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Accueil du site > Equipe Nanochimie et bioimagerie > Fluorescent molecular probes

Solvatochromic and fluorogenic dyes

Mise à jour 17 janvier 2018

Fig. 1. Simplified Jablonski diagram and the key mechanisms involved in the environment-sensitive probes together with examples of dyes developed/used in our group.

Fluorescent environment-sensitive probes are specially designed dyes that change their fluorescence intensity (fluorogenic) or color (e.g. solvatochromic) in response to change in their microenvironment polarity, viscosity and molecular order 1. The studies of the last decade, including those of our group, have shown that these molecules become universal tools in fluorescence sensing and imaging. In fact, any biomolecular interaction or change in biomolecular organization results in modification of the local microenvironment, which can be directly monitored by these types of probes.

Solvatochromic dyes constitute a large class of environment-sensitive probes which change their color in response to polarity. Generally, they are push-pull dyes undergoing intramolecular charge transfer. Emission of their highly polarized excited state shifts to the red in more polar solvents. The typical examples of dyes, we worked on in our group, are push-pull fluorene (FR0) 2, pyrene (PA) 3, and 3-methoxychromone (3MC-2) 4 and Nile Red 5. FR0, originally developed by us in 2010 is one of the most polarity-sensitive fluorescent dyes developed to date 2. Excited-state intramolecular proton transfer is the second key concept to design efficient solvatochromic dyes, which responds to the microenvironment by changing relative intensity of the two emissive tautomeric forms. This was a subject of our earlier studies, where we were particularly interested in 3-hydrohyflavones, 3-hydroxychromones and 3-hydroxyquinolones, dyes presenting solvent-dependent dual emission 6,7. Owing to sensitivity to polarity and hydration, solvatochromic dyes have been successfully applied by us to biological membranes for studying lipid domains (rafts), apoptosis and endocytosis. We also applied solvatochromic dyes as fluorescent labels, showing that they can detect practically any type of biomolecular interactions, involving proteins, nucleic acids and biomembranes, because the binding event excludes local water molecules from the interaction site.

On the other hand, fluorogenic probes usually exploit intramolecular rotation (conformation change) as a design concept, with molecular rotors being main representatives. In this series, we recently reported a push-pull deoxaborine dye DXB Red, which combined properties of a molecular rotor and a solvatochromic dye 8. The alternative mechanism, we exploited to achieve a fluorogenic response was aggregation-caused quenching, which uses disruption of the self-quenched dimers and nano-assemblies of dyes in less polar environments of lipid membranes and biomolecules 9. Both type of probes were successfully applied by us (in collaboration with a group of D. Bonnet, Strasbourg) for detection of G protein coupled receptors (oxytocin).

Overall, solvatochromic and fluorogenic probes enable background-free bioimaging in wash-free conditions as well as quantitative analysis when combined with advanced microscopy, such as fluorescence lifetime (FLIM) and ratiometric imaging. Our current developments in fluorescent environment-sensitive probes are directed to address the following challenges : (i) improving optical properties, especially brightness, photostability and far-red to near-infrared operating range ; (ii) minimizing non-specific interactions of the probes in biological media ; (iii) their adaptation for advanced microscopies, notably for superresolution and in vivo imaging.

References

1. Klymchenko AS. Solvatochromic and Fluorogenic Dyes as Environment-Sensitive Probes : Design and Biological Applications. Acc Chem Res. 2017, 50, 366-375.

2. Kucherak, O.A. ; Didier, P. ; Mély, Y. ; Klymchenko, A.S. Fluorene analogues of Prodan with superior fluorescence brightness and solvatochromism. J. Phys. Chem. Lett. 2010, 1, 616-620.

3. Kucherak, O. A. ; Richert, L. ; Mély, Y. ; Klymchenko, A. S. Dipolar 3-methoxychromones as bright and highly solvatochromic fluorescent dyes. Phys. Chem. Chem. Phys. 2012, 14, 2292 - 2300.

4. Yosuke Niko, Pascal Didier, Yves Mely, Gen-ichi Konishi, Andrey S. Klymchenko, Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes, Scientific Reports (Nature publishing group), 2016, 6, 18870.

5. Kucherak, O. ; Oncul, S. ; Darwich, Z. ; Yushchenko, D. A. ; Arntz, Y. ; Didier, P. ; Mély, Y. ; Klymchenko, A. S. Switchable Nile Red-based probe for cholesterol and lipid order at the outer leaflet of biomembranes. J. Am. Chem. Soc. 2010, 132, 4907–4916.

6. Demchenko, A. P. ; Mely, Y. ; Duportail, G. ; Klymchenko, A. S. Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes. Biophys. J. 2009, 96, 3461-3470.

7. Klymchenko, A. S. ; Demchenko, A. P. : Multiparametric probing of intermolecular interactions with fluorescent dye exhibiting excited state intramolecular proton transfer. Physical Chemistry Chemical Physics 2003, 5, 461-468.

8. Karpenko, I. A. ; Niko, Y. ; Yakubovskyi, V. P. ; Gerasov, A. O. ; Bonnet, D. ; Kovtun, Y. P. ; Klymchenko, A. S. : Push-pull dioxaborine as fluorescent molecular rotor : far-red fluorogenic probe for ligand-receptor interactions. Journal of Materials Chemistry C 2016, 4, 3002-3009.

9. Karpenko, I. A. ; Niko, Y. ; Yakubovskyi, V. P. ; Gerasov, A. O. ; Bonnet, D. ; Kovtun, Y. P. ; Klymchenko, A. S. : Push-pull dioxaborine as fluorescent molecular rotor : far-red fluorogenic probe for ligand-receptor interactions. Journal of Materials Chemistry C 2016, 4, 3002-3009.

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