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.
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-FTIR). 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.
Onodera
Kota
K
Laboratory for Nanoelectronics and Spintronics, Research Institute of Electrical Communication, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Japan.
Hirano-Iwata
Ayumi
A
Miyamoto
Ko-ichiro
K
Kimura
Yasuo
Y
Kataoka
Masatoshi
M
Shinohara
Yasuo
Y
Niwano
Michio
M
D001152:Arsenicals / Q000737:chemistry*; D005708:Gallium / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry*
D001152:Arsenicals; D010455:Peptides; D011506:Proteins; D014867:Water; C043055:gallium arsenide; D005708:Gallium