Photogeneration of quinone methide from adamantylphenol in an ultrafast non-adiabatic dehydration reaction.

The ultrafast photochemical reaction of quinone methide (QM) formation from adamantylphenol was monitored in real time using femtosecond transient absorption spectroscopy and fluorescence upconversion in solution at room temperature. Experiments were complemented by theoretical studies simulating the reaction pathway and elucidating its mechanism. Excitation with sub-20 fs UV pulses and broadband probing revealed ultrafast formation of the long-lived QM intermediate directly in the ground state, occurring with a time constant of around 100 fs. UV-vis transient absorption data covering temporal dynamics from femtoseconds to hundreds of milliseconds revealed persistence of the absorption band assigned to QM and partially overlapped with other contributions tentatively assigned to triplet excited states of the adamantyl derivative and the phenoxyl radical that are clearly distinguished by their evolution on different time scales. Our data, together with the computations, provide evidence of a non-adiabatic photodehydration reaction, which leads to the formation of QM in the ground state via a conical intersection, circumventing the generation of a transient QM excited state.

Population branching in the conical intersection of the retinal chromophore revealed by multipulse ultrafast optical spectroscopy.

The branching ratio of the excited-state population at the conical intersection between the S(1) and S(0) energy surfaces (Φ(CI)) of a protonated Schiff base of all-trans retinal in protic and aprotic solvents was studied by multipulse ultrafast transient absorption spectroscopy. In particular, pump-dump-probe experiments allowed to isolate the S(1) reactive state and to measure the photoisomerization time constant with unprecedented precision. Starting from these results, we demonstrate that the polarity of the solvent is the key factor influencing the Φ(CI) and the photoisomerization yield.

Heterogeneity and relaxation dynamics of the photoexcited retinal Schiff base cation in solution.

We present steady-state and broadband femtosecond fluorescence spectra of the protonated Schiff base of retinal in various protic and aprotic solvents, as a function of the excitation wavelength. A detailed spectral decomposition of the time-resolved fluorescence spectra allows us to isolate three spectral components: (i) the vibrationally relaxed S(1) fluorescence, (ii) a vibrationally hot S(1) fluorescence, and (iii) a higher-lying emission that undergoes spectral evolution on a time scale of 300-400 fs, which we assign to S(2) fluorescence. The vibrationally "cold" S(1) fluorescence exhibits three decay components upon 400 nm excitation (except in acetonitrile, which has two), but only two of them upon 570 nm excitation. These components clearly demonstrate the heterogeneity of the S(1) state in the sense that emission stems from several shallow potential surface minima. We discuss a connection between these decay channels and reactive and nonreactive excited-state paths on the basis of their solvent-dependent population and of previous high-performance liquid chromatography studies. There is no clear trend of the fluorescence decay times with solvent properties. Rather, a solvent effect manifests itself in acetonitrile, in that the number of fluorescence decay channels is smaller and that the quenching of the hot fluorescence seems more efficient. This effect has to do with the population pathways leading to the fluorescent states. These observations stress the heterogeneity of excited retinal Schiff base, influencing the decay but also the population channels. They also reinforce the claim that steric effects play an important role in the dynamics of the protein.

Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin.

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.

Application of functionalized lanthanide-based nanoparticles for the detection of okadaic acid-specific immunoglobulin G.

Marine biotoxins are widespread in the environment and impact human health via contaminated shellfish, causing diarrhetic, amnesic, paralytic, or neurotoxic poisoning. In spite of this, methods for determining if poisoning has occurred are limited. We show the development of a simple and sensitive luminescence resonance energy transfer (LRET)-based concept which allows the detection of anti-okadaic acid rabbit polyclonal IgG (mouse monoclonal IgG1) using functionalized lanthanide-based nanoparticles. Upon UV excitation, the functionalized nanoparticles were shown to undergo LRET with fluorophore-labeled anti-okadaic acid antibodies which had been captured and bound by okadaic acid-decorated nanoparticles. The linear dependence of fluorescence emission intensity with antigen-antibody binding events was recorded in the nanomolar to micromolar range, while essentially no LRET signal was detected in the absence of antibody. These results may find applications in new, cheap, and robust sensors for detecting not only immune responses to biotoxins but also a wide range of biomolecules based on antigen-antibody recognition systems. Further, as the system is based on solution chemistry it may be sufficiently simple and versatile to be applied at point-of-care.

Antibody-based donor-acceptor spatial reconfiguration in decorated lanthanide-doped nanoparticle colloids for the quantification of okadaic acid biotoxin.

With the increasing movement away from the mouse bioassay for the detection of toxins in commercially harvested shellfish, there is a growing demand for the development of new and potentially field-deployable tests in its place. In this direction we report the development of a simple and sensitive nanoparticle-based luminescence technique for the detection of the marine biotoxin okadaic acid. Photoluminescent lanthanide nanoparticles were conjugated with fluorophore-labelled anti-okadaic acid antibodies which, upon binding to okadaic acid, gave rise to luminescence resonance energy transfer from the nanoparticle to the organic fluorophore dye deriving from a reduction in distance between the two. The intensity ratio of the fluorophore: nanoparticle emission peaks was found to correlate with okadaic acid concentration, and the sensor showed a linear response in the 0.37-3.97 µM okadaic acid range with a limit of detection of 0.25 µM. This work may have important implications for the development of new, cheap, and versatile biosensors for a range of biomolecules and that are sufficiently simple to be applied in the field or at point-of-care.

Dependence of photochemical reactivity of the all-trans retinal protonated Schiff base on the solvent and the excitation wavelength.

An all-optical experimental technique aimed at measuring photoisomerization quantum yield (phi) of the all-trans protonated Schiff base of retinal in solution has been implemented. Upon the increase in the excitation wavelength from 400 to 540 nm a slight increase in phi from 0.16 +/- 0.03 to 0.20 +/- 0.02 is observed in the chromophore dissolved in methanol, whereas the phi value of the one dissolved in acetonitrile varies only from 0.22 +/- 0.03 (400 nm) to 0.23 +/- 0.04 (540 nm). The results suggest that dissipation of the excited-state vibrational energy excess, along with environment-induced modifications of the potential energy surfaces are necessary for an efficient retinal photoisomerization in both solvent and protein environment.

Ultrafast excited state dynamics of the protonated Schiff base of all-trans retinal in solvents.

We present a comparative study of the ultrafast photophysics of all-trans retinal in the protonated Schiff base form in solvents with different polarities and viscosities. Steady-state spectra of retinal in the protonated Schiff base form show large absorption-emission Stokes shifts (6500-8100 cm(-1)) for both polar and nonpolar solvents. Using a broadband fluorescence up-conversion experiment, the relaxation kinetics of fluorescence is investigated with 120 fs time resolution. The time-zero spectra already exhibit a Stokes-shift of approximately 6000 cm(-1), indicating depopulation of the Franck-Condon region in < or =100 fs. We attribute it to relaxation along skeletal stretching. A dramatic spectral narrowing is observed on a 150 fs timescale, which we assign to relaxation from the S(2) to the S(1) state. Along with the direct excitation of S(1), this relaxation populates different quasistationary states in S(1), as suggested from the existence of three distinct fluorescence decay times with different decay associated spectra. A 0.5-0.65 ps decay component is observed, which may reflect the direct repopulation of the ground state, in line with the small isomerization yield in solvents. Two longer decay components are observed and are attributed to torsional motion leading to photo-isomerization. The various decay channels show little or no dependence with respect to the viscosity or dielectric constant of the solvents. This suggests that in the protein, the bond selectivity of isomerization is mainly governed by steric effects.

Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: protein versus solvent environments.

Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing "locked" chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these six systems is compared and spectral signatures of isomerization and solvation are discussed. We find that the ultrafast red-shifting of fluorescence is associated mostly with solvation dynamics, whereas isomerization manifests itself as quenching of fluorescence. The observed multiexponential quenching of the protein samples differs from the single-exponential lifetimes of the chromophores in solution. The locked chromophore in the protein environment decays faster than in solution. This is due to additional channels of excited-state energy dissipation via the covalent and hydrogen bonds with the protein environment. The observed large dispersion of quenching timescales observed in the protein samples that contain the native pigment favors both an inhomogeneous model and an excited-state barrier for isomerization.