量子點(diǎn)熒光探針細(xì)胞標(biāo)記的研究
發(fā)布時(shí)間：2018-09-11 21:37
【摘要】：量子點(diǎn)作為一種新型的熒光納米材料，具有優(yōu)于其他熒光材料如有機(jī)熒光染料、熒光蛋白的光學(xué)性質(zhì)。近年來，量子點(diǎn)已被廣泛的應(yīng)用于細(xì)胞標(biāo)記，組織及活體成像等領(lǐng)域。當(dāng)量子點(diǎn)被用于生物成像標(biāo)記時(shí)，常被修飾上各種特異性的生物分子，，因而評(píng)價(jià)修飾后的量子點(diǎn)熒光探針是很必要的。基于此，本論文用三種方法制備了量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針，并搭建了一套系統(tǒng)用于評(píng)價(jià)三種量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針細(xì)胞標(biāo)記效果，論文具體完成的工作如下： （1）用靜電連接、偶聯(lián)劑EDC偶聯(lián)和變性轉(zhuǎn)鐵蛋白包覆三種方法制備了CdTe/CdSe量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針，并通過不同的手段如發(fā)射光譜、毛細(xì)管電泳、測(cè)量其zeta電位等對(duì)其進(jìn)行了表征，結(jié)果發(fā)現(xiàn)相對(duì)于單純的量子點(diǎn)，靜電連接、偶聯(lián)劑EDC偶聯(lián)制備的量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針的熒光強(qiáng)度均有所降低，而變性轉(zhuǎn)鐵蛋白包覆制備的量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針熒光強(qiáng)度略有增強(qiáng)，而這三種方法制備的量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針zeta電位的值均降低，這說明量子點(diǎn)-轉(zhuǎn)鐵蛋白探針膠體體系變得不穩(wěn)定。且將這三種量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針進(jìn)行了細(xì)胞標(biāo)記實(shí)驗(yàn)，通過CCD細(xì)胞成像及實(shí)時(shí)熒光光譜對(duì)標(biāo)記效果進(jìn)行了研究。 （2）本文建立了一種在毛細(xì)管中壓力驅(qū)動(dòng)液流的細(xì)胞標(biāo)記效率檢測(cè)簡(jiǎn)便方法。該方法利用微流注射泵將待檢測(cè)的標(biāo)記細(xì)胞組注射到毛細(xì)管的進(jìn)樣端，在壓力的驅(qū)動(dòng)下，細(xì)胞逐個(gè)通過檢測(cè)窗口，利用雙通道光譜儀監(jiān)測(cè)細(xì)胞的一系列熒光信號(hào)峰，之后對(duì)信號(hào)進(jìn)行統(tǒng)計(jì)分析就可得到標(biāo)記的效率。我們利用這一套系統(tǒng)對(duì)上述三種不同方法制備的量子點(diǎn)-轉(zhuǎn)鐵蛋白熒光探針標(biāo)記HeLa細(xì)胞效率進(jìn)行了考察，這三種方法制備的探針標(biāo)記效率依次為78.86±9.57%，85.55±3.88%，40.09±10.2%，表明EDC偶聯(lián)的探針標(biāo)記效率最高，靜電連接的探針標(biāo)記效率其次，變性轉(zhuǎn)鐵蛋白包裹的探針標(biāo)記效率最低，這一規(guī)律與同時(shí)進(jìn)行的流式細(xì)胞儀驗(yàn)證的結(jié)果相符。該方法有望在熒光探針的研究中得到應(yīng)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 鹵9.57 and 85.55 鹵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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08;TB383.1
本文編號(hào)：2237925
[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 鹵9.57 and 85.55 鹵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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08;TB383.1
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本文編號(hào)：2237925
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