Label-Free Detection of Protein-Protein Interactions at the GaAs/Water Interface through Surface Infrared Spectroscopy: Discrimination between Specific and Nonspecific Interactions by Using Secondary

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• 作者：Kota Onodera ; Ayumi Hirano-Iwata ; Ko-ichiro Miyamoto ; Yasuo Kimura ; Masatoshi Kataoka ; Yasuo Shinohara ; Michio Niwano
• 刊名：Langmuir
• 出版年：2007
• 出版时间：November 20, 2007
• 年：2007
• 卷：23
• 期：24
• 页码：12287 - 12292
• 全文大小：254K
• 年卷期：v.23,no.24(November 20, 2007)
• ISSN：1520-5827

文摘

Here, we propose a label-free detection of protein-protein interactions that enables simultaneous qualitative analysisof target proteins by using Fourier transform infrared (FTIR) absorption spectroscopy in multiple internal reflectiongeometry (MIR-FTIR). Using this method, the target proteins were detected based on the peak height of the amideI and amide II bands, while discrimination of specific and nonspecific signals is made based on the secondary structureof the analytes, which is determined through second-derivative analysis of the amide I band. As a model system, anantigen peptide was immobilized on the surface of GaAs, which was transparent to mid-infrared light, and the interactionwith its antibody was examined in aqueous media. We demonstrated that the binding of the antibody to the antigenimmobilized on a GaAs surface selectively gave rise to -sheet amide I vibrations (1639 and 1690 cm^-1), while nostructurally related signals were induced by nonspecifically adsorbed proteins. The peak height of the -peak (1639cm^-1) in the amide I band linearly increased with the antiserum concentration as well as that of the amide II band.The detection limit (S/N = 3) was a 1:36 000 dilution for the amide I signal. In addition, through the use of surface-sensitive MIR-FTIR, the present sensor selectively detected the antigen-antibody interactions at the surfaces withoutbeing affected by the presence of bulk species, enabling rapid and wash-free detection. Our method provides not onlyrapid label-free detection of protein-protein interactions but a more accurate discrimination between specific andnonspecific interactions through the use of the secondary structure of the target proteins as a measure for the specificsignals.