School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Nanlu, 250100 Jinan, Shandong, PR China.
Anal Chim Acta. 2010 Mar 10;662(2):170-6. doi: 10.1016/j.aca.2010.01.014. Epub 2010 Jan 18.
A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0x10(-14)-3.0x10(-12) mol L(-1) was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.
本文提出了一种使用量子点和荧光显微镜技术对表面结合的抗体进行单分子计数的方法。通过在玻璃基底上修饰羧基基团，得到一个亲水表面，该表面与抗体的氨基反应，从而实现抗体的共价固定。首先研究了单分子在修饰表面上的非特异性吸附。然后，将量子点与表面固定的抗体分子形成复合物，并用作单分子成像的荧光探针。这里选择荧光显微镜作为单分子荧光检测的工具。通过电子倍增电荷耦合器件获取产生的荧光信号，并发现其与样品浓度成正比。在最佳条件下，通过单分子计数方法，在单分子数量和样品浓度之间获得了 5.0x10(-14)-3.0x10(-12) mol L(-1)的线性响应范围。
