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Special Issue "Fluorescence Spectroscopy of Biomolecules"

A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Analytical Chemistry".

Deadline for manuscript submissions: closed (31 January 2021).

Special Issue Editor

Dr. Thomas Gustavsson
E-Mail Website
Guest Editor
LIDYL, Laboratoire Interactions, Dynamiques et Lasers
CEA, CNRS, Université Paris-Saclay
CEA Saclay, 91191 Gif-sur-Yvette France

Interests: fluorescence spectroscopy, femtochemistry and femtobiology; electronic and structural dynamics; photovoltaics

Special Issue Information

Dear Colleagues,

It is my great pleasure to invite you to submit an article for a high-profile Special Issue on the “Fluorescence Spectroscopy of Biomolecules” to be published in Molecules.

This Special Issue aims to highlight research on biomolecules using fluorescence spectroscopy in a very broad sense.

It concerns steady-state as well as time-resolved fluorescence studies, addressing the structural and dynamic properties of the excited biomolecules. Experimental and theoretical studies in solution and in the gas phase aiming at the characterization of the emitting excited state are welcome.

More specifically, it concerns the fluorescence of natural biomolecules, such as nucleic acids, enzymes and proteins, as well as flavins and haemoglobin, in a non-exhaustive list. It also deals with modified fluorescent nucleobases and amino acid analogues, aimed for use as biomarkers. In addition, it also treats studies of fluorescent ligands and drugs interacting with natural biomolecules. In this sense, it addresses fundamental processes such as energy and charge transfer phenomena.

Review articles and perspectives from experts in the field are also welcome.

Dr. Thomas Gustavsson
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Molecules is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2000 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • steady-state fluorescence
  • time-resolved fluorescence
  • excited states
  • femtochemistry and femtobiology
  • electronic and structural dynamics
  • relaxation and reaction mechanisms
  • energy transfer
  • electron and charge transfer
  • proton and hydrogen transfer
  • potential energy surfaces
  • conical intersections

Published Papers (8 papers)

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Research

Article
Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide
Molecules 2021, 26(8), 2206; https://doi.org/10.3390/molecules26082206 - 12 Apr 2021
Cited by 1 | Viewed by 458
Abstract
Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent [...] Read more.
Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity
Molecules 2021, 26(2), 420; https://doi.org/10.3390/molecules26020420 - 14 Jan 2021
Viewed by 668
Abstract
8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The [...] Read more.
8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
Nile-Red-Based Fluorescence Probe for Selective Detection of Biothiols, Computational Study, and Application in Cell Imaging
Molecules 2020, 25(20), 4718; https://doi.org/10.3390/molecules25204718 - 14 Oct 2020
Viewed by 694
Abstract
A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm [...] Read more.
A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm to 567 nm, which induces an obvious color change from blue to pink and exhibits a 35-fold fluorescence enhancement at 645 nm in red emission range. NRSH displays rapid (<1 min) response for H2S, which is faster than other biothiols (>5 min). The detection limits of probe NRSH towards biothiols are very low (22.05 nM for H2S, 34.04 nM for Cys, 107.28 nM for GSH and 113.65 nM for Hcy). Furthermore, NRSH is low cytotoxic and can be successfully applied as a bioimaging tool for real-time monitoring biothiols in HeLa cells. In addition, fluorescence mechanism of probe NRSH is further understood by theoretical calculations. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
Excited-State Proton Transfer in 8-Azapurines I: A Kinetic Analysis of 8-Azaxanthine Fluorescence
Molecules 2020, 25(12), 2740; https://doi.org/10.3390/molecules25122740 - 12 Jun 2020
Cited by 2 | Viewed by 705
Abstract
A super-continuum white laser with a half-pulse width of ~75 ps was used to observe the kinetics of a postulated excited-state proton transfer in 8-azaxanthine and its 8-methyl derivative. Both compounds exhibited dual emissions in weakly acidified alcoholic media, but only one band [...] Read more.
A super-continuum white laser with a half-pulse width of ~75 ps was used to observe the kinetics of a postulated excited-state proton transfer in 8-azaxanthine and its 8-methyl derivative. Both compounds exhibited dual emissions in weakly acidified alcoholic media, but only one band was present in aqueous solutions, exhibiting an abnormal Stokes shift (>12,000 cm−1). It was shown that long-wavelength emissions were delayed relative to the excitation pulse within alcoholic media. The rise time was calculated to be 0.4–0.5 ns in both methanol and deuterated methanol. This is equal to the main component of the fluorescence decay in the short-wavelength band (340 nm). Time-resolved emission spectra (TRES) indicated a two-state photo-transformation model in both compounds. Global analysis of the time dependence revealed three exponential components in each compound, one of which had an identical rise-time, with the second attributed to a long-wavelength band decay (6.4 ns for aza-xanthine and 8.3 ns for its 8-methyl derivative). The origin of the third, intermediate decay time (1.41 ns for aza-xanthine and 0.87 ns for 8-methyl-azaxanthine) is uncertain, but decay-associated spectra (DAS) containing both bands suggest the participation of a contact ion pair. These results confirm the model of phototautomerism proposed earlier, but the question of the anomalous isotope effect remains unsolved. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
Excited State Dynamics of 8-Vinyldeoxyguanosine in Aqueous Solution Studied by Time-Resolved Fluorescence Spectroscopy and Quantum Mechanical Calculations
Molecules 2020, 25(4), 824; https://doi.org/10.3390/molecules25040824 - 13 Feb 2020
Cited by 1 | Viewed by 918
Abstract
The fluorescent base guanine analog, 8-vinyl-deoxyguanosine (8vdG), is studied in solution using a combination of optical spectroscopies, notably femtosecond fluorescence upconversion and quantum chemical calculations, based on time-dependent density functional theory (TD-DFT) and including solvent effect by using a mixed discrete-continuum model. In [...] Read more.
The fluorescent base guanine analog, 8-vinyl-deoxyguanosine (8vdG), is studied in solution using a combination of optical spectroscopies, notably femtosecond fluorescence upconversion and quantum chemical calculations, based on time-dependent density functional theory (TD-DFT) and including solvent effect by using a mixed discrete-continuum model. In all investigated solvents, the fluorescence is very long lived (3–4 ns), emanating from a stable excited state minimum with pronounced intramolecular charge-transfer character. The main non-radiative decay channel features a sizeable energy barrier and it is affected by the polarity and the H-bonding properties of the solvent. Calculations provide a picture of dynamical solvation effects fully consistent with the experimental results and show that the photophysical properties of 8vdG are modulated by the orientation of the vinyl group with respect to the purine ring, which in turn depends on the solvent. These findings may have importance for the understanding of the fluorescence properties of 8vdG when incorporated in a DNA helix. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
Excited State Lifetimes of Sulfur-Substituted DNA and RNA Monomers Probed Using the Femtosecond Fluorescence Up-Conversion Technique
Molecules 2020, 25(3), 584; https://doi.org/10.3390/molecules25030584 - 29 Jan 2020
Cited by 1 | Viewed by 1149
Abstract
Sulfur-substituted DNA and RNA nucleobase derivatives (a.k.a., thiobases) are an important family of biomolecules. They are used as prodrugs and as chemotherapeutic agents in medical settings, and as photocrosslinker molecules in structural-biology applications. Remarkably, excitation of thiobases with ultraviolet to near-visible light results [...] Read more.
Sulfur-substituted DNA and RNA nucleobase derivatives (a.k.a., thiobases) are an important family of biomolecules. They are used as prodrugs and as chemotherapeutic agents in medical settings, and as photocrosslinker molecules in structural-biology applications. Remarkably, excitation of thiobases with ultraviolet to near-visible light results in the population of long-lived and reactive triplet states on a time scale of hundreds of femtoseconds and with near-unity yields. This efficient nonradiative decay pathway explains the vanishingly small fluorescence yields reported for the thiobases and the scarcity of fluorescence lifetimes in the literature. In this study, we report fluorescence lifetimes for twelve thiobase derivatives, both in aqueous solution at physiological pH and in acetonitrile. Excitation is performed at 267 and 362 nm, while fluorescence emission is detected at 380, 425, 450, 525, or 532 nm. All the investigated thiobases reveal fluorescence lifetimes that decay in a few hundreds of femtoseconds and with magnitudes that depend and are sensitive to the position and degree of sulfur-atom substitution and on the solvent environment. Interestingly, however, three thiopyrimidine derivatives (i.e., 2-thiocytidine, 2-thiouridine, and 4-thiothymidine) also exhibit a small amplitude fluorescence component of a few picoseconds in aqueous solution. Furthermore, the N-glycosylation of thiobases to form DNA or RNA nucleoside analogues is demonstrated as affecting their fluorescence lifetimes. In aqueous solution, the fluorescence decay signals exciting at 267 nm are equal or slower than those collected exciting at 362 nm. In acetonitrile, however, the fluorescence decay signals recorded upon 267 nm excitation are, in all cases, faster than those measured exciting at 362 nm. A comparison to the literature values show that, while both the DNA and RNA nucleobase and thiobase derivatives exhibit sub-picosecond fluorescence lifetimes, the 1ππ* excited-state population in the nucleobase monomers primarily decay back to the ground state, whereas it predominantly populates long-lived and reactive triplet states in thiobase monomers. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
A Ratiometric and Colorimetric Hemicyanine Fluorescent Probe for Detection of SO2 Derivatives and Its Applications in Bioimaging
Molecules 2019, 24(21), 4011; https://doi.org/10.3390/molecules24214011 - 05 Nov 2019
Cited by 3 | Viewed by 1148
Abstract
Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO32−/HSO3. The probe displayed both colorimetric and ratiometric responses toward SO32−/HSO3. It displayed [...] Read more.
Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO32−/HSO3. The probe displayed both colorimetric and ratiometric responses toward SO32−/HSO3. It displayed a quick response (within 60 s), good selectivity and high sensitivity (a detection limit of 28 nM) towards SO32−/HSO3. The SO32−/HSO3 sensing mechanism was confirmed as the Michael addition reaction by ESI-MS. Moreover, the probe could be applied to measure the level of sulfite in real samples, like sugar and chrysanthemum, and it could also be used to detect SO32−/HSO3 in HepG2 cells through confocal fluorescence microscopy, which proved its practical application in clinical diagnosis. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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Article
A Green-emitting Fluorescent Probe Based on a Benzothiazole Derivative for Imaging Biothiols in Living Cells
Molecules 2019, 24(3), 411; https://doi.org/10.3390/molecules24030411 - 23 Jan 2019
Cited by 9 | Viewed by 1314
Abstract
A new green-emitting fluorescent probe 1 was developed for biothiol detection. The sensing mechanism was considered to be biothiol-induced cleavage of the 2,4-dinitrobenzene- sulfonate group in probe 1 and resulting inhibition of the probe’s photoinduced electron transfer (PET) process. Probe 1 exhibited favorable [...] Read more.
A new green-emitting fluorescent probe 1 was developed for biothiol detection. The sensing mechanism was considered to be biothiol-induced cleavage of the 2,4-dinitrobenzene- sulfonate group in probe 1 and resulting inhibition of the probe’s photoinduced electron transfer (PET) process. Probe 1 exhibited favorable properties such as excellent selectivity, highly sensitive (0.12 µM), large Stokes shift (117 nm) and a remarkable turn-on fluorescence signal (148-fold). Furthermore, confocal fluorescence imaging indicated that probe 1 was membrane-permeable and suitable for visualization of biothiols in living A549 cells. Full article
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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