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Fluorescent molecular probes and nanoparticles

Last update 13 October 2015

Fluorescent molecular probes and nanoparticles

We develop fluorescent probes that provide information about their nano-environment by change of their fluorescence color. For building these probes, we use mainly 3-hydroxychromone fluorophores. They undergo Excited State Intramolecular Proton Transfer (ESIPT), which generates two excited species, normal (N*) and tautomer (T*), giving rise to the two-color fluorescence. The ratio of these two bands N*/T* provides information on the polarity, hydration and electrostatics (1, 2). Fluorescence and sensor properties of these dyes can be finely tuned by proper molecular design (3).

Membranes Probes

We develop molecular probes sensitive to different properties of lipid membranes. The selectivity of the probes to a particular membrane property can be achieved by proper selection of the fluorophore and its appropriate localization in the lipid membrane (3). Recent examples are probes of hydration and surface charge (F2N12S) (4), dipole (F8N1S and PPZ8) (5, 6) and transmembrane potential (di-SFA) (7).


Apoptosis detection

Normal cell membranes exhibit strong transbilayer asymmetry, while in apoptotic cells, it is lost. The probe F2N12S, which incorporates selectively into the outer leaflet of the cell plasma membrane, can detect this loss of asymmetry by changing its N*/T* intensity ratio (4). Being ratiometric, the response of the new probe can be easily quantified by microscopy techniques, a feature that can be hardly achieved with fluorescently labeled annexin V probes. Recently, we showed that NR12S can also be applied for apoptosis detection (10).

Membrane domain imaging

Membrane domains or “rafts” are believed to be involved in various cell functions. In model membranes, these domains, defined as liquid-ordered phase, can be observed as “rafts” floating in the fluid phase. NR12S and F2N12S probes due to their high sensitivity to the membrane hydration allows visualization of lipid domains in model membranes and monitoring lipid order in cellular membranes using ratiometric imaging (8,9) or FLIM.

Probes of biomolecular interactions

Using environment-sensitive probes we develop general strategies for monitoring interactions of peptides or proteins with nucleic acids, proteins and lipid membranes (11). Interaction of a peptide with its target commonly changes its local nano-environment, which can be detected by the environment-sensitive probe though change of its emission color.

Peptide-nucleic acid interaction

We develop environment-sensitive labels based on 3HC for sensing peptide-oligonucleotide (ODN) interactions. When attached to the N-terminus of the HIV-1 NCp7 protein, the N*/T* ratio of this probe depends on the interacting ODN sequence and correlates with the 3D structure of the corresponding complexes (12). We extended this approach, by developing a 3HC-based amino acid analogue that ideally substitutes Trp residues and allows site-selectively investigating complexes with ODN (13). Other environment-sensitive fluorescent amino acids are under development.

Peptide-protein interaction

Labeling a protein with a 3HC dye allows monitoring its interaction with a target protein, through changes of the N*/T* ratio (14), and thus, constitutes a universal tool for sensing peptide-protein interactions (11). The improvement of the sensor fluorophore is in progress.

Protein-membrane interaction

We developed a 3HC-based probe to monitor peptide/membrane interactions (15). Currently, using fluorescent amino acid analogues based on this 3HC probe, we study the insertion and the orientation of various peptides (melittin, NCp7, penetratin…) in model and cellular membranes.


1) Klymchenko, A., Mely, Y., Demchenko, A., and Duportail, G. (2004) Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes. Biochim. Biophys. Acta, 1665, 6-19.

2) 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.

3) Klymchenko, A. S., Duportail, G., Ozturk, T., Pivovarenko, V. G., Mely, Y., and Demchenko, A. P. (2002) Novel two-band ratiometric fluorescence probes with different location and orientation in phospholipid membranes. Chem. Biol. 9, 1199.

4) Shynkar, V. V., Klymchenko, A. S., Kunzelmann, C., Duportail, G., Muller, C. D., Demchenko, A. P., Freyssinet, J. M., and Mely, Y. (2007) Fluorescent biomembrane probe for ratiometric detection of apoptosis. J. Am. Chem. Soc. 129, 2187.

5) Shynkar, V. V., Klymchenko, A. S., Duportail, G., Demchenko, A. P., and Mely, Y. (2005) Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes. Biochim. Biophys. Acta - Biomembranes 1712, 128.

6) Klymchenko, A. S., Duportail, G., Mely, Y., and Demchenko, A. P. (2003) Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes. Proc. Natl. Acad. Sci. USA 100, 11219.

7) Klymchenko, A. S., Stoeckel, H., Takeda, K., and Mely, Y. (2006) Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential. J. Phys. Chem. B 110, 13624.

8) Klymchenko, A. S., Oncul, S., Didier, P., Schaub, E., Bagatolli, L., Duportail, G., and Mely, Y. (2009) Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone. Biochim. Biophys. Acta, 1788, 495-499.

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

10) Darwich, Z.; Klymchenko, A. S.; Kucherak, O. A.; Richert, L.; Mély, Y. (2012) Detection of apoptosis through the lipid order of the outer plasma membrane leaflet. Biochm. Biphys. Acta, 1818, 3048–3054.

11) Klymchenko AS, Mely Y. (2013) Fluorescent environment-sensitive dyes as reporters of biomolecular interactions. Prog Mol Biol Transl Sci. 113, 35-58.

12) Shvadchak, V.V., Klymchenko, A.S., de Rocquigny, H., and Mély, Y. (2009) Sensing peptide-oligonucleotide interactions by a two-color environment-sensitive label: application to the HIV-1 nucleocapsid protein. Nucleic Acids Res, 37, e25

13) Strizhak, A.V.; Postupalenko, V.Y. Shvadchak, V.V.; Morellet, N. Guittet, E. Pivovarenko, V.G. Klymchenko, A.S.; Mély, Y. (2012) Two-color fluorescent L-amino acid mimic of tryptophan for probing peptide-nucleic acid complexes. Bioconjugate Chem. 23, 2434−2443.

14) Enander, K., Choulier, L., Olsson, A. L., Yushchenko, D. A., Kanmert, D., Klymchenko, A. S., Demchenko, A. P., Mely, Y., and Altschuh, D. (2008) A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte. Bioconjug. Chem. 19, 1864.

15) Postupalenko, V. Y.; Shvadchak, V. V.; Duportail, G; Pivovarenko, V. G.; Klymchenko, A. S.; Mely, Y. (2011) Monitoring membrane binding and insertion of peptides by two-color fluorescent label. Biochim. Biophys. Acta, 1808, 424-432.


Fluorescent organic nanoparticles.

Fluorescent nanoparticles are currently making a revolution in the field of biological imaging due to their exceptional fluorescence brightness and possible combination with other imaging and therapeutic modalities. The most established are inorganic nanoparticles, such as quantum dots and dye-doped silica nanoparticles, but their key limitation is lack of biodegradability. Our aim is to develop fluorescent organic nanoparticles of controlled size (5 to 100 nm), biodegradable and with comparable or better brightness than quantum dots. These nanoparticles are obtained by self-assembly of lipids (1), amphiphiles (2), or polymers and can respond to external biomolecular stimuli. These nanoparticles will be applied for ultra-sensitive detection of biomolecules (enzymes and nucleic acids), cellular and animal imaging with particular focus on viral and cancer diseases.

Supramolecular vectors for oligonucleotides and peptides

We also develop new concepts in preparation of nanoscale vehicles for nucleic acids and peptides, which constitute the key challenges in modern drug/gene delivery. Earlier, our team pioneered fluorescence spectroscopy and microscopy techniques for investigation of gene delivery vectors based on cationic lipids (3,4) and polymers (5,6). Recently we work on our own strategies for design and synthesis of delivery vectors. Unsymmetrical bolaamphiphiles were developed to form nanostructures encapsulating DNA and transfecting cells in vitro (7,8). Cationic amphiphilic calixarenes were also designed to form micelles that assemble with DNA into virus like particles featuring high stability and transfection efficiency (9). Currently, we extend these approaches to develop nanoscale vectors for short oligonucleotides and peptides. Fluorescence modality is also introduced to extend their applications for theranostics.