【作者】 张红艳；

【导师】 徐淑坤；

【作者基本信息】 东北大学 ， 分析化学， 2009， 硕士

【摘要】 量子点具有很多优异的性质,如紫外吸收范围宽;荧光激发范围宽而连续;荧光发射峰峰形对称,半峰宽窄;荧光发射波长可调;抗荧光漂白能力强;荧光寿命比有机荧光染料长;量子产率较高等。近年来,作为一种新型的荧光生物探针,量子点在生物分子和多色标记、生物组织和细胞的标记成像、免疫分析以及疾病诊断等领域的研究中取得了很大的进展。本项研究在水溶性CdTe/CdS量子点的合成及其作为生物探针对胰蛋白酶测定和细胞标记的应用研究方面做了一系列工作,得到一些创新性结果,主要内容如下:首先提出用半胱氨酸作修饰剂,常见无机试剂为反应原料,将水浴法和水热法结合制备CdTe/CdS量子点的新方法。此法兼备水浴法和水热法的优点,合成周期短,条件温和,量子产率高。通过控制不同的条件合成出荧光发射波长分布范围宽(500～625 nm)的水溶性CdTe/CdS量子点。光学性能表征证明量子点的核壳结构,具备半峰宽窄、峰形对称等优异的光谱性能;结构性能表征表明量子点尺寸分布均匀,为近似球形颗粒。同时研究pH值、加热温度、加热时间等对晶体生长的影响。其次,实验中对合成的量子点进行透析纯化,后利用外层包被的半胱氨酸的羧基实现了与胰蛋白酶的共价链接,链接后量子点的荧光强度明显增强,当胰蛋白酶浓度在0.02～50.0mg/L范围内时,量子点荧光强度的变化与胰蛋白酶浓度呈现良好的线性关系。线性方程为:ΔI=17.86+8.87 c(mg/L),相关系数为r=0.9993,检出限为3μg/L。对浓度为33.3 mg/L的胰蛋白酶标准溶液平行测定11次,得到的相对标准偏差为0.78%。此法简便,快速,样品不需要复杂的前处理,结果令人满意。最后,基于与上述生物大分子的作用规律,利用量子点表面的羧基与抗体分子中氨基之间的作用,实现了CdTe/CdS量子点与兔抗人CEACAM8(CD67)抗体的共价结合,通过抗原-抗体之间的特异性反应,采用直接标记法成功地对宫颈癌上皮细胞HeLa细胞进行免疫标记与成像。结果表明,通过直接标记法可以成功实现对细胞的标记,有效减少量子点在细胞表面的非特异性吸附。与现有对HeLa细胞标记的报道相比,本方法简化了标记步骤,为量子点进一步用于癌细胞的检测提供了有益的参考。更多还原

【Abstract】 Quantum dots(QDs),as novel inorganic fluorophores,have attracted more and more attentions.Compared with traditional organic fluorophores,QDs possess a number of advantages due to their excellent properties such as narrower and symmetric emission spectra, broad and continuous absorption spectra,adjustable fluorescence emission wavelength,good photostability,and higher quantum yield.Recently,QDs with excellent optical properties are more and more applied in the bio-analysis,environmental analysis and detection of clinical medicine,instead of organic fluorescent dyes.Therefore,it is the most important to prepare high-quality QDs with ideal optical properties,good biocompatibility and less cytotoxicity directly in aqueous medium.In this study,a great deal of work was done on preparing high-quality QDs,applying QDs as biological probes in bio-labeling and cell imaging.The main content is as follows:The new method combining water-bathing and hydrothermal heating for the synthesis of a series of high-quality L-Cysteine(L-Cys) capped CdTe/CdS QDs was firstly introduced. This method possesses both advantages of water-bathing and hydrothermal methods to get high-quality QDs,such as markedly reduced synthesis time,higher quality of the QDs than water-bathing and better stability than a lone hydrothermal method.L-Cys was used as the stabilizer because it’s less toxicity.In the study,the core/shell CdTe/CdS QDs with different emission wavelength(500～625 nm) were prepared with different reaction time.The fluorescence and UV-vis absorption spectra of QDs indicated that CdS shell was capped on the CdTe core and the fluorescence of CdTe/CdS QDs was enhanced by the CdS overcoating.The transmission electronic microscopy(TEM) and powder X-ray diffraction(XRD) of as prepared QDs indicated that QDs dispersed well in aquous solution and the shape was approximately spherical.Meanwhile,the effect of pH value,reaction tempreture,hydrothermal time etc on the growth of QDs was studied.For the applications of QDs in analytical and biological fields,the as-prepared L-Cys capped CdTe/CdS QDs were conjugated with Trypsinase,in which the interactions rules between QDs and biological macromolecules such as proteins and enzymes were obtained. The as-prepared QDs were firstly purified by semipermeable merbrance.Then the as purified QDs were able to be conjuncted with trypsinase by the conjugation between the carboxylic groups on the surface of QDs and the amino groups on trypsinase,leading to the obvious enhancement in fluorescence intensity.The experimental results for the condition optimization suggested that the fluorescence of as prepared QDs responded selectively to trypsinase.A good linearity between the fluorescence intensity and the concentration of trypsinase in the range of 0.02～50.0 mg/L was obtained with a correlation coefficient of 0.9993 and the linear regression equation is△I=17.86+8.87 c(mg/L).The detection limit,calculated following the 3o IUPAC criteria,of 3μg/L was obtained with the RSD of 0.78%(33.3 mg/L,n=11).The method was relatively simple and sensitive.Finally,the as-prepared QDs were conjugated with rabbit anti-CEACAM8(CD67) antibody with the similar linking method as described above.By the reaction between antibody and antigen,QDs-antibody probes were successfully used to label HeLa cells.Experimental results indicated that the non-specific adsorption was not observed in the direct labeling method.Compared with other methods,the direct labeling procedure was relatively simpler.It can be expected that the CdTe/CdS QDs prepared here could be used as probes to get further application in immuno-assay,immuno-labeling and other biological areas.更多还原