Study On The Synthesis Of Fluorescent Carbon Dots And The Applications In Pharmaceutical Analysis

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.