荧光碳点的合成、表征及其在生化分析中的应用研究
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摘要
碳点作为新型荧光碳纳米材料,不仅具有优良的光学性能与小尺寸特性,而且还具有低细胞毒性。近年来,量子点以其优异的荧光性能已经在生物,医疗等领域取得了广泛应用,但是研究发现量子点中的重金属离子具有较强的细胞毒性,同时量子点还具有光眨眼现象,这使得其实际应用受到了一定程度的限制。而碳点可以克服量子点细胞毒性及光眨眼现象的问题。因此,碳点是替代量子点的良好选择。然而,目前碳点的制备与应用还处于初级阶段,其荧光量子产率还尚未达到量子点的水平。因此,探索高荧光量子产率碳点的制备方法以及寻找其新的应用就显得尤为迫切。本文主要从荧光碳点的制备与应用两个方面出发,首先,探索出一种表面羟基化的荧光碳点合成新方法,实验经济便捷,且所得碳点具有优良的性能;其次,将表面羟基化的荧光碳点应用于金属离子的检测并探讨与金属离子的作用研究;最后,参考文献合成出了表面羧基功能化的荧光碳点,并将其成功应用于溶菌酶含片中溶菌酶的检测。论文的主要内容概括如下:
     (1)建立一种以蜡烛灰作碳源,水热法一步合成荧光碳点的新方法。合成的碳点具有良好的荧光特性、好的水溶性、抗光漂白性、抗盐及低细胞毒性。透射电子显微镜(TEM)结果显示所合成碳点均匀分布,粒径为3.1±0.5 nm,同时采用动态光散射(DLS)进一步表征了碳点在水溶液状态下的水合粒径约为10nm,且分布均匀;红外光谱表征结果显示现碳点表面有大量羟基和少量的其它基团。此外,测得碳点荧光光谱对称,最大激发与发射分别在310nm与450nm,且荧光量子产率达5.5%。然而,在以蜡烛灰作碳源经硝酸处理得到表面羧基化碳点的一系列实验中发现,即使通过复杂的分离、提纯或钝化过程,其荧光量子产率也小于3.0%。据此,我们推测,羧基是强吸电子基团,当其直接与碳核相连时会不利于碳点荧光的发射。然而,羟基具有一定的供电子能力,故比较有利于碳点荧光发射。
     (2)研究了表面羟基化的碳点与金属离子的作用。氢氧化物溶度积极小的金属离子,易与碳点表面上的羟基结合,导致碳点聚集而发生荧光猝灭。基于此原理建立以碳点为荧光探针,实现对金属离子Cr3+、Fe3+、Al3+的痕量检测,方法的检测限分别为3.1、5.6、5.1 ng/mL。此外,实验还发现碳点的荧光会随着Al3+浓度的逐渐增大,出现先逐渐猝灭再逐渐恢复的现象。实验结果表明荧光猝灭过程为静态猝灭,荧光恢复过程为静态与动态相结合的猝灭;DLS测定结果表明荧光猝灭过程碳点逐渐聚集,而荧光恢复过程碳点又逐渐分散。我们推测这一特殊现象可能是由于Al3+是两性金属离子所导致。
     (3)利用共振光散射(RLS)技术研究了碳点与溶菌酶的相互作用。在弱碱性条件下,带负电荷的碳点与带正电荷的溶菌酶通过静电作用相结合,形成大颗粒复合物,具有较强的共振光散射信号,并且信号不易受酸度和离子强度影响。在优化条件下,增强的RLS强度与溶菌酶浓度在71.5μg/L~14.3 mg/L范围内呈良好的线性关系,检出限为37.8μg/L(3σ)。此方法成功应用于溶菌酶含片中溶菌酶含量的检测,回收率在95.8%~98.9%之间,相对标准偏差小于4.4%。
Carbon dots (CDs), as a new fluorescence carbon nanomaterial, possess unique optical properties and small size effects, with good biocompatibility and without cytotoxicity. In recent years, quantum dots (QDs) have been widely applied to the fields of biology and medicine owing to their unique fluorescent properties. Regrettably, the heavy metals in the QDs, such as cadmium, have serious cytotoxicity, at the same time, QDs have light blinking phenomenon, which limited their practical applications. While CDs could overcome the shortcomings of QDs, and may be a nice substitute for QDs. To date, however, the preparation and application of CDs are at primary stage, and the fluorescence quantum yield is worse than that of QDs. So, exploring good ways to prepare CDs with high fluorescence quantum yield and searching their new applications are quite significant. In this dissertation, we mainly describe the preparation and application of CDs. Firstly, we explored one new route to synthesize hydroxyls-coated CDs. The obtained CDs have good fluorescence properties. Moreover, the process of synthesis is convenient and economic. Secondly, the hydroxyls-coated CDs were applied to the detection of metal ions. Lastly, carboxyls-functionalized CDs were synthesized according to the reference, and were successfully applied to the detection of lysozyme in Lysozyme Buccal Tablets.
     The main contents of the dissertation consist of the following three parts:
     (1) The hydroxyls-coated CDs were facilely synthesized by hydrothermal reaction of candle soot in sodium hydroxide aqueous solution. They possessed excellent properties, such as good fluorescence performance, good water-solubility, anti-photobleaching, high salt tolerance, and low cytotoxicity. Transmission electron microscope (TEM) showed that CDs were uniformly distributed with the diameter of 3.1±0.5 nm. For further analysis the size in aqueous solution, dynamic light scattering (DLS) was employed and the results showed that they were also uniformly distributed with an average diameter of about 10 nm. With fourier transform infrared (FT-IR) spectrum, we observed that the surface of CDs had many hydroxyls and a few other groups. The CDs reported here have narrow and symmetry excitation and emission spectra, with the peaks at 310 nm and 450 nm, respectively. Furthermore, the CDs have nice florescence property with a fluorescence quantum yield of about 5.5%. However, the quantum yield of the CDs with lots of carboxyls on the surface, obtained by refluxing candle soot and nitric acid, is below 3.0% even by complicated separation or modification process. Based on this, we speculate that carboxyls, with strong electron drawing ability, are not beneficial to the fluorescence emission of CDs when binding to the surface of carbon core directly, while hydroxyls, with strong electron donated ability, are very favourable to the fluorescence emission.
     (2) We applied the hydroxyls-coated CDs in the detection of metal ions and studied the interaction between them. Some metal ions, whose hydroxides have extremely low solubility product constants, are very easy to combine with the hydroxyls of CDs and link the neighboring CDs. As a consequence, CDs get aggregated, following the fluorescence quenching. According to this, we established a new method for trace amount detection of Cr3+, Al3+ and Fe3+ with CDs as a fluorescent probe, with the detection limits of 3.1,5.1,5.6 ng/mL, respectively. In addition, we found that Al3+ has different phenomenon with the other metal ions. Upon the addition of rising amounts of Al3+, the fluorescence of CDs firstly got quenched and then recovered gradually. The experiment results showed that the quenching process was static quenching wile the recovery process was static and dynamic quenching; DLS results showed that the size of CDs increased in the quenching process, while decreased in the recovery process. We propose that the special phenomenon may be ascribed to the fact that Al3+ is a zwitter ion.
     (3) The interaction between CDs and lysozyme was studied with resonance light scattering (RLS) technique. In weak alkaline conditions, the negatively charged CDs could bind with positively charged lysozyme through electrostatic attractions, forming a large complex with strong RLS signals which could not be easily influenced by pH and ionic strength. Under the optimum conditions, the enhanced RLS intensity was found to be linearly correlated with the concentrations of lysozyme in the range of 71.5μg/L~14.3 mg/L, with the detection limit of 37.8μg/L (3a). This method was successfully applied to the detection of lysozyme in Lyszome Buccal Tablets with the recovery from 95.8% to 98.9% and the RSD lower than 4.4%.
引文
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