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 Structure Analysis

摘要

Here, we propose a label-free detection of protein—protein interactions that enables simultaneous qualitative analysis of target proteins by using Fourier transform infrared (FTIR) absorption spectroscopy in multiple internal reflection geometry (MIR-FTTR). Using this method, the target proteins were detected based on the peak height of the amide I and amide II bands, while discrimination of specific and nonspecific signals is made based on the secondary structure of the analytes, which is determined through second-derivative analysis of the amide I band. As a model system, an antigen peptide was immobilized on the surface of GaAs, which was transparent to mid-infrared light, and the interaction with its antibody was examined in aqueous media. We demonstrated that the binding of the antibody to the antigen immobilized on a GaAs surface selectively gave rise to beta-sheet amide I vibrations (1639 and 1690 cm~(-1)), while no structurally related signals were induced by nonspecifically adsorbed proteins. The peak height of the beta-peak (1639 cm~(-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 without being affected by the presence of bulk species, enabling rapid and wash-free detection. Our method provides not only rapid label-free detection of protein—protein interactions but a more accurate discrimination between specific and nonspecific interactions through the use of the secondary structure of the target proteins as a measure for the specific signals.