Our research group is called ‘Photochemistry and Photobiology’. We are interested in the use of fluorescence techniques to better understand physical processes and the application of this knowledge in Biophysics and Biological studies.
Our main research lines are:
DNA labeling fluorescent dyes. Xanthene succinimidyl ester derivates have been synthesized as amine-reactive derivative able to yield stable covalently labelled biopolymers. The spectralchanges associated with its neutral-anion transition allows to be used as a nucleic acid probe in a homogeneous assay.
Excited-state proton transfer reactions.Our research in the last few years involved the kinetic characterization of excited-state proton transfer reactions and their effect on fluorophores’ properties. We determined the relevant rate constants of the ESPT reaction and the spectral parameters related to absorption and emission, kinetic equations and expressions for the fluorescence decay surface. For this, we made use a new global compartmental analysis approach.
Fluorescence resonance energy transfer (FRET) in DNA. The next research line is based in exploiting FRET as a versatile tool to study biological systems. We are developing sensors for the detection of specific DNA sequences that may be related to certain genes. Different approaches and strategies have been used to study FRET between new and improved fluorescein derivatives labelling DNA probes and DNA intercalators. The combination of these approaches with FLIM (fluorescence lifetime imaging microscopy) shows their applicability to biological systems in vivo.
Synthesis and photophysical study of novel xanthene derivatives. We are synthesizing novel fluorescein derivatives in which the carboxylic group, formerly believed to be indispensable, is replaced by other chemical substituents. This modification reduces the number of possible negative charges, as well as prevents lactonization of the chromophore in solution. This kind of new fluoresceinsretains both the high extinction coefficient and fluorescence quantum yield of fluorescein in alkaline media, while the fluorescence quantum yield is close to zero when they are dissolved in acidic media. These features are highly suitable for pH probes.
Probing amyloidogenic protein-protein interactions using single molecule fluorescence spectroscopy. The mechanism of the early stages of formation of amyloid fibrils, proteinaceus fibrils related to neurodegenerative diseases like Alzheimer’s and Parkinson’s, is not well understood. We take advantage of state-of-the-art single molecule fluorescence methodologies to probe direct interactions between single proteins in the earlyformation of pre-fibrillar oligomers. This will provide a better understanding of the mechanisms ofamyloid fibril formation.
Photophysical study of novel xanthene derivatives by single-molecule fluorescence spectroscopy (FCS). We have shown how the presence of buffer-mediated proton transfer reactions also affects the ground state protonation state. This has important implications when these fluorophores are employed in fluorescence correlation spectroscopy (FCS). Thus, buffer-mediated proton transfer reactions make the fluorescence autocorrelation function from the xanthene dyes highly sensitive to some buffers concentration. The analysis of FCS curves provided the kinetic parameters of buffer-mediated proton transfer reactions in the ground state . These are the same than those for the reaction in excited state, confirming the equal nature of the process either in the ground- or excited-state. This implicates that the ESPT reaction is promoted when a sufficiently high concentration of buffer makes the reaction fast enough to compete with fluorescence emission.
