Química Biofísica

Permanent URI for this collectionhttps://hdl.handle.net/10347/34378

O obxectivo do noso grupo é estudar a dinámica de procesos físicos e químicos en materiais moleculares, sistemas organizados e biolóxicos. Utilizamos principalmente técnicas espectroscópicas (fluorescencia con resolución temporal, detección de moléculas individuales, resonancia magnética nuclear) e cálculos mecanocuánticos para desentrañar a dinámica de variados procesos, obtendo tamén información estrutural dos sistemas investigados.

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Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines
(MDPI, 2025) Arana Díaz, Álvaro Jesús; Veiga Rúa, Sara; Cora Calvo, Diego; González Gómez, Manuel Antonio; Seijas Cerceda, Ana; Carballeda Álvarez, Maialen; Polo Montero, David; Cuesta, Alberto; Piñeiro Redondo, Yolanda; Rivas Rey, José; Novo, Mercedes; Al-Soufi, Wajih; Martínez Portela, Paulino; Sánchez Piñón, Laura; Robledo Sánchez, Diego; Universidade de Santiago de Compostela. Departamento de Química Física; Universidade de Santiago de Compostela. Departamento de Zooloxía, Xenética e Antropoloxía Física; Universidade de Santiago de Compostela. Departamento de Física Aplicada
Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies—electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)—for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture
Early Detection of Alzheimer’s Disease via Amyloid Aggregates: A Systematic Review of Plasma Spectral Biomarkers and Machine Learning Approaches
(MDPI, 2025) Hernández, Stella; Valladares Rodríguez, Sonia María; Novo, Mercedes; Al-Soufi, Wajih; Universidade de Santiago de Compostela. Departamento de Química Física; Universidade de Santiago de Compostela. Departamento de Electrónica e Computación
Background: Early diagnosis of Alzheimer’s disease (AD) is constrained by invasive and costly tests. Aggregation of 𝛽-amyloid and the Aβ 42/Aβ 40 ratio in cerebrospinal fluid (CSF) and blood are key biomarkers. Fluorescent probes can report aggregate states, and artificial intelligence (AI) can extract subtle patterns from spectral and blood data. This review synthesizes how probes and AI can identify aggregates and assess the Aβ 42/Aβ 40 ratio in body fluids to facilitate earlier AD diagnosis. Methods: PRISMA-compliant searches were conducted in Scopus, PubMed, Web of Science, and IEEE Xplore. Results: Twenty-eight studies met inclusion criteria. Plasma Aβ 42/Aβ 40 was lower in PET-positive individuals by ∼7–18%, with higher performance for mass spectrometry (mean AUC ≈ 0.80) than immunoassays (AUC ≈ 0.71). CSF Aβ 42/Aβ 40 showed larger group differences (∼50% reductions in PET+) and stronger PET concordance, outperforming plasma. Fluorescent probes—including AN-SP and CRANAD-28—were sensitive to early aggregates and showed in vivo imaging potential, but evidence is largely preclinical or from small cohorts. AI/ML approaches frequently achieved within-study accuracies >90% (e.g., 94–100% in spectral tasks), yet external validation and head-to-head tests of ratio alone versus ratio + AI remain scarce. Conclusions: Plasma Aβ 42/40 —particularly by mass spectrometry—currently provides the most reproducible fluid approximation to amyloid PET (mean AUC ≈ 0.80). Fluorescent probes sensitively detect oligomeric Aβ species and show in vivo potential, but evidence remains largely preclinical or from small cohorts. AI/ML methods can extract additional signal from spectral and multivariate blood data, yet consistent incremental gains over optimized Aβ42/40 assays have not been demonstrated due to limited external validation and head-to-head comparisons
CRISPR/Cas9 Delivery Systems to Enhance Gene Editing Efficiency
(MDPI, 2025) Seijas Cerceda, Ana; Cora Calvo, Diego; Novo, Mercedes; Al-Soufi, Wajih; Sánchez Piñón, Laura; Arana Díaz, Álvaro Jesús; Universidade de Santiago de Compostela. Departamento de Química Física; Universidade de Santiago de Compostela. Departamento de Zooloxía, Xenética e Antropoloxía Física
CRISPR/Cas9 has revolutionized genome editing by enabling precise and efficient genetic modifications across multiple biological systems. Despite its growing therapeutic potential, key challenges remain in mitigating off-target effects, minimizing immunogenicity, and improving the delivery of CRISPR components into target cells. This review provides an integrated analysis of physical, viral, and non-viral delivery systems, highlighting recent advances in the use of lipid nanoparticles, polymeric carriers, and hybrid platforms. We also examine an often overlooked factor: the aggregation behavior of the Cas9 protein, which may interfere with cellular uptake, the encapsulation efficiency, and nuclear localization. By comparing delivery platforms and their reported editing outcomes, we identify critical physicochemical parameters that influence therapeutic success. Finally, we propose standardized methods to assess Cas9 encapsulation and aggregation and discuss translational barriers such as manufacturing scalability and regulatory requirements. These insights aim to guide the development of safer and more effective CRISPR/Cas9-based therapies
2-Aminopyrimidine derivatives as potential fluorescent chemosensors for trivalent cations: Structural characterization, photophysical properties, and live-cell imaging
(Elsevier, 2025-09-22) Covarrubias, Alejandra A.; Rodríguez-Prieto, Flor; Mellado, Marco; Universidade de Santiago de Compostela. Departamento de Química Física
Variations in metallic cation levels have been associated with chronic non-communicable diseases such as Alzheimer’s and Parkinson’s (e.g., Al(III) and Fe(III)) and diabetes (e.g., Cr(III)). In this context, fluorescent chemosensors are valuable tools for detecting cation concentrations in complex matrices. Herein, we report the synthesis of two fluorescent pyrimidines: 4-(4-(dimethylamino)phenyl)-6-phenylpyrimidin-2-amine (PY-H) and 4-(4-(dimethylamino)phenyl)-6-(4-fluorophenyl)pyrimidin-2-amine (PY-4F). The crystal structure of PY-4F reveals a 30.06° torsion arrangement between the pyrimidine core and the -C₆H₄N(CH₃)₂ ring. Photophysical studies demonstrate selective fluorescence quenching in the presence of trivalent cations (Al(III), Cr(III), Fe(III)). Detection limits range from 0.060 to 0.570 µM, all below the thresholds established for drinking water by international agencies such as the World Health Organization and Environmental Protection Agency. To elucidate the sensing mechanism, a computational study using DFT and TD-DFT methods was conducted. Cytotoxicity assays in SH-SY5Y cells revealed low toxicity, and successful cell labeling was achieved using epifluorescence and confocal microscopy. These results suggest that PY-H and PY-4F are promising fluorescent probes for detecting Al(III), Cr(III), and Fe(III) in cellular environments and potentially in other complex systems.
Intrinsic visible emission of amyloid-β oligomers: a potential tool for early alzheimer's diagnosis
(Royal society of chemistry, 2025-07-21) Novo, Mercedes; Illodo Brea, Sara; Seijas Cerceda, Jesús; Moraes, Stella Hernández Faria De; Rodríguez-Prieto, Flor; Al-Soufi, Wajih; Universidade de Santiago de Compostela. Departamento de Química Física; Universidade de Santiago de Compostela. Centro de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS)
Alzheimer's disease (AD) is a major public health challenge, with its onset occurring years before symptoms appear. Soluble amyloid-β (Aβ) oligomers are key species in AD pathogenesis and diagnosis, highlighting the need for early detection. This study investigates the intrinsic fluorescence of Aβ(1–40) (Aβ40) as a label-free approach to detecting early-stage oligomers. Aβ40 exhibits autofluorescence dominated by tyrosine emission, which undergoes a strong blue shift and quenching during oligomerization. Additionally, aggregation-induced emission (AIE) in the visible spectral region emerges, correlating with Aβ oligomer concentration and providing a means to detect and quantify oligomers. At the critical aggregation concentrations cac1 = 0.5 μM and cac2 = 19 μM, distinct aggregation behaviours are observed. By employing steady-state fluorescence spectroscopy, a widely accessible technique, these findings establish a direct link between early Aβ aggregation and intrinsic fluorescence changes. This approach eliminates the need for extrinsic probes, simplifying experimental procedures and reducing artefacts. Although further studies are required to develop a robust quantitative correlation for potential diagnostic applications, Aβ autofluorescence represents a promising strategy for investigating early aggregation processes in the context of AD
Firefly luciferin precursor 2-cyano-6-hydroxybenzothiazole: Fluorescence à la carte controlled by solvent and acidity
(Elsevier, 2020) Jadhav, Ankita S.; Carreira Blanco, Carlos; Fernández Rodríguez, Berta; González Fernández, Sonia; Malkhede, Dipalee D.; Mosquera González, Manuel; Ríos Rodríguez, María del Carmen; Rodríguez-Prieto, Flor; Universidade de Santiago de Compostela. Centro de Investigación en Química Biolóxica e Materiais Moleculares; Universidade de Santiago de Compostela. Departamento de Química Física
2-Cyano-6-hydroxybenzothiazole (CBTOH) is a key intermediate in the biosynthesis of D-luciferin, also used for its chemical synthesis and as luciferin bioluminescence precursor for chemical analysis. We characterized CBTOH and its O-methylated derivative by UV–vis absorption, by steady-state and time-resolved fluorescence spectroscopy, and by density functional theory (DFT) quantum mechanical calculations. Both the acidity of the hydroxyl group and the basicity of the thiazole nitrogen strongly increase upon electronic excitation, triggering solvent- and acidity-dependent deprotonation and protonation processes, with concomitant changes in the fluorescence properties. DFT and time-dependent DFT calculations revealed the existence of a charge displacement that takes place in the excited state from the phenol moiety to the cyanothiazole group for the neutral and the cationic forms of CBTOH. This study reveals CBTOH as the first member of a new class of small dyes with strong photoacid and photobase character, its rich excited-state behavior allowing modulation of its fluorescence properties through solvent and acidity changes. These features make CBTOH a potentially useful fluorescent probe
A surfactant concentration model for the systematic determination of the critical micellar concentration and the transition width
(MDPI, 2021) Al-Soufi, Wajih; Novo, Mercedes; Universidade de Santiago de Compostela. Departamento de Química Física
The critical micellar concentration (cmc) is a fundamental property of surfactant solutions. Many proposed methods for the definition and determination of the cmc from property-concentration plots yield values, which depend on the studied property, on the specific technique used for its analysis and in many cases on the subjective choice of the chosen type of plot and concentration interval. In this focus review, we revise the application of a surfactant concentration model we proposed earlier that defines the cmc directly based on the surfactant concentration. Known equations for the concentration-dependence of different surfactant properties can then be combined with this concentration model and fitted to experimental data. This modular concept makes it possible to determine the cmc and the transition width in a systematic and unambiguous way. We revise its use in the literature in different contexts: the determination of the cmc of surfactants and their mixtures from different properties (electrical conductivity, NMR chemical shift, self-diffusion, surface tension, UV-Vis absorption, fluorescence intensity and fluorescence correlation). We also revise the dependence of the width of the transition region on composition, detailed studies of the properties of fluorescent probes and the aggregation of non-surfactant systems, namely amyloid peptides
Spectroscopic characterization of mitochondrial G-quadruplexes
(MDPI, 2022) Illodo Brea, Sara; Pérez González, Cibrán; Barcia, Ramiro; Novo, Mercedes; Rodríguez-Prieto, Flor; Al-Soufi, Wajih; Universidade de Santiago de Compostela. Centro de Investigación en Química Biolóxica e Materiais Moleculares; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Departamento de Química Física
Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies
Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents
(Nature Publishing Group, 2019) Dalgarno, Paul A.; Juan-Colás, José; Hedley, Gordon J.; Piñeiro Maseda, Lucas; Pérez González, Cibrán; Samuel, Ifor D. W.; Leake, Mark C.; Johnson, Steven; Al-Soufi, Wajih; Penedo, J. Carlos; Quinn, Steven D.; Novo, Mercedes; Universidade de Santiago de Compostela. Departamento de Química Física
The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions
Competition between tunnel- and viscosity-effects on bimolecular hydrogen-transfer reaction
(International Union of Pure and Applied Chemistry, 1993) Kensy, Uwe; Grellmann, Karl-Heinz; Mosquera González, Manuel; Universidade de Santiago de Compostela. Departamento de Química Física
The photocyclization of a methylsubstituted diphenylamine results in the formation of a transient zwitterionic dihydrocarbazole. The transient converts into a stable dihydrocarbazole by an intramolecular, sigmatropic hydrogen shift and by an intermolecular hydrogen exchange reaction. The rates of both reactions are governed by hydrogen tunnel effects. The intermolecular hydrogen exchange is the first example for a bimolecular reaction where tunnel effects have been observed. The role of solvent viscosity is discussed
Fluorescence of methylated derivatives of hydroxyphenylimidazopyridine. Resolution of strongly overlapping spectra and a new ESIPT dye showing very efficient radiationless deactivation
(Royal Society of Chemistry, 2011) Brenlla, Alfonso; Veiga, Manoel; Ríos Rodríguez, María del Carmen; Mosquera González, Manuel; Rodríguez-Prieto, Flor; Universidade de Santiago de Compostela. Departamento de Química Física
The ground- and excited-state behaviour of the isomeric species 2-(2′-methoxyphenyl)imidazo[4,5-b]pyridine (1-OMe) and 2-(2′-hydroxyphenyl)-4-methylimidazo[4,5-b]pyridine (1-NMe) in neutral and acid media has been studied by UV-vis absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy. The new dye 1-NMe is non-fluorescent in neutral media except in trifluoroethanol, where it shows a very weak fluorescence. 1-NMe also exhibits highly solvent-dependent fluorescence intensity in acidic media. We propose that the neutral species experiences a fast excited-state intramolecular proton transfer (ESIPT), relaxing afterwards by intramolecular twisting associated with internal charge transfer (TICT) and subsequent very fast internal conversion of the proton-transferred TICT structure. The behaviour of 1-NMe in acidic media is explained by the existence of a ground-state tautomeric equilibrium between species with intramolecular hydrogen bonds N–H⋯OH and N⋯HO. The first type of tautomers dissociates at the hydroxyl group in water and ethanol, but fluoresces in acetonitrile and trifluoroethanol due to the inability of these solvents to accept the proton. The second type of tautomers is non-emissive due to fast radiationless deactivation through an ESIPT-TICT process. The fluorescence of 1-OMe was investigated in neutral and acidic media, demonstrating the photobasic character of the pyridine nitrogen. A ground-state equilibrium between pyridinium and imidazolium cations was found for this species, showing overlapping absorption and fluorescence spectra. We devised a method to resolve the spectra by applying principal component global analysis to a series of excitation spectra taken at different emission wavelengths, which allowed estimation of the equilibrium constant between the cations
A model for monomer and micellar concentrations in surfactant solutions: application to conductivity, NMR, diffusion, and surface tension data
(Elsevier, 2012) Al-Soufi, Wajih; Piñeiro Maseda, Lucas; Novo, Mercedes; Universidade de Santiago de Compostela. Departamento de Química Física
An empirical model for the concentrations of monomeric and micellized surfactants in solution is presented as a consistent approach for the quantitative analysis of data obtained with different experimental techniques from surfactant solutions. The concentration model provides an objective definition of the critical micelle concentration (cmc) and yields precise and well defined values of derived physical parameters. The use of a general concentration model eliminates subjective graphical procedures, reduces methodological differences, and thus allows one to compare directly the results of different techniques or to perform global fits. The application and validity of the model are demonstrated with electrical conductivity, surface tension, NMR chemical shift, and self-diffusion coefficient data for the surfactants SDS, CTAB, DTAB, and LAS. In all cases, the derived models yield excellent fits of the data. It is also shown that there is no need to assume the existence of different premicellar species in order to explain the chemical shifts and self-diffusion coefficients of SDS as claimed recently by some authors
Photophysical study of Thioflavin T as fluorescence marker of amyloid fibrils
(Elsevier, 2014) Freire Rodríguez, Sonia; Araujo, Marcus H. de; Al-Soufi, Wajih; Novo, Mercedes; Universidade de Santiago de Compostela. Departamento de Química Física
Thioflavin T is a highly sensitive fluorescent marker of amyloid fibrils that has been widely used for in vitro biomedical assays. However, neither its complex photophysical behavior nor its binding mode to amyloid fibrils are still well understood. We present a detailed analysis of the photophysical properties of Thioflavin T in various media, including solvents and solvent mixtures of different viscosities as well as fibrillar and globular proteins. We propose a model that explains the strong wavelength dependency of the Thioflavin T fluorescence and the large fluorescence enhancement in certain environments. We determine the binding affinities and the fluorescence properties of Thioflavin T bound to amyloid-β (1–42) fibrils and to bovine serum albumin and discuss the sensitivity and the specificity of this probe to amyloid aggregates. These results allow us to assess the suitability of Thioflavin T for quantitative determinations in biomedical studies
Femtosecond pump/supercontinuum-probe spectroscopy: Optimized setup and signal analysis for single-shot spectral referencing
(AIP Publishing, 2010) Dobryakov, A.L.; Kovalenko, Sergey A.; Weigel, A.; Pérez Lustres, José Luis; Lange, J.; Müller, A.; Ernsting, N.P.; Universidade de Santiago de Compostela. Departamento de Química Física
A setup for pump/supercontinuum-probe spectroscopy is described which (i) is optimized to cancel fluctuations of the probe light by single-shot referencing, and (ii) extends the probe range into the near-uv (1000–270 nm). Reflective optics allow 50 μm spot size in the sample and upon entry into two separate spectrographs. The correlation γsame between sample and reference readings of probe light level at every pixel exceeds 0.99, compared to γconsec<0.92 reported for consecutive referencing. Statistical analysis provides the confidence interval of the induced optical density, ΔOD. For demonstration we first examine a dye (Hoechst 33258) bound in the minor groove of double-stranded DNA. A weak 1.1 ps spectral oscillation in the fluorescence region, assigned to DNA breathing, is shown to be significant. A second example concerns the weak vibrational structure around t=0 which reflects stimulated Raman processes. With 1% fluctuations of probe power, baseline noise for a transient absorption spectrum becomes 25 μOD rms in 1 s at 1 kHz, allowing to record resonance Raman spectra of flavine adenine dinucleotide in the S0 and S1 state
In Vivo Theranostics at the Peri-Infarct Region in Cerebral Ischemia
(Ivyspring International Publisher, 2014) Agulla Freire, Jesús; Brea López, David; Campos Pérez, Francisco; Sobrino Moreiras, Tomás; Argibay González, Bárbara; Al-Soufi, Wajih; Blanco González, Miguel; Castillo Sánchez, José Antonio; Ramos Cabrer, Pedro; Universidade de Santiago de Compostela. Departamento de Química Física
The use of theranostics in neurosciences has been rare to date because of the limitations imposed on the free delivery of substances to the brain by the blood-brain barrier. Here we report the development of a theranostic system for the treatment of stroke, a leading cause of death and disability in developed countries. We first performed a series of proteomic, immunoblotting and immunohistological studies to characterize the expression of molecular biomarkers for the so-called peri-infarct tissue, a key region of the brain for stroke treatment. We confirmed that the HSP72 protein is a suitable biomarker for the peri-infarct region, as it is selectively expressed by at-risk tissue for up to 7 days following cerebral ischemia. We also describe the development of anti-HSP72 vectorized stealth immunoliposomes containing imaging probes to make them traceable by conventional imaging techniques (fluorescence and MRI) that were used to encapsulate a therapeutic agent (citicoline) for the treatment of cerebral ischemia. We tested the molecular recognition capabilities of these nano-platforms in vitro together with their diagnostic and therapeutic properties in vivo, in an animal model of cerebral ischemia. Using MRI, we found that 80% of vectorized liposomes were located on the periphery of the ischemic lesion, and animals treated with citicoline encapsulated on these liposomes presented lesion volumes up to 30% smaller than animals treated with free (non-encapsulated) drugs. Our results show the potential of nanotechnology for the development of effective tools for the treatment of neurological diseases
Ultrafast deactivation of bilirubin: dark intermediates and two-photon isomerization
(Royal Society of Chemistry, 2016) Carreira Blanco, Carlos; Singer, Patrick; Diller, Rolf; Pérez Lustres, José Luis; Universidade de Santiago de Compostela. Centro de Investigación en Química Biolóxica e Materiais Moleculares; Universidade de Santiago de Compostela. Departamento de Química Física
Bilirubin is a neurotoxic product responsible for neonatal jaundice, which is generally treated by phototherapy. The photoreaction involves ultrafast internal conversion via an elusive intermediate and Z–E isomerization with minor yield (less than 3% in solution). The structure of the intermediate remains unclear. Here, the combination of UV-vis and mid-IR ultrafast transient absorption spectroscopy reports a comprehensive picture of the mechanism and provides essential structural information about the intermediate species. Thus, spectral dynamics during the earliest ps unveils a wavepacket travelling from the Franck–Condon region to the crossing point with a dark state. The latter shows a tighter molecular skeleton than the ground state and decays with 15 ps time constant. Remarkably, the relative contribution of a non-decaying component increases linearly with pump energy, suggesting that Z–E isomerization could also be triggered by two-photon excitation. Implications for the photochemistry of protein-bound open tetrapyrroles are discussed
Host-Guest Complexation Studied by Fluorescence Correlation Spectroscopy: Adamantane–Cyclodextrin Inclusion
(MDPI, 2010) Granadero, Daniel; Bordello Malde, Jorge; Pérez Alvite, María Jesús; Novo, Mercedes; Al-Soufi, Wajih; Universidade de Santiago de Compostela. Departamento de Química Física; Universidade de Santiago de Compostela. Departamento de Química Orgánica
The host-guest complexation between an Alexa 488 labelled adamantane derivative and β-cyclodextrin is studied by Fluorescence Correlation Spectroscopy (FCS). A 1:1 complex stoichiometry and a high association equilibrium constant of K = 5.2 × 104 M-1 are obtained in aqueous solution at 25 °C and pH = 6. The necessary experimental conditions are discussed. FCS proves to be an excellent method for the determination of stoichiometry and association equilibrium constant of this type of complexes, where both host and guest are nonfluorescent and which are therefore not easily amenable to standard fluorescence spectroscopic methods
Fluorescence Emission of Pyrene in Surfactant Solutions
(Elsevier, 2015) Piñeiro Maseda, Lucas; Novo, Mercedes; Al-Soufi, Wajih; Universidade de Santiago de Compostela. Departamento de Química Física
The systematic description of the complex photophysical behavior of pyrene in surfactant solutions in combination with a quantitative model for the surfactant concentrations reproduces with high accuracy the steady-state and the time resolved fluorescence intensity of pyrene in surfactant solutions near the cmc, both in the monomer and in the excimer emission bands. We present concise model equations that can be used for the analysis of the pyrene fluorescence intensity in order to estimate fundamental parameters of the pyrene-surfactant system, such as the binding equilibrium constant K of pyrene to a given surfactant micelle, the rate constant of excimer formation in micelles, and the equilibrium constant of pyrene-surfactant quenching. The values of the binding equilibrium constant KTX100 = 3300 103 M-1 and KSDS = 190 103 M-1 for Triton X-100 (TX100) and SDS micelles, respectively, show that the partition of pyrene between bulk water and micelles cannot be ignored, even at relatively high surfactant concentrations above the cmc. We apply the model to the determination of the cmc from the pyrene fluorescence intensity, especially from the intensity ratio at two vibronic bands in the monomer emission or from the ratio of excimer to monomer emission intensity. We relate the finite width of the transition region below and above the cmc with the observed changes in the pyrene fluorescence in this region
Critical aggregation concentration for the formation of early Amyloid-β (1–42) oligomers
(Nature Publishing Group, 2018-01-29) Novo, Mercedes; Freire Rodríguez, Sonia; Al-Soufi, Wajih; Universidade de Santiago de Compostela. Departamento de Química Física
The oligomers formed during the early steps of amyloid aggregation are thought to be responsible for the neurotoxic damage associated with Alzheimer’s disease. It is therefore of great interest to characterize this early aggregation process and the aggregates formed, especially for the most significant peptide in amyloid fibrils, Amyloid-β(1–42) (Aβ42). For this purpose, we directly monitored the changes in size and concentration of initially monomeric Aβ42 samples, using Fluorescence Correlation Spectroscopy. We found that Aβ42 undergoes aggregation only when the amount of amyloid monomers exceeds the critical aggregation concentration (cac) of about 90 nM. This spontaneous, cooperative process resembles surfactants self-assembly and yields stable micelle-like oligomers whose size (≈50 monomers, R h ≈ 7–11 nm) and elongated shape are independent of incubation time and peptide concentration. These findings reveal essential features of in vitro amyloid aggregation, which may illuminate the complex in vivo process
Fluorescence quenching of the N-methylquinolinium cation by pairs of water or alcohol molecules
(2018) Rodríguez-Prieto, Flor; Costa Corbelle, Carlos; Fernández Rodríguez, Berta; Ríos Rodríguez, María del Carmen; Mosquera González, Manuel; Universidade de Santiago de Compostela. Centro de Investigación en Química Biolóxica e Materiais Moleculares; Universidade de Santiago de Compostela. Departamento de Química Física
N-Methylquinolinium cation (MQ+) in its first-excited singlet state is a strong oxidant commonly used as a photosensitizer, whose fluorescence is therefore quenched by electron donors. Interestingly, the fluorescence of MQ+ is also quenched by hydroxy compounds such as water and alcohols, more difficult to oxidize. We investigated the quenching mechanism of MQ+ fluorescence by small amounts of water and alcohols in acetonitrile solution. The fluorescence intensities and lifetimes exhibited a nonlinear dependence on the quencher concentration. We found evidence that emissive exciplexes MQ+*-ROH are formed between the excited quinolinium and the hydroxy compounds. An accurate quantitative description of the results was obtained with a model in which the exciplex reacts with a second molecule of the hydroxy compound, which quenches the fluorescence. The rate constant of this process increased as the quencher ionization energy decreased. We showed also that a low basicity of the hydroxy compound inhibits the quenching process. These results are consistent with the existence of a concerted photoinduced proton-coupled electron transfer (PCET) involving an intermediate complex of the excited quinolinium with a H-bonded molecular pair of the hydroxy compounds. In these pairs, a water or an alcohol molecule is able to donate an electron to the photoexcited quinolinium cation and a proton to the second H-bonded hydroxy molecule, showing an enhanced reducing power in comparison with the isolated molecule. The structure of the intermediate complex was investigated using high-level quantum mechanical calculations. At high water concentrations in acetonitrile/water mixtures, the quenching process is slowed down, indicating that higher water aggregates are less effective for a PCET process. The results obtained may be relevant to the study of water oxidation and electron transfer in biological systems

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