Surface Plasmon Field-Enhanced Fluorescence Spectroscopy Studies of the Interaction between an Antibody and Its Surface-Coupled Antigen

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• 作者：Fang Yu ; Danfeng Yao ; and Wolfgang Knoll
• 刊名：Analytical Chemistry
• 出版年：2003
• 出版时间：June 1, 2003
• 年：2003
• 卷：75
• 期：11
• 页码：2610 - 2617
• 全文大小：163K
• 年卷期：v.75,no.11(June 1, 2003)
• ISSN：1520-6882

文摘

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagneticfield of a surface plasmon mode for the excitation ofsurface-confined fluorophores. The ability to simultaneously monitor the interfacial refractive index changesand the fluorescence signals in real time offers a hugepotential for applications of SPFS in surface immunoreaction detection. In this study, gold surfaces were functionalized by mixed self-assembled monolayers exposingan antigen (biotin) at a density that was varied over a widerange. Specific antibody-antigen interactions were observed for anti-biotin antibody solutions passing over thesurfaces with a rather high flow speed driven by a home-built liquid-handling system. First, the use of the fluorophores Cy5 and Alexa Fluor 647 in SFPS-based immunoassays was investigated. It was found that Cy5 exhibitsstrong self-quenching, which makes it rather unsuitablefor quantitative measurements. For the in situ measurement of the binding kinetics, an angular "detuning" effectwas confirmed to negatively interfere with the fluorescencesignal in cases where large SPR signals were detected.An in-depth comparison between the SPR and the fluorescence signal reveals that the fluorescence yield of thedyes depends strongly on the separation distance fromthe gold surface. And finally, we stress the ability of SPFSto detect binding to surfaces containing extremely dilutedantigen density, where the SPR signal failed to follow.