构建酚醛树脂为基质的荧光探针应用于细胞中核酸的检测

Nucleic acids exist in almost all organisms. Abnormal concentration of them indicate that disease occurs. Thus, it is important for quantitative detection of nucleic acids in organism. Dye labeled DNA fragment have often been designed as the fluorescence probe for nucleic acid detection in recent years. However, it is difficult for some dye-based probes to pass through plasma membrane. Furthermore, some dye-based probes can be degraded by enzymes in the cells. To resolve these problems, small sized phenol formaldehyde resin (PFR) nanoparticles will be taken as the fluorescence material and specific DNA fragment will be used to labeled PFR nanoparticles. Another end of the DNA fragment will be connected to the large sized gold nanoparticles. By this way, many small sized PFR nanoparticles will be bound onto the surface of Au nanoparticles through the specific DNA fragment. Due to the fluorescent quench from Au nanoparticles to PFR nanoparticles, the fluorescence intensity of PFR nanoparticles becomes lower. However, the DNA fragment will leave Au nanoparticles in the present of the target nucleic acid which can mate with the base sequence, making the distance between PFR nanoparticles and Au nanoparticles much further. Therefore, the fluorescence intensity of PFR nanoparticles will recover. Compared to dyes, the fluorescence probe consisting of PFR nanopartilces has the advantage of better material protection from enzymatic cleavage and lower biotoxicity, which can be applied for the detection of multiple nucleic acids in cells with better sensitivity and effectivity.

核酸几乎存在于所有的生物体内，其浓度的异常往往是一些疾病的预兆，因而定量检测生物体内核酸含量十分重要。针对目前出现的以有机染料构建的荧光探针在实际应用中存在难以进入细胞、易被细胞内酶或一些细胞液所降解、检测活细胞内核酸灵敏度低等问题，本项目利用小尺寸的酚醛树脂纳米颗粒作为荧光材料，对目标核酸具有特异性的DNA片段进行标记，并将该片段的另一端与大尺寸金纳米粒子连结，构建短链DNA“捆绑”酚醛树脂到金纳米粒子表面的复合材料。由于金纳米粒子对酚醛树脂的荧光淬灭作用，构建的该类复合材料具有低的荧光发射强度。当目标核酸存在时，由于特殊DNA片段对目标核酸分子的匹配性，标记有酚醛树脂的DNA片段从金纳米颗粒表面脱落，拉远了酚醛树脂与金纳米粒子的距离，酚醛树脂的荧光恢复。与有机染料相比，该类以酚醛树脂为基质构建的荧光探针具有更好的酶切保护作用和更低的生物毒性，能够实现细胞内多个核酸的更灵敏、更有效检测。

酚醛树脂 (PFR) 内部和表面具有丰富的羟基官能团，能够包覆和还原金属离子从而制备多功能复合材料。本课题通过水热合成方法，合成酚醛树脂纳米粒子，通过直接还原Ag+，在酚醛树脂表面原位合成Ag纳米粒子负载酚醛树脂复合材料，运用于细胞成像和大肠杆菌的抗菌；通过包覆四氧化三铁纳米粒子，制备核壳结构的Fe3O4@PFR纳米球，并进一步负载到石墨烯表面，制备具有磁性、荧光性和光热转换性能的复合材料，应用于细胞成像和肿瘤细胞的光热治疗中；运用对氯苯酚代替苯酚，合成掺杂氯的PFR纳米粒子，荧光发射强度增大，粒径可控在40 nm左右；在PFR的合成过程中加入硫脲，制备掺杂硫的PFR纳米粒子，提高材料的发光强度，用于三聚氰胺的分析检测;通过羧氨反应, 将四羧基酞菁偶联到对氨基苯酚上，运用水热方法，合成了酞菁锌功能化的酚醛树脂纳米粒子，研究发现由于酞菁锌的引入，酚醛树脂荧光发射具有很大的改善，该复合材料运用于蛋白质的灵敏检测中；水热合成酞菁锌复合的酚醛树脂量子点，并将其包覆到50 nm左右介孔硅中，通过修饰相应的DNA碱基链，可望用于细胞成像和细胞中核酸的检测；通过包覆上转换纳米粒子，合成粒径在50 nm左右的包覆有上转换纳米颗粒的酚醛树脂纳米球，研究结果表明，由于上转换材料的包覆，酚醛树脂荧光发射提高了近3倍左右。通过修饰DNA碱基链，应用于目标核酸的检测，效果显著。在本项目资助下，发表SCI论文5篇，授权国家发明专利3项。培养硕士研究生4名，其中一名顺利毕业，3名在读。共投入经费21万，支出7.48万元，各项支出基本与预算相符。剩余经费计划用于本项目的后续支出。