Vanessa Trouillet

One-Pot Synthesis of Near-Infrared Fluorescent Gold Clusters for Cellular Fluorescence Lifetime Imaging†

A facile strategy to synthesize water-soluble fluorescent gold nanoclusters (Au NCs) stabilized with the bidentate ligand dihydrolipoic acid (DHLA) is reported. The DHLA-capped Au NCs are characterized by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. The Au NCs possess many attractive features including ultrasmall size, bright near-infrared luminescence, high colloidal stability, and good biocompatibility, making them promising imaging agents for biomedical and cellular imaging applications. Moreover, their long fluorescence lifetime (>100 ns) makes them attractive as labels in fluorescence lifetime imaging (FLIM) applications. As an example, the internalization of Au NCs by live HeLa cells is visualized using the FLIM technique.

Facile preparation of water-soluble fluorescent gold nanoclusters for cellular imaging applications

We report a facile strategy to synthesize water-soluble, fluorescent gold nanoclusters (AuNCs) in one step by using a mild reductant, tetrakis(hydroxymethyl)phosphonium chloride (THPC). A zwitterionic functional ligand, D-penicillamine (DPA), as a capping agent endowed the AuNCs with excellent stability in aqueous solvent over the physiologically relevant pH range. The DPA-capped AuNCs displayed excitation and emission bands at 400 and 610 nm, respectively; the fluorescence quantum yield was 1.3%. The effect of borohydride reduction on the optical spectra and X-ray photoelectron spectroscopy (XPS) results indicated that the AuNC luminescence is closely related to the presence of Au(I) on their surfaces. In a first optical imaging application, we studied internalization of the AuNCs by live HeLa cells using confocal microscopy with two-photon excitation. A cell viability assay revealed good biocompatibility of these AuNCs. Our studies demonstrate a great potential of DPA-stabilized AuNCs as fluorescent nanoprobes in bioimaging and related applications.

Effect of Protein Adsorption on the Fluorescence of Ultrasmall Gold Nanoclusters

The interaction of proteins with ultrasmall gold nanoclusters (Au NCs) is investigated. Upon protein association, the fluorescence of Au NCs is significantly enhanced and, concomitantly, their luminescence lifetime is prolonged. The results stress the importance of investigating the behavior of fluorescent metal NCs in complex biological environment for advancing their bio-nanotechnology applications.

Ultrasmall Fluorescent Silver Nanoclusters: Protein Adsorption and Its Effects on Cellular Responses

AbstractUltrasmall silver nanoclusters (AgNCs) are a novel type of fluorescent nanoprobes that have aroused a great deal of interest in recent years. In view of many promising applications in biological research, it is of great importance to explore their behavior in the complex biological environment. In this study, interactions of AgNCs with a model protein, human serum albumin (HSA), have been systematically investigated by using a variety of techniques including absorption spectroscopy, steady-state and time-resolved fluorescence, as well as circular dichroism spectroscopy. The results show that the physicochemical properties of both proteins and AgNCs undergo changes upon their interactions; however, it appears that the overall conformation of HSA remains essentially unaffected in the complex. Binding of HSA to AgNCs was assessed by measuring tryptophan fluorescence quenching of HSA by AgNCs. Furthermore, biological implications of protein adsorption were quantitatively explored by evaluating responses of HeLa cells to AgNC exposure through live-cell fluorescence microscopy and a cytotoxicity test, revealing that protein adsorption has a significant effect on the biological response to AgNC exposure.

Grafting efficiency of synthetic polymers onto biomaterials: A comparative study of grafting- from versus grafting- to

In the present study, the two grafting techniques grafting-from - by activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) - and grafting-to - by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) - were systematically compared, employing cellulose as a substrate. In order to obtain a meaningful comparison, it is crucial that the graft lengths of the polymers that are grafted from and to the substrates are essentially identical. Herein, this was achieved by utilizing the free polymer formed in parallel to the grafting-from reaction as the polymer for the grafting-to reaction. Four graft lengths were investigated, and the molar masses of the four free polymers (21 ≤ M(n) ≤ 100 kDa; 1.07 ≤ Đ(M) ≤ 1.26), i.e. the polymers subsequently employed in the grafting-to reaction, were shown to be in the same range as the molar masses of the polymers grafted from the surface (23 ≤ M(n) ≤ 87 kDa; 1.08 ≤ Đ(M) ≤ 1.31). The molecular weights of the chains grafted from the surface were established after cleavage from the cellulose substrates via size exclusion chromatography (SEC). High-resolution Fourier transform infrared microscopy (FT-IRM) was employed as an efficient tool to study the spatial distribution of the polymer content on the grafted substrates. In addition, the functionalized substrates were analyzed by X-ray photoelectron spectroscopy (XPS), contact angle (CA) measurements, and field-emission scanning electron microscopy (FE-SEM). For cellulose substrates modified via the grafting-from approach, the content of polymer on the surfaces increased with increasing graft length, confirming the possibility to tailor not only the length of the polymer grafts but also the polymeric content on the surface. In comparison, for the grafting-to reaction, the grafted content could not be controlled by varying the length of the preformed polymer: the polymer content was essentially the same for the four graft lengths. Consequently, the obtained results, when employing cellulose as a substrate and under these conditions, suggest that the grafting-from approach is superior to the grafting-to technique with respect to controlling the distribution of the polymeric content on the surface.

Photo‐Patterning of Non‐Fouling Polymers and Biomolecules on Paper

Functional cellulose substrates with tetrazole moieties are generated to serve as universal platforms for the spatio-temporal immobilization of synthetic ultra-low fouling polymer brushes and protein species via a nitrile imine-mediated tetrazole-ene cycloaddition (NITEC)-based protocol. Poly(carboxybetaine acrylamide) brushes are grafted from initiators photo-patterned by NITEC utilizing single electron transfer living radical polymerization. Streptavidin is photo-immobilized with remarkable efficiency, opening the possibility to generate new materials for biomedical and biosensing applications.

Temperature responsive cellulose-graft-copolymers via cellulose functionalization in an ionic liquid and RAFT polymerization

Well-defined cellulose-graft-polyacrylamide copolymers were synthesized in a grafting-from approach by reversible addition-fragmentation chain transfer polymerization (RAFT). A chlorine moiety (degree of substitution DS(Cl) ≈ 1.0) was introduced into the cellulose using 1-butyl-3-methylimidazolium chloride (BMIMCl) as solvent before being substituted by a trithiocarbonate moiety resulting in cellulose macro-chain transfer agents (cellulose-CTA) with DS(RAFT) of 0.26 and 0.41. Poly(N,N-diethylacrylamide) (PDEAAm) and poly(N-isopropylacrylamide) (PNIPAM) were subsequently grafted from these cellulose-CTAs and the polymerization kinetics, the molecular weight characteristics and the product composition were studied by nuclear magnetic resonance spectroscopy, X-ray photoelectron spectroscopy, and size exclusion chromatography of the polyacrylamides after cleavage from the cellulose chains. The number-average molecular weights, Mn, of the cleaved polymers ranged from 1100 to 1600 g mol(-1) for PDEAAm (dispersity Đ = 1.4-1.8) and from 1200 to 2600 g mol (-1) for PNIPAM (Đ = 1.7-2.1). The LCST behavior of the cellulose-graft-copolymers was studied via the determination of cloud point temperatures, evidencing that the thermoresponsive properties of the hybrid materials could be finely tuned between 18 and 26 °C for PDEAAm and between 22 and 26 °C for PNIPAM side chains.

Preparation of Reactive Three-Dimensional Microstructures via Direct Laser Writing and Thiol-ene Chemistry

Three-dimensional microstructures are fabricated employing the direct laser writing process and radical thiol-ene polymerization. The resin system consists of a two-photon photoinitiator and multifunctional thiols and olefins. Woodpile photonic crystals with 22 layers and a rod distance of 2 μm are fabricated. The structures are characterized via scanning electron microscopy and focused ion beam milling. The thiol-ene polymerization during fabrication is verified via infrared spectroscopy. The structures are grafted in a subsequent thiol-Michael addition reaction with different functional maleimides. The success of the grafting reaction is evaluated via laser scanning microscopy and X-ray photoelectron spectroscopy. The grafting density is calculated to be close to 200 molecules μm(-2) .

Benzylguanine thiol self-assembled monolayers for the immobilization of SNAP-tag proteins on microcontact-printed surface structures.

The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O(6)-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (microCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.

Spatially controlled photochemical peptide and polymer conjugation on biosurfaces

An efficient phototriggered Diels-Alder conjugation is utilized to graft in an effective and straightforward approach poly(trifluoro ethyl methacrylate) (Mn = 3700 Da, Đ = 1.27) and a model peptide (GIGKFLHS) onto thin hyaluronan films and cellulose surfaces. The surfaces were functionalized with an o-quinodimethane moiety - capable of releasing a caged diene - via carbodiimide mediated coupling. The o-quinodimethane group is employed as a photoactive linker to tether predefined peptide/polymer strands in a spatially controlled manner onto the biosurface by photoenol ligation. An in-depth characterization employing XPS, ToF-SIMS, SPR, ellipsometry, and AFM was conducted to evidence the effectiveness of the presented approach.