碳基量子点的制备与斑马鱼荧光成像的应用及相关毒理的研究

发布时间：2018-05-24 09:04

本文选题：碳量子点 + 石墨烯量子点　；参考：《郑州大学》2017年硕士论文

【摘要】：荧光碳基量子点作为新兴的有机碳材料,因其具有特异的荧光性能、稳定的物理化学性质、高的生物相容性等优异性能而跻身于材料界的“翘楚”。近年来,碳基量子点的广泛研究越来越贴近生物和环境等相关领域,使得我们在材料制备和应用的过程中,都更加关心其生物相容性和毒理风险性的探索。本论文以两种生物质(头发和猪皮)以及一种低毒无害的物质柠檬酸为原料制备了三种绿色低毒、高生物相容性的碳量子点,并将其应用于斑马鱼荧光成像的研究。另外,我们从现在研究很热门的石墨烯材料出发,制备了一种荧光性能优越的还原性氧化石墨烯量子点,将其应用于斑马鱼荧光成像和相关毒理学的研究。首先,以两种生物质头发和猪皮为原料,采用微波辅助碳化的方法高效合成了基于头发和猪皮这两种富含蛋白的生物质的荧光碳量子点。既没有外加掺杂剂,也没有使用任何有毒试剂,直接将生物质原材料通过微波辅助碳化的方法快速制备了头发量子点(HCDs)和猪皮量子点(SCDs)。同时,我们也通过化学水热合成法以柠檬酸和尿素作为原材料合成了柠檬酸碳量子点,并与前两种以生物质为原料的量子点做对照,探索三种不同碳量子点的荧光性能及其他各项性质的差异。三种碳量子点都是在10 nm以内的纳米颗粒,原材料的不同导致了其化学组成和荧光性能的不同,HCDs和SCDs在紫外光范围内主要为蓝色荧光而CCDs主要为绿色荧光,且三种量子点的量子产率QY也有较大差异,分别为86.06%(HCDs),51.35%(SCDs)和19.73%(CCDs)。三种碳量子点都具有激发依赖性的宽光谱荧光,能够被用作荧光标记物。其次,采用改进的Hummer的方法将石墨粉在高比例的氧化剂(KMnO4)下氧化成氧化石墨烯(GO),并通过后续DMF热处理的方法获得氮掺杂的高效荧光的还原性氧化石墨烯量子点(rGOQDs)。以GO为前体,通过DMF水热修饰的方法制备了还原性氧化石墨烯量子点rGOQDs,其中DMF被用作氮源和还原剂来提高rGOQDs的光致发光性能。经过DMF水热修饰后,GO被还原和氮掺杂而成为rGOQDs,该rGOQDs被环氧基团,羰基,羟基和C-N等功能化基团修饰,大大改善了其荧光性能,使得其量子产率由6.53%上升至24.62%,最终所得的rGOQDs拥有激发依赖性的宽波长荧光性能。然后,成功将HCDs,SCDs和CCDs三种碳量子点应用于斑马鱼活体荧光成像,使用一系列不同的浓度来测试不同量子点在斑马鱼体内的荧光成像效果,比较不同量子点荧光成像效果的差异,并分析碳量子点在斑马鱼体内的吸收、分布、代谢和排泄(ADME)的影响。通过对斑马鱼活体内长达15天的荧光效果研究,结果表明,在量子点溶液浸泡的48小时内,胚胎的成像效果良好。而随着时间的延长,碳量子点的荧光效果越来越差,直至最终完全观测不到荧光。通过向碳量子点溶液中加入生物体内含有的物质ATP,NADH和Fe3+离子,进行一系列浓度的猝灭测试以模拟生物活体内碳量子点的衰败过程,结果表明三种物质均能不同程度地猝灭碳量子点。最后,对于rGOQDs,主要研究了其在斑马鱼活体动物模型上的荧光成像,以及其相应的发育毒理学,基因毒理学研究。从其孵化率、死亡率、心率、体长和畸形等指标上反应其发育毒性,各指标都有一定的减弱,表明rGOQDs对斑马鱼有一定的发育毒性。为了研究rGOQDs对斑马鱼基因表达量扰动性,我们选择斑马鱼的芳香烃受体相关的基因cyp1a,cyp1c和cyp7a1以及对外界压力敏感的基因hsp70四个基因来进行研究。实时荧光定量PCR的结果表明,四种基因cyp1a,cyp1c,cyp7a1和hsp70分别明显上调了8.49,4.65,5.93和3.63倍,表明rGOQDs确实对斑马鱼的芳香烃受体(AhR)信号传导通路有一定的扰动性。同时,运用Tg(cyp1a:gfp)斑马鱼对rGOQDs进行研究,研究结果表明rGOQDs暴露后的Tg(cyp1a:gfp)斑马鱼荧光强度显著增加。此外,Western Blot的结果进一步证明了绿色荧光蛋白随着暴露浓度增加而高表达的状况,与荧光成像及实时荧光定量PCR的结果相符。这些关于石墨烯材料风险性的研究,将对石墨烯类材料的后续生物学应用具有重要的借鉴意义和参考价值。
[Abstract]:As a new organic carbon material, fluorescent carbon based quantum dots have become the "top" of the material industry because of their specific fluorescence properties, stable physical and chemical properties, high biocompatibility and other excellent properties. In recent years, the extensive research of carbon based quantum dots is becoming more and more close to the related fields of raw materials and environment. In this paper, two kinds of biomass (hair and pigskin) and a kind of low toxic and harmless citric acid are used as raw materials to prepare three kinds of carbon quantum dots with low toxicity and high biocompatibility, and apply them to the study of zebrafish fluorescence imaging. We have prepared a highly fluorescent reductive graphene oxide quantum dot from the very popular graphene material, and applied it to the study of fluorescence imaging and related toxicology of zebrafish. First, two kinds of biomass hair and pigskin were used as raw materials and microwave assisted carbonization was used to efficiently synthesize the hair based on hair. The fluorocarbon quantum dots (CDS) of two protein rich biomass, such as pigskin and pigskin, have been prepared by microwave assisted carbonization of biomass (HCDs) and quantum dots (SCDs) by microwave assisted carbonization, and we also use the chemical hydrothermal synthesis of citric acid by chemical hydrothermal synthesis. The carbon quantum dots of citrate were synthesized with urea as raw materials, and compared with the first two quantum dots with biomass as raw materials, the difference between the fluorescence and other properties of the three different carbon quantum dots was explored. The three carbon quantum dots were within 10 nm. The difference in the raw material resulted in the chemical composition and fluorescein The difference in energy is that HCDs and SCDs are mainly blue fluorescence in the ultraviolet range and CCDs is mainly green fluorescence, and the quantum yield QY of the three quantum dots are also different, 86.06% (HCDs), 51.35% (SCDs) and 19.73% (CCDs), respectively. The three kinds of carbon quantum dots all have wide spectral fluorescence that excite dependence, and can be used as fluorescent labels. Second, Graphite powder was oxidized to graphene oxide (GO) under a high proportion of oxidant (KMnO4) by improved Hummer method, and the reduced graphite oxide quantum dots (rGOQDs) with high efficiency fluorescence of nitrogen doping was obtained by subsequent DMF heat treatment. The amount of reductive graphene oxide was prepared by DMF hydrothermal modification with GO precursor. Subpoint rGOQDs, in which DMF is used as a nitrogen source and reducing agent to improve the photoluminescence properties of rGOQDs. After DMF hydrothermal modification, GO is reduced and nitrogen doped to become rGOQDs. The rGOQDs is modified by functional groups such as epoxy groups, carbonyl, hydroxyl and C-N, greatly improving their fluorescence properties, making the quantum yield rise from 6.53% to 24.62%. The final rGOQDs has excitation dependent wide wavelength fluorescence performance. Then, three carbon quantum dots (HCDs, SCDs and CCDs) are successfully applied to zebrafish living fluorescence imaging, and a series of different concentrations are used to test the fluorescence imaging effects of different quantum dots in zebrafish, and compare the difference of fluorescence imaging effects at different quantum dots. The effects of carbon quantum dots on the absorption, distribution, metabolism and excretion (ADME) in zebrafish were analyzed. The results of 15 days' fluorescence in zebrafish body were studied. The results showed that the imaging effect of the embryos was good within 48 hours of soaking in the quantum dots solution. A series of concentrations of ATP, NADH and Fe3+ ions were added to the carbon quantum dots solution to simulate a series of quenching tests to simulate the decay process of carbon quantum dots in living organisms. The results showed that the three substances could all quenched carbon quantum dots in varying degrees. Finally, for rGOQDs, the main research was The fluorescence imaging of the zebrafish living animal model and its corresponding developmental toxicology and gene toxicology study on the developmental toxicity of its hatchability, mortality, heart rate, body length and malformation have been weakened to a certain extent, indicating that rGOQDs has certain developmental toxicity to zebrafish. In order to study the rGOQDs pair of spots. We chose four genes, CYP1A, cyp1c and CYP7A1, and HSP70 gene HSP70, which were related to the aromatics receptor related genes of zebrafish. The results of real-time quantitative PCR showed that the four genes, CYP1A, cyp1c, CYP7A1, and HSP70, were significantly up to 8.49,4.65,5.93 and 3.63 times higher than that of cyp1c, CYP7A1 and HSP70, respectively. RGOQDs did have a certain disturbance to the signal transduction pathway of the aromatic hydrocarbon receptor (AhR) of zebrafish. At the same time, Tg (cyp1a:gfp) zebrafish was used to study rGOQDs. The results showed that the fluorescence intensity of Tg (cyp1a:gfp) zebrafish after rGOQDs exposure was significantly increased. Moreover, the results of Western Blot further proved that the green fluorescent protein was followed by the results. The high expression of exposure concentration is consistent with the results of fluorescence imaging and real-time fluorescent quantitative PCR. The research on the risk of graphene materials will have important reference and reference value for the follow-up biological application of graphene materials.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;TS254.7

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