Didier Casanova

Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells

Low concentrations of reactive oxygen species, notably hydrogen peroxide (H(2)O(2)), mediate various signalling processes in the cell. Production of these signals is highly regulated and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H(2)O(2) generation in living cells. The Y(0.6)Eu(0.4)VO(4) nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H(2)O(2) with a 30-s temporal resolution and a dynamic range of 1-45 microM. The differences in the timing of intracellular H(2)O(2) production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H(2)O(2), in many signalling processes H(2)O(2) is, however, the dominant oxidant. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species.

Organic Functionalization of Luminescent Oxide Nanoparticles toward Their Application As Biological Probes

Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with alpha-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species.

Inferring maps of forces inside cell membrane microdomains.

Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g., organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show its general relevance for membrane compartmentation.

Optical in situ size determination of single lanthanide-ion doped oxide nanoparticles

We show that the size of a lanthanide-ion doped nanoparticle can be accurately determined from its luminosity. The optically determined size distribution is in very good agreement with the distribution obtained from transmission electron microscopy. These data confirm that single nanoparticles are visualized in microscopy experiments. Nanoparticles as small as 13nm are detectable with integration times of 500ms.

Luminescent lanthanide-ion doped nanoparticles as single-biomolecule labels and oxidant sensors

We report on the single-particle properties of lanthanide-ion doped oxide nanoparticles. We have demonstrated that their size can be accurately determined from their luminosity. The optically determined size distribution is in very good agreement with the distribution obtained from transmission electron microscopy (TEM). We also showed that the photobleaching of these nanoparticles is related to a reduction process and that we can use it to sense in a concentration-dependent manner the presence of an oxidant like H2O2. Finally, we propose a way to perform nanoparticle-protein coupling and to determine the protein-nanoparticle ratio at the single-particle level.

Functionalized luminescent oxide nanoparticles for sodium channel imaging at the single molecule level

Lanthanide-ion doped oxide nanoparticles were functionalized for use as fluorescent biological labels. These nanoparticles are synthesized directly in water which facilitates their functionalization, and are very photostable without emission intermittency. Nanoparticles functionalized with guanidinium groups act as artificial toxins and specifically target sodium channels. They are individually detectable in cardiac myocytes, revealing a heterogeneous distribution of sodium channels. Functionalized oxide nanoparticles appear as a novel tool particularly well adapted to long-term single-molecule tracking.

New Biological Labels Based on Functionalized YVO4:Eu Nanoparticles

Abstract : Lanthanide-ion doped oxide (YVO4:Eu) nanoparticles were synthesized as aqueous colloids and functionalized by a bioactive silane shell to be used as fluorescent biological labels. Nanoparticles functionalized with guanidinium groups were able to act as artificial toxins which specifically target Na+ channels. They were individually detectable in live cardiac myocytes. Functionalized oxide nanoparticles appear as a new interesting tool, especially attractive for long-term single-molecule tracking due to their photo-stability and long luminescence lifetime.

Spatio-Temporal Patterns of Reactive Oxygen Species Production in PDGF Signaling Revealed by Nanoparticle Imaging in Living Cells

Signaling by PDGF (Platelet Derived Growth Factor) is involved in cell migration, for metastasis formation or reparation of vascular lesions. Hydrogen peroxide (H2O2) is a known second messenger in this pathway and its intracellular concentration regulates the cell response. However, conventional methods are unable to measure quantitatively its temporal evolution. Here, we propose a new approach based on the imaging of YVO4:Eu nanoparticles. Their luminescence is indeed modulated by the oxidation state of doping europium ions. After photoinduced reduction, their chemical oxidation by H2O2 can be monitored by the nanoparticle imaging. We demonstrated in vitro that these particles are efficient probes to dynamically and quantitatively measure H2O2 concentration. By internalizing these nanoparticles in mamallian cells, we measured the oxidant response to a PDGF stimulation. We revealed the temporal pattern of H2O2 production and determined the effective affinity of PDGF receptors for their ligand. We thus proved that a persistent stimulation was necessary to trigger a significant H2O2 production: this intrinsic filtering could be of major physiological interest for understanding reliable cell migration. This response implies transactivation of EGF receptors, which we proved to be dominant at short times. The comparison of normal and tumoral cells revealed a faster and more important H2O2 production in tumoral cells. This likely relies on the different expression levels of proteins of the signaling cascade and points to the potential role of signal transduction dynamics for the regulation of metastasis formation.This work proposes the first quantitative measurements of the oxidant signaling in cells by the use of new nanoprobes. It more generally opens new perspectives for the study of the spatio-temporal organization of the cell response and its physiological importance.

Light Emission Properties and Biological Applications of Lanthanide Doped Oxide Nanoparticles

Rare-earth doped oxides as bulk materials are well known for their numerous applications in light emitting devices. Emission properties of nanoparticles, in association with their small size, open the way to new applications such as the elaboration of transparent luminescent devices or new biological labels. The key issue for such applications is the control of the surface state of the particles in order to preserve their dispersion state, to guarantee a strong emission and/or ensure strong interactions with specific target sites. Our work in this field mainly concerns yttrium vanadate particles (YVO4:Ln with Ln=Eu, Dy and Yb/Er) that are obtained as aqueous suspensions through a simple reaction of coprecipitation [1]. As compared to the bulk material, these particles (10-40 nm in diameter) exhibit the characteristic emission from the lanthanide dopant but with a lower efficiency (quantum yield of 15% and emission lifetime of 0.7 ms). The first part of our work is devoted to the improvement of the emission properties of particles. Our results show that the emission process is altered either by surface hydroxyl groups or by the poor cristallinity of the particles. We show that large improvement can be obtained following an original process which allows recovering the particles as colloidal dispersions after their thermal treatment at 1000°C. In the case of Eu3+ doped particles, quantum yield and emission lifetime were increased up to 40% and 0.8 ms respectively without notable increase of particle size. Moreover, the emission spectrum, either from colloidal suspensions or from single particles fits almost perfectly to the one from the bulk material. The second part of our work is devoted to the surface derivatization of the particles for applications as biological probes [2]. We chose a general scheme involving the coating of the nanoparticles with a thin layer of amino-silane. This process was chosen in order to allow further versatile grafting reactions trough the surface amino groups. This strategy will be detailed in the case of the coupling between our particles and a protein through the use of a homo-bifunctional cross-linker. The quantification of the number of attached proteins was achieved using dual-color microscopy and fluorescently-tagged proteins, by observing the step-like photobleaching of the organic fluorescent tag. The observation of labelled toxins interacting with living cells shows the high potentiality of rare-earth-doped oxide particles as new biological probes.

Fluorescence resonance energy transfer using lanthanide-ion doped oxide nanoparticles as donors

We have demonstrated fluorescence resonance energy transfer (FRET) between lanthanide-ion doped oxide nanoparticles acting as donors and organic acceptor molecules (Cy5). Due to the long nanoparticle lifetime and the large Stokes shift between nanoparticle absorption and emission, unambiguous and precise FRET measurements can be performed despite the presence of large free acceptor oncentrations. We determined FRET efficiencies as a function of Cy5 concentration which are in very good agreement with a multiple acceptor-multiple donor calculation.