生物素诱导的共振能量转移荧光增强及其在生物测定中的应用。

Biotin induced fluorescence enhancement in resonance energy transfer and application for bioassay.

机构信息

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.

出版信息

Talanta. 2009 Dec 15;80(2):454-8. doi: 10.1016/j.talanta.2009.07.011. Epub 2009 Jul 10.

DOI:10.1016/j.talanta.2009.07.011

PMID:19836503

Abstract

A novel method to significantly enhance fluorescence resonance energy transfer (FRET) signal which occurred from fluoresceine isothiocyanate (FITC) to Dylight 549 was studied in this paper. Streptavidin was labeled with the donor fluorophore FITC and biotinamide was conjugated to the acceptor Dylight 549. When biotinamide bound to streptavidin, FRET would occur from FITC to Dylight 549 while a remarkable fluorescence enhancement of streptavidin-FITC was observed. The fluorescence enhancement of streptavidin-FITC in the presence of biotin was utilized in the FRET system to obtain higher fluorescence signal. Increase of fluorescence intensity of FITC and decrease of Dylight 549 depended on the concentration of competitive biotin. A homogeneous analysis method was established based on the fluorescence recovery of FITC in the FRET system with fluorescence enhancement. This method is highly sensitive and simple to determine the concentration of biotin. The detection limit for biotin was 0.5 nM and the linear range of the assay was 0.8-9.8 nM. The response time is no more than 15 min during the one-step assay due to the high affinity between streptavidin and biotin.

摘要

本文研究了一种显著增强荧光共振能量转移（FRET）信号的新方法，该信号源于异硫氰酸荧光素（FITC）到 Dylight 549。链霉亲和素被供体荧光团 FITC 标记，生物素酰胺与受体 Dylight 549 连接。当生物素酰胺与链霉亲和素结合时，FRET 会从 FITC 转移到 Dylight 549，同时观察到链霉亲和素-FITC 的荧光显著增强。在 FRET 系统中利用链霉亲和素-FITC 的荧光增强来获得更高的荧光信号。FITC 的荧光强度增加和 Dylight 549 的荧光强度降低取决于竞争性生物素的浓度。基于 FRET 系统中 FITC 荧光恢复的荧光增强，建立了一种均相分析方法。该方法具有较高的灵敏度和简单性，可用于测定生物素的浓度。生物素的检测限为 0.5 nM，测定范围为 0.8-9.8 nM。由于链霉亲和素和生物素之间的高亲和力，在一步测定中，响应时间不超过 15 分钟。