核酸是最基本的生命物质之一，分为脱氧核糖核酸(DNA)和核糖核酸(RNA)，在遗传信息的储存、复制、传递以及蛋白质的合成中发挥着重要作用。实时原位监测活细胞及生物体内DNA和RNA的分离、聚集和裂解等结构动态变化对理解核酸转录、蛋白表达、细胞凋亡等细胞行为和研究遗传性疾病具有重要意义。然而，由于目前的成像探针存在生物膜穿透能力弱、无法同步区分DNA和RNA、自身光稳定性差等局限性，在活细胞，尤其是生物体内，实时原位的观察DNA和RNA结构的动态变化仍然面临很大的挑战。碳量子点由于其细胞毒性小，光稳定性好，易于进行表面功能化修饰等特点，在细胞及活体原位影像分析中发挥了重要作用。本论文中，我们通过对碳量子点进行化学修饰，提高碳量子点表面的正电荷，得到了具有高效生物膜穿透能力的阳离子碳量子点探针(carbon quantum dot，cQD)，实现了细胞及线虫体内DNA和RNA结构的同步原位荧光成像。本论文具体研究内容如下： (1)设计合成了一种具有高效生物膜穿透能力的阳离子cQD探针。首先，通过硝酸氧化剥离导电炭黑颗粒得到了尺寸均一的含羧基碳量子点。进一步，通过对苯二胺修饰，得到了绿色荧光碳量子点。最后，将4-羧丁基三苯基溴化膦通过对苯二胺的氨基连接到碳量子点上，得到带正电的cQD探针。cQD探针与规则双螺旋的dsDNA和柔性折叠的ssRNA具有不同的结合方式，从而实现对二者的光谱区分。 (2)实现了cQD探针对活细胞内DNA和RNA的同步原位成像与动态影像分析。合成的阳离子cQD探针能够有效的穿过细胞膜／细胞核膜，与细胞内的DNA和RNA结合，在488 nm激发下，细胞核发出很强的绿色荧光，而在543nm激发下，细胞质和核仁发出红色荧光，证明了cQD探针对活细胞内DNA和RNA的特异性识别能力。进一步，由于该探针强的光稳定性和低毒性，实现了对细胞有丝分裂过程中DNA和RNA结构动态变化的影像分析，并且通过STED超高分辨成像，实现了对染色体和核仁结构的3D重构。 (3)最后，我们探讨了该cQD探针对活的线虫体内DNA和RNA的原位成像分析。将cQD探针加入到秀丽隐杆线虫的培养液中，我们发现，cQD不仅进入线虫的肠道，而且继续穿过肠道壁、性腺壁等一系列组织器官，最终进入线虫性腺内，与生殖细胞DNA和RNA结合，与细胞结果类似，在488 nm和543 nm激光激发下分别发出绿色和红色荧光。这一结果证实了cQDs在体内跟踪DNA和RNA的能力，更重要的是证明了其穿过肠道壁、组织间质、性腺囊壁、生殖细胞膜／细胞核膜等组织细胞水平膜屏障的高效生物膜穿透能力。 关键词：碳量子点 膜穿透 核酸 生物体 荧光成像

 Nucleic acids, both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are one of the most basic life materials. They play an important role in the storage, copy and transfer of genetic information, and the synthesis of protein. Direct monitoring the structure changes of DNA and RNA in real-time, such as the segregation, aggregation, and cleavage, is of great significance for understanding the behavior of cell transcription, division, protein expression and apoptosis. However, due to the lack of probe photostable enough to distinguish DNA and RNA, and more importantly, to cross multiple membrane barriers ranging from cell-organelle to tissue-organ levels, it is still a great challenge to real-time observe the dynamic structure changes of DNA and RNA in living cells, especially in vivo. Compared to traditional semiconductor quantum dots and organic dyes, carbon quantum dots possess superior properties in terms of non-toxic, high resistance to photobleaching, easy modification and so on, making them proficient for real-time in situ imaging analysis in living cells and organisms. Here, we report the discovery of a cationic carbon quantum dot (cQD) probe with the high efficiency of membrane-penetrating by chemical modification to improve the positive charge of the cQD. The cQD probe emits spectrally distinguishable fluorescence upon binding with double-stranded DNA and single-stranded RNA, achieving synchronously real-time in situ fluorescent imaging of the dynamic structure changes of DNA and RNA in living cells and Caenorhabditis elegans (C. elegans). The main research contents are as follows: 1.We report the discovery of a cationic cQD probe with the high efficiency of membrane-penetrating. First, The cQD with uniform size was made by oxidizing the conductive carbon nanoparticles in refluxing HNO₃ to add a large number of carboxyl groups to the edges of cQDs. Then, further modification with p-phenylendiamine (pda) through acylation produces cQD with modest green fluorescence. Finally, 4-carboxybutyl triphenylphosphonium (PPh₃⁺) bromide was chemically linked to cQD through the pda sites to obtain the cationic cQD probe. The cQD interacts with double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) differently, producing spectrally distinguishable fluorescence. 2.We achieved the synchronous in-situ imaging of DNA and RNA and their dynamic changes of structure in live cells by using the cQD probe. The cQD probe can effectively cross the cell membrane and nuclear membrane to bind with the intracellular DNA and RNA. Under excitation of 488 nm, the nucleus emits strong green fluorescence, while under excitation of 543nm, the cytoplasm and nucleoli emit red fluorescence, demonstrating the specific recognition ability of the cQD probe to DNA and RNA in living cells. Furthermore, due to the strong photostability and low toxicity of the probe, we achieved imaging the dynamic structure changes of DNA and RNA during cell division and also the 3D structure reconstruction of the chromosome and nucleolus using stimulated emission depletion microscopy (STED). 3.Finally, we explored the possibility of in-situ imaging of DNA and RNA in live organisms. The C. elegans were fed with cQD in culture media. As expected, cQD initially entered the intestinal pathway, but surprisingly, it can go through interstitial tissues, gonad vesicle with the continuous incubation, and finally the cQD probe permeate trough the germ cell membrane and nuclear membrane into germ cell nuclear. Similar to the results in living cells, the cQD probe labelling DNA and RNA in germ cells also emits green and red fluorescences, respectively. All the results showed the cQD probe possess the ability of selectivity towards dsDNA and ssRNA, and in particular, the unique ability to penetrate through various biological barriers in cells and live organisms. Key words: Carbon quantum dots, Membrane-penetrating, Nucleic acid, Live organisms, Fluorescent imaging