一种基于量子点荧光微球的高灵敏度免疫层析技术初步研究

学校代码：10270 学号：072200928

硕士学位论文

论文题目一种基于量子点荧光微球的高灵敏度

免疫层析技术初步研究

学院生命与环境科学学院

专业生物化学与分子生物学

研究方向纳米检测技术

研究生姓名白亚龙

指导教师魏新林王元凤

完成日期 2010年4月

论文独创性声明

本论文是我个人在导师指导下进行的研究工作及取得的研究成果。论文中除了特别加以标注和致谢的地方外，不包含其他人或机构已经发表或撰写过的研究成果。其他同志对本研究的启发和所做的贡献均已在论文中做了明确的声明并表示了谢意。

作者签名：日期：

论文使用授权声明

本人完全了解上海师范大学有关保留、使用学位论文的规定，即：学校有权保留送交论文的复印件，允许论文被查阅和借阅；学校可以公布论文的全部或部分内容，可以采用影印、缩印或其它手段保存论文。保密的论文在解密后遵守此规定。

作者签名：导师签名：日期：

上海师范大学硕士学位论文摘要

摘要

量子点具有宽的吸收峰，窄而对称的发射峰，且发射峰（即发光颜色）随尺寸可调，以及较高的荧光强度、较强的抗光漂白能力等特点，与传统有机染料相比具有更优越的性能，是一种极具潜力的荧光探针制备物。近年来在生物与医学免疫检测方面得到飞速的发展，同时，在食品安全检测方面也不断渗透漫延。因其具有优越的光学性能，有望突破胶体金免疫层析检测，制备出更为灵敏的量子点免疫层析快速检测试纸条。

本项工作围绕量子点的免疫检测体系，主要开展以下方面的研究：

1. 制备MPA稳定的水溶性CdTe量子点，探讨了最佳的合成条件，随着回流时间的延长，得到一系列由绿色过渡到红色的CdTe量子点，并对不同时间取得的样品进行了紫外-可见吸收光谱、荧光光谱、TEM等表征。对其纯化和稳定性能也做了相应的探讨，发现透析不适合量子点纯化，体系pH8-10有利于量子点的稳定。

2. 以红色CdTe量子点作为代表，小鼠IgG作为模式抗体，用免疫层析法探讨了量子点与抗体的最佳偶联方式，先后对比了直接偶联、EDC与NHS单独最为偶联剂、EDC 与NHS混合作为偶联剂这三种方法制备量子点探针，免疫层析结果显示EDC与NHS 混合作为偶联剂（质量比3：4）制备得到的探针具有优越的性能。直接偶联与以NHS 作为偶联剂制备的探针性能较差，不适合实际应用。

3. 为了得到更高荧光强度的荧光粒子，从两个方面入手提高量子点荧光强度：第一，制备核壳型量子点以期提高量子点荧光强度；第二，制备CdTe@SiO2纳米粒子以期提高单个纳米粒子的荧光强度。通过实验结果来看，水相制备的核壳型纳米粒子荧光强度并未得到改善，反而降低。反相微乳液制备的CdTe@SiO2纳米粒子因为实验试剂等影响，荧光强度大幅度下降，更不适合于检测分析用。

4. 制备高强度的SiO2/CdTe纳米粒子，用反相微乳液或史道伯法合成50-500 nm的二氧化硅纳米粒子，用硅烷偶联剂APTES使其氨基化，然后用偶联剂EDC与NHS将其与CdTe量子点偶联，制备得到高强度的荧光微球。

5. 经过一系列优化实验，以小鼠IgG与羊抗小鼠互为抗原抗体组装荧光微球免疫层析体系，经过与胶体金免疫层析对比，初步判断灵敏度比胶体金免疫层析体系高4-20倍。

关键词：CdTe量子点；偶联；抗体；探针；二氧化硅；免疫层析

ABSTRACT

Quantum dots (QDs), also named semiconductor nanocrystals, are mainly composed of group II–VI or III–V elements. Compared with conventional fluorophores, the nanoparticles have unique optical properties such as size tunable fluorescence and broad absorption spectra, narrow and symmetric emission spectra, strong fluorescence intensity and excellent anti-photobleaching property.

The aqueous-compatible QDs show great potentials as a new type of probe material in various fields, such as cellular imaging, clinical diagnoses, environmental pollutant. Especially, in the field of food safety detection, because of the superior optical property, the QDs probe is expected to exceed GICA and be used to assemble a more sensitive rapid detection immuno-chromatography stripe.

This study mainly focused on the following areas.

1. The water-soluble CdTe QDs were synthesized for different refluxing time with MPA as stabilizer. Various reaction conditions that affected the optical properties of CdTe QDs were studied, including the time of reaction and the pH value. Furthermore, these nanoparticles were characterized by UV-Vis spectroscopy, fluorescence spectra and TEM. Purification method and stability were studied, it is found that dialysis was not suitable for purification of CdTe QDs and pH8-10 was conducive to the stability of CdTe QDs.

2. The red-emitting CdTe QDs and mouse IgG were taken as the representative to study the conjugation behavior of QDs and antibody by a novel immunochromatographic method. After comparing to several methods, i.e., direct conjugation, EDC-mediated conjugation, NHS-mediated conjugation, EDC/NHS-mediated conjuation,

1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC) and

N-hydroxysulfo-succinimide (NHS) were selected together as coupling agents to conjugate QDs with antibody efficiently. The immunochromatographic strip was a superior method to study the conjugation of the fluorophore and antibody.

3. In order to obtain a higher fluorescence intensity of the nanoparticles, two schemes were applied: the first one, it is expected to add a shell to enhance the fluorescence intensity, that is, to prepare the core-shell QDs; the second one, in order to enhance the fluorescence