对不同细胞中目标蛋白的荧光成像实验，初步探索疾病与蛋白表达之间的关系，为蛋白质功能研究和疾病诊断与治疗提供帮助。

特定结构蛋白质、小分子硫醇、二氧化硫、甲醛等均是重要的生物分子，其在生物体的异常浓度与某些疾病息息相关。高灵敏度检测上述生物分子对于疾病的诊断和治疗意义重大。荧光检测技术因其实时、灵敏等优点在生物成像、高灵敏特异性生物分子检测等领域至关重要。本项目以香豆素、氟硼吡硌、罗丹明、1,8-萘酰亚胺等为荧光团；2,4-二硝基苯磺酸基、马来酰亚胺、苯硼酸、,-不饱和酮为受体，生物素、-半乳糖、吲哚美欣为肿瘤细胞的靶向基团，设计、合成了四个系列、十多个高选择性、高灵敏度、具有较长发射波长的荧光探针实现对血清白蛋白、硫醇、亚硫酸盐、甲醛等生物分子的检测以及对细胞内上述分析物的荧光成像。将荧光探针共价键合到纳米SiO2表面，实现对糖和氢离子的检测以及探针的回收。共聚焦荧光成像实验结果表明合成的吲哚美欣类和生物素类荧光探针能够特异性地检测肿瘤细胞，而-D-半乳糖类荧光探针可以实现对HepG-2细胞内源性甲醛的荧光成像，上述结果可望为生物活性分子的生理功能研究和疾病诊断及治疗提供帮助。通过核磁、质谱以及高效液相色谱探讨了探针与目标分析物的反应过程，并利用计算机模拟和分子对接研究了探针对目标分析物的响应机理，初步探索了分子结构与性能的关系，为进一步提高探针分子的性能以及新探针的开发提供依据。