Ab initio CASPT2//CASSCF relaxation path computations are employed to determine the intrinsic (e.g., in vacuo) mechanism underlying the rise and decay of the luminescence of the anionic form of the green fluorescent protein (GFP) fluorophore. Production and decay of the fluorescent state occur via a two-mode reaction coordinate. Relaxation along the first (totally symmetric) mode leads to production of the fluorescent state that corresponds to a planar species. The second (out-of-plane) mode controls the fluorescent state decay and mainly corresponds to a barrierless twisting of the fluorophore phenyl moiety. While a "space-saving" hula-twist conical intersection decay channel is found to lie only 5 kcal mol(-1) above the fluorescent state, the direct involvement of a hula-twist deformation in the decay is not supported by our data. The above results indicate that the ultrafast fluorescence decay observed for the GFP chromophore in solution is likely to have an intrinsic origin. The possible effects of the GFP protein cavity on the fluorescence lifetime of the investigated chromophore model are discussed.

Vibrational activities in the Raman and resonance Raman spectra of the cationic, neutral, and anionic forms of 4'-hydroxybenzylidene-2,3-dimethyl-imidazolinone, a model compound for the green fluorescent protein chromophore, have been obtained from quantum-chemical calculations in vacuo and with the inclusion of solvent effects through the polarizable continuum model. It is found that inclusion of solvent effects improves slightly the agreement with experimental data for the cationic and neutral forms, whose spectra are qualitatively well-described already by calculations in vacuo. In contrast, inclusion of solvent effects is crucial to reproduce correctly the activities of the anionic form. The structural effects of solvation are remarkable both in the ground and in the lowest excited state of the anionic chromophore and influence not only the vibrational activity but also the photodynamics of the lowest excited state. CASPT2//CASSCF photoreaction paths, computed by including solvent effects at the CASSCF level, indicate a facile torsional deformation around both exocyclic CC bonds. Rotation around the exocyclic CC double bond is shown to lead to a favored radiationless decay channel, more efficient than that in gas phase, and which explains the ultrafast fluorescence decay and ground-state recovery observed in solution. Conversely, rotation around the exocyclic CC single bond accounts for the bottleneck observed in the ground-state recovery cycle. It is also speculated that the ultrafast radiationless decay channel would be hampered in protein for unfavorable electrostatic interactions and steric reasons.

Electrochemiluminescence (ECL) is a powerful transduction technique with a leading role in the biosensing field due to its high sensitivity and low background signal. Although the intrinsic analytical strength of ECL depends critically on the overall efficiency of the mechanisms of its generation, studies aimed at enhancing the ECL signal have mostly focused on the investigation of materials, either luminophores or coreactants, while fundamental mechanistic studies are relatively scarce. Here, we discover an unexpected but highly efficient mechanistic path for ECL generation close to the electrode surface (signal enhancement, 128%) using an innovative combination of ECL imaging techniques and electrochemical mapping of radical generation. Our findings, which are also supported by quantum chemical calculations and spin trapping methods, led to the identification of a family of alternative branched amine coreactants, which raises the analytical strength of ECL well beyond that of present state-of-the-art immunoassays, thus creating potential ECL applications in ultrasensitive bioanalysis.

Evidence of the biradicaloid and polyenic character of quinoidal oligothiophenes is reported by proving at the CASSCF//CASPT2 computational level the presence of a low-lying double exciton state responsible for the weak features observed in the NIR absorption region of the longest members of this class of molecules. The energy lowering of this state, accompanying the length increase in the oligomers, causes a displacement of the ground-state equilibrium geometry toward more biradicaloid structures because of the more efficient S0-S1 state mixing. Furthermore, it is shown that the doubly excited state is strongly coupled to the ground electronic state, and the coupling is mediated by a collective mode dominated by the out-of-phase stretching of adjacent CC bonds, recently shown to govern the Raman activity. All together, this evidence offers a unified view of the low-lying electronic states for quinoidal oligothiophenes and polyenes.