Microtech 2011

Single molecule SERS leads to the observation of the intermediate form of green fluorescent protein chromophore

N.Z. Shafiq, Md. Shafayet Khurshid, A. Kaan Kalkan
Oklahoma State University, US

Keywords: single molecule, SERS, green fluorescent protein, GFP

Abstract:

The dominant absorption band at 395 nm of the green fluorescent protein (GFP) is attributed to the protonated form of the chromophore. Photo excitation forms an excited intermediate anion from this excited protonated form and relaxes back to the intermediate ground state, which eventually repopulates the ground state protonated form of the chromophore. Our unique study reports the observation of this excited state proton movement cycle (i.e. Förster Cycle) of the GFP chromophore by acquiring vibrational Raman spectra of single GFP molecules using “nanometal-on-semiconductor” SERS substrates and applying a “pump (405 nm) and probe (532 nm)” technique. We observe a new Raman peak at 1510 cm-1 and ascribe this as a marker for the intermediate form of the chromophore. Statistical analysis of the GFP population indicates a significantly higher probability of capturing this 1510 cm-1 peak with the protonation (1560 cm-1) Raman marker over the probability of finding both the 1510 cm-1 and deprotonation (1530 cm-1) peak in one “spectral jump”. We recognize that strong optical pumping due to the surface enhanced fields by the nanoparticles can produce favorable conditions for observing the intermediate form at single molecule resolution.
 

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