摘要
量子点(QDs)是近年发展起来的一种新型荧光纳米材料,与传统有机荧光染料相比,具有许多优良的荧光性能,在生物分析化学、荧光免疫学、基因组学、蛋白质组学和细胞生物学等领域显示出广阔的应用前景,已经引起人们的广泛关注。
本论文在简要综述量子点技术发展的基础上,以量子点制备、纯化、性能表征及在农药和兽药残留检测中应用为主线,主要开展以下几个方面工作。
一、基于量子点和羊抗小鼠二抗的偶联物,建立一种灵敏、快速检测饮用水中毒死蜱的荧光免疫分析法(cFLISA)。
基于量子点和二抗偶联物的荧光免疫分析法(cFLISA)中的线性检测范围为15.2-205.5ng mL-1,灵敏度(IC50)为50.2ng mL-1,检出限(LOD)为8.2ng mL-1。
荧光免疫分析法(cFLISA)与传统酶联免疫分析法(ELISA)相比,不仅灵敏度提高1.5倍,检测时间也缩短0.5h。
在实际水样检测中,荧光免疫分析法(cFLISA)的检测结果与酶联免疫分析法(ELISA)、高效液相色谱方法(HPLC)的检测结果相符,表明该方法可用于环境和食品中农药残留的检测和大规模样品筛查。
二、利用水相法合成碲化镉量子点,并制备相应量子点-链霉亲和素荧光探针(QDs-SA),建立基于QDs-SA偶联物的新型荧光免疫分析方法(QDs-SA-cFLISA)并应用于毒死蜱的检测。
采用水相合成法,以巯基丙酸为稳定剂,制备表面带羧基的水溶性碲化镉量子点(CdTe QDs);其激发光谱宽且连续,发射光谱比较窄而对称,粒径比较均一荧光性质稳定,具有良好的光学性能。
将表面带羧基的水溶性碲化镉量子点(CdTe QDs)和链霉亲和素(SA)偶联,制备量子点-链霉亲和素荧光探针(QDs-SA),作为信号系统和信号放大系统,建立新型的荧光免疫分析方法(QDs-SA-cFLISA)。
荧光免疫分析方法(QDs-SA-cFLISA)与常规酶联免疫分析法(ELISA)相比,量子点-链霉亲和素荧光探针(QDs-SA)取代常规酶联免疫分析法(ELISA)中的酶标抗体,灵敏度提高5.5倍,检测时间缩短1h。
在实际水样检测中,荧光免疫分析方法(QDs-SA-cFLISA)的检测结果与酶联免疫分析法(ELISA、高效液相色谱方法(HPLC)的检测结果相符,表明该方法可用于饮用水中毒死蜱残留的检测。
三、小分子直接包被技术和量子点(QDs)纳米材料相结合,建立灵敏、快速的免疫分析方法并用于虾仁中氯霉素的检测。
酶标板表面用戊二醛(GA)处理,引入醛基与氨基化氯霉素直接偶联,将氯霉素小分子直接包被在酶标板上,并结合生物素化一抗和量子点-链霉亲和素荧光探针(QDs-SA),建立基于小分子半抗原直接包被方式和量子点(QDs)荧光探针结合的新型荧光免疫分析方法(QDs-SA-direct Hap coated cFLISA)。
荧光免疫分析方法(QDs-SA-direct Hap coated cFLISA)的直接包被模式与传统酶联免疫分析法(ELISA)的完全抗原包被模式相比,抗原-抗体结合效率高且稳定性强,不仅灵敏度提高5倍,检测时间也缩短1h。
在实际样品虾仁检测中,荧光免疫分析方法(QDs-SA-direct Hap coated cFLISA)的检测结果与酶联免疫分析法(ELISA、高效液相色谱方法(HPLC)的检测结果相吻合,表明该方法可作为传统酶联免疫分析法(ELISA)重要的补充,也用于食品和环境中兽药残留的检测。
Quantum dots (QDs) are somewhat spherical nanocrystals in the size range of 1-10 nm diameter. These QDs are a brand new class of luminescent inorganic fluorophores, which have several important advantages over conventional fluorescent dyes. In recent years, QDs have gained increasing attention and played important roles in the fields of biochemistry, fluorescence immunoassay, genomics, proteome, cell biology and molecular biology, etc. Aiming at this important research direction, the development of QDs was summarized, and then the following several works have mainly performed by taking QDs preparation, QDs characterization and QDs applications in pesticide and veterinary drugs residue analysis as the line of this dissertation. 1. A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of chlorpyrifos in drinking water has been developed.
Based on QDs as the fluorescence label coupled with secondary antibody (Ab2), the cFLISA allowed for chlorpyrifos determination in a liner working range of 15.2-205.5ng-mL-1. The 50% inhibition value (IC50) and the limit of detection (LOD) of the cFLISA were 50.2ng mL-1 and 8.4ng mL-1 respectively. In water sample analysis, the results of cFLISA were similar to those obtained from cELISA and HPLC, while the detection time by cFLISA was reduced 0.5h compared with ELISA. It showed that cFLISA could be used as a new screening method for the detection of pesticide residue in food and envirement.
2. Water-soluble CdTe QDs were synthesized directly and coupled with streptavidin (SA) as the fluorescent probe. Based on QDs-SA conjugate and ELISA, a novel fluoroimmunoassay (QDs-SA-cFLISA) has been developed for the detection of chlorpyrifos in drinking water.
Water-soluble CdTe QDs were prepared in aqueous solution by using 3-mercaptopropyl acid as the stabilizer and then characterized by fluorescence spectroscopy and TEM, respectively. The results show that these QDs have good optical properties, such as narrow spectral line widths and continuous absorption profiles, which provide a powerful foundation for further applications. A rapid and sensitive competitive fluorescence-linked immunosorbent assay (QDs-SA-cFLISA) based on QDs-SA conjugate has been developed for the detection of chlorpyrifos in drinking water. The sensitivity of QDs-SA-cFLISA increased by 5.5 folds and the detection time reduced 1h compared to conventional ELISA. In real sample analysis, the results of QDs-SA-cFLISA were similar to those obtained from a ELISA and a HPLC. This study showed that QDs-SA-cFLISA was more rapid and sensitive than conventional ELISA. Therefore, it can be used as a novel screening method for the detection of pesticide residue.
3. Based on direct coating of small molecular hapten (Hap) on the surface of microtiter plates and QDs-SA fluorescent probe, a new fluoroimmunoassay (QDs-SA-direct Hap coated cFLISA) has been developed for the detection of chloramphenicol (CAP).
In this Hap directly coated assay, the surface of microtiter plates was treated with a glutaraldehyde (GA) polymer network to introduce aldehyde groups for crosslinking of CAP with amino groups. Compared with the plates coated with Hap-carrier protein conjugate, the modified plates presented significantly high antibody and antigen (Ab-Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and QDs-SA conjugate were employed in this assay for amplification of signals. The sensitivity of QDs-SA-direct Hap coated cFLISA increased by approximate 5 folds and the stability improved greatly. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and HPLC. Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.
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