【作者】 刘慧；

【导师】 黄承志；

【作者基本信息】 西南大学 ， 分析化学， 2013， 博士

【摘要】 碳点(Carbon Dots, CDs)作为新型荧光碳纳米材料,具有类似量子点优良的光致发光性能,同时具有化学惰性和良好的生物相容性。因此,·碳点是潜在的可以替代量子点的良好选择。然而,碳点的制备与应用还有一定的挑战,主要体现在以下两方面：(1)高荧光量子产率的碳点,其发射光颜色大多为蓝色或绿色,与生物体的背景荧光相近,不利于成像研究；而发射光为红色的碳点,其荧光量子产率都较低。(2)由于碳点表面基团的不确定性和缺乏多样性,其应用依然受到限制。因此,如何充分利用荧光碳点的优良性质,将其应用在更多的领域还需进一步研究。本文主要从荧光碳点的制备出发,研究了其生物相容性,并最终将其应用于药物分析中。论文的主要内容包括三个部分,概括如下：第1部分：荧光碳点合成新方法研究。以血晶素为碳源,比较了煅烧和水热法制备碳点的发光性质,并最终采用水热法一步合成荧光碳点。研究发现,血晶素的碱性水溶液在聚四氟乙烯反应釜中,250℃下反应4h得到碳点,荧光量子产率达8.3%。合成的碳点不仅具有良好的光致发光特性、还有较好的水溶性和光化学稳定性。其形貌通过透射电子显微镜(TEM)表征,结果显示所合成碳点为球形或类球形,粒径分布在2-5nm,平均粒径约为3.23nm；另外,碳点在水溶液状态下的水合粒径采用动态光散射(DLS)进一步表征,约为10nm。Zeta电位分析表明碳点表面带有负电荷。高分辨透射电镜(HRTEM)成像显示其具有完整的晶体结构,晶格参数为0.22nm。X射线电子能谱与红外光谱表征结果显示碳点表面主要由C和O两种元素组成,带有羟基和羧基官能团。此外,该碳点具有良好的光致发光性能,激发与发射光谱较为对称,其荧光发射随激发波长红移而红移且强度会发生变化,最大激发与发射分别在290nm和390nm。第2部分：荧光碳点在药物分析中的应用。这一部分主要基于碳点荧光信号的改变检测药物,包括四个内容：1.基于小檗碱类猝灭CDs荧光检测小檗碱类。小檗碱类是一类广泛存在于自然界中的异奎宁生物碱,具有广泛的药理活性。研究发现小檗碱类的五种成分均可以猝灭CDs的荧光,且猝灭程度随着小檗碱类浓度的增大而增大。基于此,将CDs分别用于检测这五种小檗碱类,并在优化的条件下分别建立了线性关系。利’用此种方法检测盐酸小檗碱片中的盐酸小檗碱含量,结果与标示量一致。对猝灭机理进行研究,发现CDs的荧光发射与五种小檗碱类的吸收均有重叠,猝灭是因为CDs与它们发生能量转移。同时由于反应前后CDs荧光量子产率没有发生变化,说明CDs与小檗碱类药物之间发生了辐射能量转移而导致CDs荧光的猝灭。2.基于药根碱致CDs发射红移检测药根碱。研究发现,药根碱在碱性条件下使CDs发射峰红移,且红移程度随着药根碱浓度增大而增大。药根碱浓度在2.30μM到414μM范围内,位移值与浓度对数值呈良好的线性关系,方程式：Δλem=17.3log c-6.64(μM), r=0.995(n=10)。药根碱致CDs发射红移的机理同样是基于辐射能量转移,主要是由于药根碱的羟基在碱性条件下解离,导致吸收发生变化,与CDs的荧光发射重叠部分发生变化,不同波长处猝灭程度不同,于是发射峰红移。3.基于支链聚乙烯亚胺钝化的碳点(PEI-CDs,参考文献制备)荧光恢复检测乙酰半胱氨酸。因为铜离子能与PEI-CDs表面钝化剂上的氨基发生配位作用,从而猝灭其荧光。但如果有乙酰半胱氨酸存在,其结构上的巯基能与铜离子作用将Cu2+还原为Cu+同时自身形成二硫键,从而破坏了Cu2+与PEI-CDs表面氨基之间的作用,使得PEI-CDs荧光恢复,以此建立了PEI-CDs免标记快速、灵敏检测乙酰半胱氨酸的方法。在优化条件下,荧光恢复效率与乙酰半胱氨酸浓度在5.56μM～277.8μM范围内呈良好的线性关系,线性方程为：I/I0=0.88+0.013c(μM)，r=0.997(n=10)。4.在PEI-CDs表面偶联上FITC,基于荧光比率法检测pH。通过荧光光谱表征证明PEI-CDs上偶联了FITC。在pH6.4-7.2范围内对pH有响应,线性方程如下：I520/I450=0.550+0.0252pH, r=0.986。第3部分：CDs生物相容性的研究。首先,研究了血晶素制备的CDs及支链聚乙烯亚胺钝化的碳点(PEI-CDs,根据文献制备)和CTAB-CDs(本实验室以富勒烯为碳源,CTAB为钝化剂制备)的细胞毒性。结果发现由血晶素制备的羟基碳点浓度高达5mg/mL时也不会对细胞造成明显毒性；,但PEI-CDs浓度大于2mg/mL可见明显的细胞毒性,且细胞存活率比同浓度PEI存在时低,这是由于在PEI-CDs制备过程中有受热,可能导致PEI进一步聚合从而增大了其毒性；CTAB作为阳离子表面活性剂,其细胞毒性为众人所知,CTAB-CDs的毒性相对最强,达到0.003mg/mL细胞存活率即降低至30%。说明碳点本身细胞毒性很低,其细胞毒性最终取决于CDs表面钝化剂的毒性作用。另外以斑马鱼和豆芽为模式生物,考察了CDs和PEI-CDs对于动植物活体生物生长的影响。在0.05mg/mL CDs和PEI-CDs溶液中,均未发现有斑马鱼胚胎孵化延迟效应和胚胎发育畸形；通过数据分析发现在0.05mg/mL CDs和PEI-CDs溶液中斑马鱼仔鱼死亡率与对照组没有明显差异。CDs浓度小于0.005mg/mL时不会对豆芽的生长产生明显的阻碍,而浓度为20mg/mL时培养的豆芽根茎长则与对照组有明显差异。PEI-CDs浓度小于0.05mg/mL没有明显差异,大于0.2mg/mL则培养的豆芽根茎长与对照组有明显的差异。这些结果说明,碳点本身具有良好的生物相容性,其毒性最终取决于CDs表面钝化剂的毒性强弱。总而言之,本论文新制备了荧光碳点,并基于碳点荧光信号的改变将该碳点与另外一种PEI钝化碳点用于药物分析；最后研究了该碳点和其他两种碳点的生物相容性。更多还原

【Abstract】 Carbon dots (CDs), a new fluorescent carbon nanomaterial, not only possess unique optical properties, but also have good biocompatibility. So they are considered as a potential substitute for quantum dots. However, there remain some challenges in the preparation and application of CDs. Firstly, fluorescence quantum yield of the red CDs are always too low, while the CDs with high fluorescence quantum yield are blue or green, which is similar to the fluorescence of biological sample. Secondly, uncertainty of the surface groups and the lack of diversity of CDs lead to the limitation of their application. As a result, the development of novel preparation of CDs with excellent fluorescence and how to make it applicable in more and more fields are quite important. In this thesis, we mainly state the preparation, biological compatibility of CDs, as well as their applications in the pharmaceutical analysis.The main contents of the thesis consist of the following three parts:Part I. Study on the preparation of CDs. By comparation the photoluminescence performance of CDs synthesized from hemin by calcination and hydrothermal reaction, we prepared CDs by hydrothermal reaction from hemin. The hydroxyls-coated CDs were facilely synthesized by hydrothermal reaction from hemin, which possesses excellent photoluminescence property, good water-solubility and optochemical stability. Transmission electron microscope (TEM) imaging showed that CDs were uniformly distributed with the diameter of2-5nm, and the average diameter is3.23nm. Dynamic light scattering (DLS) analysis showed that the average hydrodynamic size of CDs was about10nm and the surface of CDs was negative charged. High resolution transmission electron microscopy (HRTEM) measurement revealed the CDs have integrated crystalline structure, and the lattice parameter was0.22nm. The X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) results implied the existence.of hydroxyl and carboxyl groups on the surface of the CDs. The CDs had good photoluminescence properties with quantum yield of8.3%.Part Ⅱ. The application of CDs in the pharmaceutical analysis. Based on the fluorescence change, the CDs were applied in the pharmaceutical analysis. This section includes four elements.1. Detection of the berberines (Bers) based on fluorescence quenching of CDs. The emission of the CDs can be quenched obviously with the addition of five kinds of Bers respectively. The fluorescence intensity of CDs decreases with increasing amount of Bers. Under optimum experimental conditions, good linear correlations between the quenching intensity (ΔI) and the logarithm of the concentration of Bers was obtained and a simple spectrofluorometric method of various Bers was established. The concentration of Ber in berberine hydrochloride tablets determined by the proposed method relates well with that claimed by the label. To understand the mechanism of the experiment, we found that the emission of CDs and the absorption of Bers overlapped, and we speculated energy transfer may happen between CDs and Bers. Furthermore, the fluorescence lifetime of CDs have no variation before and after the addition of Bers, indicating quenching radiation energy transfer occured.2. Detection of jatrorrhizine (Jat) based on the emission red shift of CDs. It was found that, in BR buffer at pH10, the addition of Jat may lead to the quench of CDs fluorescence intensity and induce the red shift of the emission of CDs. And the redshift presented a good linear relationship with the logarithm of the concentration of Jat from2.30μM to414uM with the the equation of Δλem=17.3log c-6.64(μM), correlation coefficients of0.995(n=10). It should be noted that the other four Bers are not able to change the emission peak of CDs. It was speculated the red-shift is owing to the radiation energy transfer. Under this condition, higher pH induces further dissociation of hydroxyl of Jat, and then the absorption of Jat changed, which lead to the change of overlap between CDs and Jat.3. Detection of N-Acetylcysteine (NAC) based on the fluorescence of CDs passivated by branched polyethylene imine (PEI-CDs, prepared according to literature) off-on. Cu2+ions can be captured by the amino groups of PEI-CDs and form an absorbent complex at the surface of CDs, resulting in fluorescence quenching of the PEI-CDs. While in the present of NAC, thiol group of NAC can reduce cupric to cuprous and form disulfur bond, thus the fluorescence of PEI-CDs resumes. Herein, we presented a non-label, fast detection-of NAC. Under the optimum condition, enhanced PL intensity was found to be linearly correlated with the concentrations of NAC, and a linear equation of I/I0=0.88+0.013c (μM) is followed in the range of5.56μM～277.8μM with the correlation coefficients of0.997(r, n=10).4. FITC was coupled on the surface of PEI-CDs for pH testing based on fluorescence ratio method. Fluorescence spectroscopy showed the achievement of the coupling of FITC on PEI-CDs, which can detect the slight changes of pH from6.4to7.2.Part Ⅲ. Biocompatibility study of the proposed CDs. Firstly, cytotoxicity of the CDs prepared with hemin, CDs passivated by branched polyethylene imine (PEI-CDs, prepared according to literature) and CTAB-CDs (fullerenes as carbon source, CTAB as the passivation) was achieved in this section. The results showed that more than5mg/mL hydroxylated CDs prepared with hemin can not lead to significant toxicity to the cells; while PEI-CDs are toxic even at the concentration of2mg/mL, and the cell viability is lower than that in the presence of only PEI at the same concentration. We speculated that during the preparation of PEI-CDs, high temperature may induce the polymerization of PEI, which lead to the increased toxicity. Cytotoxicity of CTAB-CDs is the highest among the three kinds of CDs in this study because of the toxicity of CTAB, which is widely known as a cationic active agent, and0.003mg/mL of CTAB-CDs can make the cell viability reduce to30%. In a word, CDs itself is not toxic, and the corresponding cytotoxicity depends on the passivation agent on the surface of CDs. Zebrafish and bean sprouts were employed as the model organisms to investigate the biocompatibility of CDs and PEI-CDs to live plants and animals. In0.05mg/mL of CDs or PEI-CDs solution, no delayed embryo hatching effects and embryo malformations were observed, and the analysis demonstrated larval mortality in these two groups is not obvious diferent from the control. Further study showed that less than5mg/mL of CDs solution didn’t significantly impede the growth of sprouts, while the concentration increased to20mg/mL the length of sprouts roots were distinct different from the control group. Less than0.05mg/mL, PEI-CDs has no effect on the length of sprouts rhizome, but when the concentration of PEI-CDs is higher than0.2mg/mL, the length of sprouts rhizome are obviously different compared to the control. These study indicated that within a certain range, CDs have good biocompatibility, and the corresponding cytotoxic depends on the passivation agent on the surface of CDs.In summary, we explored a new method to synthesize CDs. And then, CDs were applied in pharmaceutical analysis. Lastly, we studied the biocompatibility of CDs preparation from hemin and the PEI-CDs.更多还原