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Cagri Topal1 Kaan Kalkan1

1, Oklahoma State Univ, Stillwater, Oklahoma, United States

Once the chromophore of green fluorescent protein (GFP) is optically pumped (~395 nm) at its protonated form (A to A*), it undergoes a series of spontaneous steps, downhill in energy. In several picoseconds, it deprotonates to an excited intermediate state, I*, which subsequently de-excites in few nanoseconds to a ground intermediate state, I, by fluorescence (509 nm). Finally, the chromophore protonates and converts back to its A-state in less than a nanosecond. The cycle, A to A* to I* to I and then back to A, is known as the Förster cycle. So far, the I-state has only been identified on the basis of time-resolved fluorescence spectroscopy. The optical absorption spectrum of GFP at its I-state is difficult to acquire due to its short lifetime in the Förster cycle. Here, by Resonant Energy Transfer (RET) from a single Ag nanoparticle (AgNP) to adsorbed GFP molecules, we report capturing the optical absorption band of the I-state (~490 nm) under 405 nm optical pumping. RET is observed in the form of quenching dips on the Mie scattering spectrum of a single AgNP. However, we acquire the correct absorption spectrum by dividing RET spectrum by plasmon scattering lineshape and multiplying it with the fourth power of the photon energy, following the basic RET physics. GFP is conjugated to AgNPs through histidine-Ag binding. Without 405 nm optical pumping, we acquire two absorption bands, at 476 nm and 443 nm, which are precisely indicative of the B-state of GFP (stable deprotonated form) and denatured GFP, respectively. Apparently, a fraction of the GFP denatures during the 10-min scattering acquisition under intense broadband excitation. Although the major GFP population is expected to be at the A-state, the A-state marker band (395 nm) is out range of the measurement. Here, we rely on multiple attributes of a plasmonic AgNP to observe the I-state of GFP chromophore. First, the intense Mie scattering is measurable from a single particle. Second, oscillator strength of RET is much stronger than that of optical absorption. Third, by plasmon-enhanced (surface-enhanced) optical pumping, the dynamic GFP population can be significantly shifted from the A-state toward its excited states, A*, I* and I, in the Förster cycle. Thereby, RET from a single AgNP to the I-state GFP adsorbates can be resolved from Mie scattering.

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