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Thomas Pons

Publications and source records attributed to Thomas Pons.

9 recordsLinked to original sources
PubMed2006

Solution-phase single quantum dot fluorescence resonance energy transfer.

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.

Cadmium Compounds↗
PubMed2006

Hydrodynamic dimensions, electrophoretic mobility, and stability of hydrophilic quantum dots.

Luminescent semiconductor quantum dots (QDs) have great potential for use in biological assays and imaging. These nanocrystals are capped with surface ligands (bifunctional molecules, amphiphilic polymers, phospholipids, etc.) that render them hydrophilic and provide them with functional properties. These coatings alters their hydrodynamic radii and surface charge, which can drastically affect properties such as diffusion within the cell cytoplasm. Heavy atom techniques such as transmission electron microscopy and X-ray scattering probe the inorganic core and do not take into account the ligand coating. Herein we use dynamic light scattering to characterize the hydrodynamic radius (R(H)) of CdSe-ZnS QDs capped with various hydrophilic surface coatings (including dihydrolipoic acid and amphiphilic polymers) and self-assembled QD-protein bioconjugates. Experiments were complemented with measurements of the geometric size and zeta potential using agarose gel electrophoresis and laser Doppler velocimetry. We find that the effects of surface ligands on the hydrodynamic radius and on the nanoparticle mobility are complex and strongly depend on a combination of the inorganic core size and nature and lateral extension of the hydrophilic surface coating. These properties are critical for the design of QD-based biosensing assays as well as QD bioconjugate diffusion in live cells.

Biosensing Techniques↗
PubMed2006

Designer variable repeat length polypeptides as scaffolds for surface immobilization of quantum dots.

We demonstrate the use of a series of engineered, variable-length de novo polypeptides to discretely immobilize luminescent semiconductor nanocrystals or quantum dots (QDs) onto functional surfaces. The polypeptides express N-terminal dicysteine and C-terminal hexahistidine residues that flank a variable number (1, 3, 5, 7, 14, 21, 28, or 35) of core beta-strand repeats, with tyrosine, glutamic acid, histidine, and lysine residues located at the turns. Polypeptides have molecular weights ranging from 4 to 83 kDa and retain a rigid structure based on the antiparallel beta-sheet motif. We first use a series of dye-labeled polypeptides to test and characterize their self-assembly onto hydrophilic CdSe-ZnS QDs using fluorescence resonance energy transfer (FRET). Results indicate that peptides maintain their beta-sheet conformation after self-assembly onto the QD surfaces, regardless of their length. We then immobilize biotinylated derivatives of these polypeptides on a NeutrAvidin-functionalized substrate and use them to capture QDs via specific interactions between the peptides' polyhistidine residues and the nanocrystal surface. We found that each of the polypeptides was able to efficiently capture QDs, with a clear correlation between the density of the surface-tethered peptide and the capacity for nanocrystal capture. The versatility of this capture strategy is highlighted by the creation of a variety of one- and two-dimensional polypeptide-QD structures as well as a self-assembled surface-immobilized FRET-based nutrient sensor.

Amino Acid Sequence↗
PubMed2005

Effects of (multi)branching of dipolar chromophores on photophysical properties and two-photon absorption.

To investigate the effect of branching on linear and nonlinear optical properties, a specific series of chromophores, epitome of (multi)branched dipoles, has been thoroughly explored by a combined theoretical and experimental approach. Excited-state structure calculations based on quantum-chemical techniques (time-dependent density functional theory) as well as a Frenkel exciton model nicely complement experimental photoluminescence and one- and two-photon absorption findings and contribute to their interpretation. This allowed us to get a deep insight into the nature of fundamental excited-state dynamics and the nonlinear optical (NLO) response involved. Both experiment and theory reveal that a multidimensional intramolecular charge transfer takes place from the donating moiety to the periphery of the branched molecules upon excitation, while fluorescence stems from an excited state localized on one of the dipolar branches. Branching is also observed to lead to cooperative enhancement of two-photon absorption (TPA) while maintaining high fluorescence quantum yield, thanks to localization of the emitting state. The comparison between results obtained in the Frenkel exciton scheme and ab initio results suggests the coherent coupling between branches as one of the possible mechanisms for the observed enhancement. New strategies for the rational design of NLO molecular assemblies are thus inferred on the basis of the acquired insights.

Absorption↗
PubMed2003

Mechanisms of membrane potential sensing with second-harmonic generation microscopy.

We characterize the transmembrane voltage response of a novel second-harmonic generation (SHG) marker using a screening protocol with giant unilamellar vesicles. Two mechanisms are found to contribute to the voltage response: (1) an electro-optic-induced alteration of the molecular hyperpolarizability and (2) an electric-field-induced alteration of the degree of molecular alignment. We quantify the relative weights and of these contributions and provide an upper limit to their response time, which is found to be submillisecond. The identification of two voltage response mechanisms leads to new strategies for the molecular design of membrane potential markers.

Cell Membrane↗
PubMed2002

Photoinduced flip-flop of amphiphilic molecules in lipid bilayer membranes.

We demonstrate localized photoinduced flip-flop of stilbazolium markers in model lipid bilayer membranes. The flip-flop mechanism and dynamics are determined by combined two-photon excited fluorescence and second-harmonic generation microscopy. Upon illumination of labeled membranes with a femtosecond laser beam, two-photon absorption induced photoisomerization provokes a significant increase in the cis- marker population whose flip-flop rate was determined to be at least a thousand times faster than that for transmarkers, allowing the possibility of fast targeted control of the local interleaflet distribution of markers in biological membranes.

Journal Article↗
PubMedDate not supplied

Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery.

We demonstrate the use of self-assembled luminescent semiconductor quantum dot (QD)-peptide bioconjugates for the selective intracellular labeling of several eukaryotic cell lines. A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a terminal polyhistidine tract was synthesized and used to facilitate the transmembrane delivery of the QD bioconjugates. The polyhistidine sequence allows the peptide to self-assemble onto the QD surface via metal-affinity interactions while the oligoarginine sequence allows specific QD delivery across the cellular membrane and intracellular labeling as compared to nonconjugated QDs. This peptide-driven delivery is concentration-dependent and thus can be titrated. Upon internalization, QDs display a punctate-like staining pattern in which some, but not all, of the QD signal is colocalized within endosomes. The effects of constant versus limited exposure to QD-peptide conjugates on cellular viability are evaluated by a metabolic specific assay, and clear differences in cytotoxicity are observed. The efficacy of using peptides for selective intracellular delivery is highlighted by performing a multicolor QD labeling, where we found that the presence or absence of peptide on the QD surface controls cellular uptake.

Animals↗