Two-color sum-frequency generation spectroscopy (2C-SFG) is used to probe the molecular and electronic properties of an adsorbed layer of the green fluorescent protein mutant 2 (GFPmut2) on a platinum (111) substrate. First, the spectroscopic measurements, performed under different polarization combinations, and atomic force microscopy (AFM) show that the GFPmut2 proteins form a fairly ordered monolayer on the platinum surface. Next, the nonlinear spectroscopic data provide evidence of particular coupling phenomena between the GFPmut2 vibrational and electronic properties. This is revealed by the occurrence of two doubly resonant sum-frequency generation processes for molecules having both their Raman and infrared transition moments in a direction perpendicular to the sample plane. Finally, our 2C-SFG analysis reveals two electronic transitions corresponding to the absorption and fluorescence energy levels which are related to two different GFPmut2 conformations: the B (anionic) and I forms, respectively. Their observation and wavelength positions attest the keeping of the GFPmut2 electronic properties upon adsorption on the metallic surface.
We show that sum-frequency generation spectroscopy performed in the total internal
reflection configuration (TIR–SFG) combined with a dense gold nanoparticles monolayer
allows us to study, with an excellent signal to noise ratio and high signal to background
ratio, the conformation of adsorbed molecules. Dodecanethiol (DDT) was used as probe
molecules in order to assess the potentialities of the approach. An enhancement of more
than one order of magnitude of the SFG signals arising from the adsorbed species is
observed with the TIR geometry compared to the external reflection one while
the SFG non-resonant contribution remains the same for both configurations.
Although further work is required to fully understand the origin of the SFG process
on nanoparticles, our work opens new possibilities for studying nanostructures.
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