Goldman Ellen R, Uyeda H Tetsuo, Hayhurst Andrew, Mattoussi Hedi
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USA.
Methods Mol Biol. 2007;374:207-27. doi: 10.1385/1-59745-369-2:207.
We have developed several conjugation strategies based on noncovalent self-assembly for the attachment of proteins and other biomolecules to water-soluble luminescent colloidal semiconductor nanocrystals (quantum dots [QDs]). The resulting QD-protein conjugates were employed in designing a variety of bioinspired applications, including single and multiplexed immunosorbent assays to detect toxins and small molecule explosives. In these studies we showed that QD fluorophores offer several important advantages. In particular, their tunable broad excitation spectra combined with narrow fluorescence emission peaks permit single-line excitation of multiple color nanocrystals, with facile signal deconvolution to extract individual contributions from each population (e.g., size) of QDs in multiplexed assays. Furthermore, the QDs strong resistance to photobleaching under continuous illumination relative to many organic dyes makes them ideal fluorophores for long-term cellular imaging studies. This chapter details the materials and methods for the synthesis of surface-functionalized CdSe-ZnS core-shell QDs, the construction and preparation of recombinant proteins, the conjugation of antibodies (and antibody fragments) to QDs, and the use of antibody-conjugated QDs in fluoroimmunoassays.
我们已经开发了几种基于非共价自组装的缀合策略,用于将蛋白质和其他生物分子连接到水溶性发光胶体半导体纳米晶体(量子点[QDs])上。所得的量子点-蛋白质缀合物被用于设计各种受生物启发的应用,包括用于检测毒素和小分子炸药的单重和多重免疫吸附测定。在这些研究中,我们表明量子点荧光团具有几个重要优势。特别是,它们可调谐的宽激发光谱与窄荧光发射峰相结合,允许对多种颜色的纳米晶体进行单线激发,在多重测定中通过简便的信号解卷积从每个量子点群体(例如尺寸)中提取各自的贡献。此外,相对于许多有机染料,量子点在连续光照下对光漂白具有很强的抗性,这使其成为长期细胞成像研究的理想荧光团。本章详细介绍了合成表面功能化的CdSe-ZnS核壳量子点的材料和方法、重组蛋白的构建和制备、抗体(及抗体片段)与量子点的缀合,以及抗体缀合量子点在荧光免疫测定中的应用。