College of Chemistry & Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiaotong University, Shanghai, P R China.
Electrophoresis. 2013 Jun;34(12):1764-71. doi: 10.1002/elps.201200649. Epub 2013 Apr 20.
The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10(4) -1.5 × 10(6) Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.
荧光量子点（QD）的生物偶联和 QD 生物偶联物的纯化在生物应用中至关重要。在本文中，我们系统地研究了使用尺寸排阻色谱（SEC）技术、荧光光谱和荧光相关光谱（FCS）分离过程中 QD 生物偶联物的纯化效率及其稳定性。在这项研究中，商业 QD 和异硫氰酸荧光素（FITC）被用作标记探针，牛血清白蛋白（BSA）和抗体（Erbitux）被用作模式样品。共价键合和静电相互作用用于生物偶联过程。我们系统地研究了柱的规模、加载体积、洗脱缓冲液和分离介质等因素对 QD 生物偶联物纯化效率的影响，通过自制的 SEC 装置优化了条件，两次 SEC 纯化可得到高纯度和单分散的 QD 生物偶联物。此外，我们通过 FCS 研究了不同偶联方式和纯化条件下 QD 生物偶联物的稳定性，发现静电结合模式下生物偶联物的稳定性较差。我们还观察到，Sephacryl S300 HR（球形蛋白的分离范围：1×10^4-1.5×10^6 Da）是纯化绝大多数 QD-生物偶联物的最佳选择。我们在这里描述的工作将有助于 QD 在生命科学中的推广和应用。
