Goran Zgrablić

Femtosecond fluorescence upconversion setup with broadband detection in the ultraviolet

We show a femtosecond fluorescence upconversion setup with broadband detection to measure time-resolved emission spectra in the 300-550 nm range, upon excitation between 250 and 300 nm, with a time resolution of 100 fs. We present time-resolved fluorescence emission spectra of 2,5-diphenyloxazole in solution, which demonstrate the capabilities of the setup.

Ultrafast energy relaxation in bacteriorhodopsin studied by time-integrated fluorescence

Time-integrated fluorescence experiments on native bacteriorhodopsin and on its non-isomerizing form bR5.12 are reported. The experimental set-up was designed such as to observe emission exclusively from the excited state intermediate I-460. We obtain the first systematic investigation of the fluorescence spectra as a function of the excitation wavelength tuned throughout the entire absorption band of bR. An important finding is that the position of the fluorescence maximum does not show a systematic shift when the excitation wavelength is shortened. For excitation with high excess energy, we observe a broadening of the blue wing of the bR fluorescence, indicating incomplete vibrational energy relaxation on the time scale of the lifetime of I-460. Due to a much longer excited state lifetime, vibrational energy relaxation is more effective in bR5.12 and the fluorescence spectra are much less dependent on excitation wavelength. The results are placed in the general framework of thermalization between the retinal chromophore and the protein environment, and are compared with information obtained by femtosecond experiments.

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.