量子点荧光探针细胞标记的研究

发布时间：2018-09-11 21:37

【摘要】：量子点作为一种新型的荧光纳米材料，具有优于其他荧光材料如有机荧光染料、荧光蛋白的光学性质。近年来，量子点已被广泛的应用于细胞标记，组织及活体成像等领域。当量子点被用于生物成像标记时，常被修饰上各种特异性的生物分子，，因而评价修饰后的量子点荧光探针是很必要的。基于此，本论文用三种方法制备了量子点-转铁蛋白荧光探针，并搭建了一套系统用于评价三种量子点-转铁蛋白荧光探针细胞标记效果，论文具体完成的工作如下：（1）用静电连接、偶联剂EDC偶联和变性转铁蛋白包覆三种方法制备了CdTe/CdSe量子点-转铁蛋白荧光探针，并通过不同的手段如发射光谱、毛细管电泳、测量其zeta电位等对其进行了表征，结果发现相对于单纯的量子点，静电连接、偶联剂EDC偶联制备的量子点-转铁蛋白荧光探针的荧光强度均有所降低，而变性转铁蛋白包覆制备的量子点-转铁蛋白荧光探针荧光强度略有增强，而这三种方法制备的量子点-转铁蛋白荧光探针zeta电位的值均降低，这说明量子点-转铁蛋白探针胶体体系变得不稳定。且将这三种量子点-转铁蛋白荧光探针进行了细胞标记实验，通过CCD细胞成像及实时荧光光谱对标记效果进行了研究。（2）本文建立了一种在毛细管中压力驱动液流的细胞标记效率检测简便方法。该方法利用微流注射泵将待检测的标记细胞组注射到毛细管的进样端，在压力的驱动下，细胞逐个通过检测窗口，利用双通道光谱仪监测细胞的一系列荧光信号峰，之后对信号进行统计分析就可得到标记的效率。我们利用这一套系统对上述三种不同方法制备的量子点-转铁蛋白荧光探针标记HeLa细胞效率进行了考察，这三种方法制备的探针标记效率依次为78.86±9.57%，85.55±3.88%，40.09±10.2%，表明EDC偶联的探针标记效率最高，静电连接的探针标记效率其次，变性转铁蛋白包裹的探针标记效率最低，这一规律与同时进行的流式细胞仪验证的结果相符。该方法有望在荧光探针的研究中得到应用。
[Abstract]:As a new kind of fluorescent nanomaterials, quantum dots have better optical properties than other fluorescent materials such as organic fluorescent dyes and fluorescent proteins. In recent years, quantum dots have been widely used in cell labeling, tissue and in vivo imaging. When quantum dots are used for biomarkers, they are often modified with various specific biomolecules, so it is necessary to evaluate the fluorescence probes of modified quantum dots. Based on this, three methods were used to prepare quantum dot-transferrin fluorescence probe, and a system was set up to evaluate the labeling effect of three quantum dot-transferrin fluorescent probes. The main works are as follows: (1) CdTe/CdSe quantum dot-transferrin fluorescence probe was prepared by electrostatic bonding, coupling agent EDC coupling and denatured transferrin coating. It was characterized by capillary electrophoresis and zeta potential measurement. It was found that the fluorescence intensity of quantum dot-transferrin fluorescence probe prepared by coupling agent EDC was decreased compared with simple quantum dot, electrostatic connection and coupling agent EDC. However, the fluorescence intensity of quantum dot-transferrin fluorescence probe prepared by denatured transferrin coating increased slightly, while the zeta potential of the three methods decreased. This indicates that the colloidal system of quantum dot-transferrin probe becomes unstable. Three kinds of quantum dot-transferrin fluorescent probes were used for cell labeling. The labeling effect was studied by CCD cell imaging and real-time fluorescence spectroscopy. (2) A simple method for the detection of cell labeling efficiency in capillary pressure driven fluid flow was established. In this method, the labeled cell group to be detected was injected into the injection end of capillary by microflow injection pump. Under the pressure, the cells passed through the detection window one by one, and a series of fluorescence peaks of the cells were monitored by dual channel spectrometer. The efficiency of marking can be obtained by statistical analysis of the signal. We used this system to investigate the efficiency of labeling HeLa cells with three different methods of quantum dot-transferrin fluorescence probe. The labeling efficiency of the three methods was 78.86 卤9.57 and 85.55 卤3.88 respectively, which indicated that the probe labeling efficiency of EDC coupling was the highest, that of electrostatic connection was the second, and that of denatured transferrin coated probe was the lowest. This rule is consistent with the results of simultaneous flow cytometry verification. The method is expected to be applied in the study of fluorescent probes.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08;TB383.1

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