Controlled Second Harmonic Generation with Optically Trapped Lithium Niobate Nanoparticles.

We report the second harmonic generation (SHG) response from a single 34 nm diameter lithium niobate nanoparticle. The experimental setup involves a first beam devoted to the optical trapping of single nanoparticles, whereas a second arm involves a femtosecond laser source leading to the SHG emission from the trapped nanoparticles. SHG operation where one to three nanoparticles are present in the optical trap is first demonstrated, highlighting the transition between coherent and incoherent SHG, the latter known as hyper-Rayleigh scattering (HRS). With a spatial light modulator moving the optical trap in and out of the focus of the femtosecond beam, the SHG intensity is switched back and forth between a low and a high level. This controlled operation opens new avenues for nanoparticle characterization and applications in sensing or communication and information technologies and constitutes the first step in the design of active substrateless metasurfaces.

Effect of Silane Monolayers and Nanoporous Silicon Surfaces on the Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Detection of Sepsis Metabolites Biomarkers Mixed in Solution.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) is a promising strategy for clinical diagnosis based on metabolite detection. However, several bottlenecks (such as the lack of reproducibility in analysis, the presence of an important background in low-mass range, and the lack of organic matrix for some molecules) prevent its transfer to clinical cases. These limitations can be addressed by using nanoporous silicon surfaces chemically functionalized with silane monolayers. In the present study, sepsis metabolite biomarkers were used to investigate the effects of silane monolayers and porous silicon substrates on MALDI-ToF MS analysis (signal-to-noise value (S/N), relative standard deviation of the S/N of triplicate samples (STDmean), and intra-substrates uniformity). Also, the impact of the physicochemical properties of metabolites, with different isoelectric points and hydrophobic-hydrophilic balances, was assessed. Four different silane molecules, with various alkyl chain lengths and head-group charges, were self-assembled in monolayers on plane and porous silicon surfaces. Their surface coverage and conformity were investigated by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The seven metabolites detected on the stainless-steel target plate (lysophosphatidylcholine, caffeine, phenylalanine, creatinine, valine, arginine, and glycerophosphocholine) are also detected on the silanized and bare, plane and porous silicon surfaces. Moreover, two metabolites, glycine and alanine, which are not detected on the stainless-steel target plate, are detected on all silanized surfaces, except glycine which is not detected on CH3 short-modified porous silicon and on the bare plane silicon substrate. In addition, whatever the metabolites (except phenylalanine and valine), at least one of the silicon surfaces allows to increase the S/N value in comparison with the stainless-steel target plate. Also, the heterogeneity of matrix crystallization features is linked to the STDmean which is poor on the NH3+ monolayer on plane substrate and better on the NH3+ monolayer on porous substrate, for most of the metabolites. Nevertheless, matrix crystallization features are not sufficient to systematically get high STDmean and uniformity in MALDI-ToF MS analysis. Indeed, the physicochemical properties of metabolites and surfaces, limitations in metabolite extraction from the pores, and improvement in metabolite desorption due to the pores are shown to significantly impact MS analysis. In particular, in the case of the most hydrophobic metabolites studied, the highest S/N values and the best STDmean and uniformity (the lowest values) are reached by using porous substrates, while in the case of the most hydrophilic metabolites studied, plane substrates demonstrated the highest S/N and the lowest STDmean. No clear trend of surface chemistry was evidenced.

Effects of Silane Monolayers on Lysophosphatidylcholine (LysoPC) Detection by Desorption Ionization on Silicon Mass Spectrometry (DIOS-MS) in Solution and Plasma.

Desorption ionization on silicon mass spectrometry (DIOS-MS) enables high throughput analysis of low-molecular-weight biomolecules. However, detection of metabolite biomarkers in complex fluids such as plasma requires sample pretreatment, limiting clinical application. Here, we show that porous silicon, chemically modified using monolayers of n-propyldimethylmethoxysilane molecules, is a good candidate for fingerprinting lysophosphatidylcholine (lysoPC) in plasma, without sample pretreatment, for DIOS-MS-based diagnosis (e.g., sepsis). Results were correlated to lysoPC molecule location inside/outside the pores, determined by time-of-flight secondary ion mass spectrometry profiling, and to physicochemical properties.

Curious Binding Energy Increase between the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein and Angiotensin-Converting Enzyme 2 Adsorbed on a Silane Monolayer from Molecular Dynamics Simulations.

In the context of the COVID-19 outbreak since December 2019, antigenic tests are widely used, for diagnosis purposes, to detect the SARS-CoV-2 spike protein in nasopharyngeal fluid through its interactions with specific antibodies. However, the SARS-CoV-2 spike protein is subject to rapid mutations yielding more and more variants that might lose their affinity toward the currently used antibodies. The virus entry into the host cell involves interactions between the angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor-binding domain. Consequently, ACE2 could be a target with limited mutation escaping possibilities. However, as the enzyme has not evolved to recognize the virus, its affinity with the spike protein receptor-binding domain is lower than that with specific antibodies. The present molecular dynamics simulations study suggests that the adsorption of the ACE2 on specific silane monolayers could increase its affinity toward the spike protein receptor-binding domain. Indeed, silane monolayers, combining silane molecules with short alkyl chains and positively charged head groups and silane molecules without charged head groups, could adsorb the ACE2 while maintaining its bioactivity (orientation compatible with the spike protein trapping, low conformational changes) and increasing its interactions with the spike protein receptor-binding domain (number of hydrogen bonds and electrostatic interactions) to lead to an increase by 20% both in the binding free energy and in the enzyme /receptor-binding domain rupture force. This work could help develop biosensing tools efficient toward any variants of the SARS-CoV-2 spike protein.

Gold-seeded Lithium Niobate Nanoparticles: Influence of Gold Surface Coverage on Second Harmonic Properties.

Hybrid nanoparticles composed of an efficient nonlinear optical core and a gold shell can enhance and tune the nonlinear optical emission thanks to the plasmonic effect. However the influence of an incomplete gold shell, i.e., isolated gold nano-islands, is still not well studied. Here LiNbO3 (LN) core nanoparticles of 45 nm were coated with various densities of gold nano-seeds (AuSeeds). As both LN and AuSeeds bear negative surface charge, a positively-charged polymer was first coated onto LN. The number of polymer chains per LN was evaluated at 1210 by XPS and confirmed by fluorescence titration. Then, the surface coverage percentage of AuSeeds onto LN was estimated to a maximum of 30% using ICP-AES. The addition of AuSeeds was also accompanied with surface charge reversal, the negative charge increasing with the higher amount of AuSeeds. Finally, the first hyperpolarizability decreased with the increase of AuSeeds density while depolarization values for Au-seeded LN were close to the one of bare LN, showing a predominance of the second harmonic volumic contribution.

Effects of the Chemical and Structural Properties of Silane Monolayers on the Organization of Water Molecules and Ions at Interfaces, from Molecular Dynamics Simulations.

Understanding the organization of the hydration layer at functionalized silica surfaces is relevant for a large range of biosensing applications or surface phenomena such as biomolecule adsorption. Silane monolayers are widely used to functionalize silica surfaces. Using molecular dynamics simulations, we have investigated the role of silane molecule head-group charge, alkyl chain length, and surface coverage in the structural organization and dynamic properties of Na+ ions, Cl- ions, and water molecules at the interface. The silane molecules studied are 3-aminopropyldimethylethoxysilane, n-propyldimethylmethoxysilane, octadecyldimethylmethoxysilane, and (dimethylamino)dimethylsilylundecanoate. Our results suggest that the distribution of interfacial ions is sensitive to the 2D dispersion of the silane-charged head groups. Also, while charged silane monolayers show a strong orientation of interfacial water molecules, which leads to a rupture in the hydrogen bond network and disturbs their tetrahedral organization, the arrangement of water molecules at the interface with uncharged silane monolayers seems to be related to the surface roughness and to alkyl chain length. In line with these results, the diffusion of ions and water molecules is higher at the CH3 long monolayer interface than at the CH3 short monolayer interface and at the charged monolayer interfaces. Also, whatever the silane molecules studied, bulk properties are recovered around 0.7 nm above the interface. The interfacial water organization is known to impact biomolecule adsorption. Therefore, these results could further help in optimizing the functionalization layers to capture analytes.

On the Reaction Pathways and Growth Mechanisms of LiNbO3 Nanocrystals from the Non-Aqueous Solvothermal Alkoxide Route.

Phase-pure, highly crystalline sub-50 nm LiNbO3 nanocrystals were prepared from a non-aqueous solvothermal process for 72 h at 230 °C and a commercial precursor solution of mixed lithium niobium ethoxide in its parent alcohol. A systematic variation of the reaction medium composition with the addition of different amounts of co-solvent including butanol, 1,3-propanediol, 1,4-butanediol, and 1,5-pentanediol resulted in the formation of nanocrystals of adjustable mean size and shape anisotropy, as demonstrated from XRD measurements and TEM imaging. Colloidal stability of ethanol- and water-based suspensions was evaluated from dynamic light scattering (DLS)/zeta potential studies and correlated with FTIR data. Thanks to the evolution in the nanocrystal size and shape distribution we observed, as well as to the available literature on the alkoxide chemistry, the reaction pathways and growth mechanisms were finally discussed with a special attention on the monomer formation rate, leading to the nucleation step. The polar, non-perovskite crystalline structure of LiNbO3 was also evidenced to play a major role in the nanocrystal shape anisotropy.

Orthogonal Chemical Functionalization of Au/SiO2/TiW Patterned Substrates.

Macropatterned and micropatterned gold/silicon dioxide/titanium tungsten (Au/SiO2/TiW) substrates were orthogonally functionalized: three different molecules (monovalent silane, thiol, and phosphonic acid) were used to specifically form organolayers on Au, SiO2, or TiW areas of patterned substrates. The orthogonality of the functionalization (i.e., selective grafting of thiol on Au, phosphonic acid on TiW, and silane on SiO2) was assessed by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), Fourier transform infrared spectroscopy (FTIR), and contact angle measurements. These results are especially promising for the selective anchoring of targets (e.g., biomolecules, nanoparticles, nanowires, nanotubes, or other nano-objects) onto patterned zones of multimaterial substrates, such as nanosensors or other nanodevices.

Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and Its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations.

Protein adsorption on surfaces is used in analytical tools as an immobilization mean to trap the analyte to be detected. However, protein adsorption can lead to a conformational change in the protein structure, resulting in a loss of bioactivity. Here, we study adsorption of a streptavidin-biotin complex on amorphous SiO2 surfaces functionalized with five different silane self-assembled monolayers by all-atom molecular dynamics simulations. We find that the streptavidin global conformational change, as well as the nature of residues with high mobility, depends on the alkyl chain length and head-group charge of silane molecules. Effects on interactions with biotin are further investigated by steered molecular dynamics (SMD) simulations, which mimics atomic force microscopy (AFM) with the biotin attached on the tip. We show the combined effects of adsorption-induced global conformational changes and of the position of residues with high mobility on the streptavidin-biotin rupture force. By comparing our results to experimental and SMD rupture forces obtained in water, without any surface, we conclude that silane with uncharged and short alkyl chains allows streptavidin immobilization, while keeping biotin interactions better than silanes with long alkyl chains or charged head groups.

Deciphering multivalent glycocluster-lectin interactions through AFM characterization of the self-assembled nanostructures.

Pseudomonas aeruginosa is a human opportunistic pathogen responsible for lung infections in cystic fibrosis patients. The emergence of resistant strains and its ability to form a biofilm seem to give a selective advantage to the bacterium and thus new therapeutic approaches are needed. To infect the lung, the bacterium uses several virulence factors, like LecA lectins. These proteins are involved in bacterial adhesion due to their specific interaction with carbohydrates of the host epithelial cells. The tetrameric LecA lectin specifically binds galactose residues. A new therapeutic approach is based on the development of highly affine synthetic glycoclusters able to selectively link with LecA to interfere with the natural carbohydrate-LecA interaction. In this study, we combined atomic force microscopy imaging and molecular dynamics simulations to visualize and understand the arrangements formed by LecA and five different glycoclusters. Our glycoclusters are small scaffolds characterized by a core and four branches, which terminate in a galactose residue. Depending on the nature of the core and the branches, the glycocluster-lectin interaction can be modulated and the affinity increased. We show that glycocluster-LecA arrangements highly depend on the glycocluster architecture: the core influences the rigidity of the geometry and the directionality of the branches, whereas the nature of the branch determines the compactness of the structure and the ease of binding.

Oxidized Titanium Tungsten Surface Functionalization by Silane-, Phosphonic Acid-, or Ortho-dihydroxyaryl-Based Organolayers.

Titanium tungsten (TiW) films (200 nm thick) were cleaned by oxygen plasma, and the resulting oxidized surfaces were functionalized by 3-aminopropylphosphonic acid (APPA), 3-ethoxydimethylsilylpropylamine (APDMES), or dopamine (DA) to form three different organolayers. The three resulting organolayers were characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and Fourier transform infrared spectroscopy analyses. The stability of each organolayer was investigated. Our results suggested that the Si-O-Ti or Si-O-W bonds formed by the reactions of APDMES with surface-oxidized TiW were rather labile, whereas the catechol layer was less labile. The APPA layer was the most stable of all tested surface modifications.

Orthogonal chemical functionalization of patterned Au/TiW substrate for selective immobilization of nanoparticles.

The evolution of nanobiosensors stresses the need for multi-material nanopatterned surfaces to enhance sensing performances. Titanium tungsten (TiW) has been mastered and routinely implemented in nanoelectronic devices, in a reproducible way and at industrial production scales. Such a material may be envisioned for use in (bio)chemical nanoelectronic sensors, but the surface functionalization of such material has yet to be studied. In the present article, the orthogonal chemical functionalization of patterned Au on TiW substrates has been explored for the first time. Surface functionalizations were assessed by x-ray photoelectron spectroscopy, polarization modulation infrared reflection-absorption spectroscopy and time-of-flight secondary ion mass spectrometry imaging. Au/TiW patterned substrates were functionalized with mercapto-undecamine. Thanks to the orthogonality of thiol/Au versus phosphonic acid/TiW reactions, only the Au features were modified leading to the amine derivatized surface. This allowed for the localizing of carboxy-functionalized nanoparticles by electrostatic interaction on Au with a selectivity above 10 when compared to TiW.

Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters.

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

The anti-adhesive effect of glycoclusters on Pseudomonas aeruginosa bacteria adhesion to epithelial cells studied by AFM single cell force spectroscopy.

The human opportunistic pathogen Pseudomonas aeruginosa (PA) is responsible for chronic infections of the respiratory epithelium in cystic fibrosis patients. PA takes advantage of an arsenal of virulence factors to infect and colonize human lungs. Among them, the lectin LecA favours epithelium invasion by interacting with host cell globotriaosylceramide (Gb3). A new therapeutic approach is based on the development of synthetic multivalent molecules (glycoclusters) targeting LecA with a higher affinity than its natural ligand. Atomic force microscopy-single cell force spectroscopy has been used to study the effect of glycoclusters on the bacteria-cell interaction. Glycoclusters have been shown to affect the detachment work and detachment force of the bacteria-cell interaction. The specificity and the efficiency of the glycocluster in targeting the lectin and destabilizing the PA-epithelial cell adhesion are demonstrated and discussed.

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa.

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis-galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (Kd ). This has led to high-affinity LecA ligands with Kd values in the low nanomolar range (Kd =22 nm for the best one).

Autoantibodies against heat shock proteins as biomarkers for the diagnosis and prognosis of cancer.

Thanks to their specificity and stability in the sera, autoantibodies (AAbs) against tumor-associated antigens (TAAs) are very attractive biomarkers for the development of less invasive serological tests for the diagnosis and prognosis of cancer. Heat shock proteins (HSP) belong to TAAs and they are over-expressed in various human cancers. Elevated HSP can stimulate the immune system to produce anti-HSP antibodies. So far, AAbs against HSP have been identified in the circulation of various cancer patients. Here we will review current literature on the use of anti-HSP antibodies for cancer diagnosis and prognosis. The challenges as well as future directions of AAbs identification in oncology are also discussed.

Direct silanization of zirconia for increased biointegration.

High-performance bioinert ceramics such as zirconia have been used for biomedical devices since the early seventies. In order to promote osseointegration, the historical solution has been to increase the specific surface of the implant through roughness. Nevertheless these treatments on ceramics may create defects at the surface, exposing the material to higher chances of early failure. In zirconia, such treatments may also affect the stability of the surface. More recently, the interest of improving osseointegration of implants has moved the research focus towards the actual chemistry of the surface. Inspired by this, we have adapted the current knowledge and techniques of silica functionalization and applied it to successfully introduce 3-aminopropyldimethylethoxy silane (APDMES) directly on the surface of zirconia (3Y-TZP). We used plasma of oxygen to clean the surface and promote hydroxylation of the surface to increase silane density. The samples were extensively characterized by means of X-ray photoelectron spectroscopy (XPS) and contact angle, mechanically tested and its cytotoxicity was evaluated through cell adhesion and proliferation tests. Additionally, aging was studied to discard negative effects of the treatment on the stability of the tetragonal phase. No adverse effect was found on the mechanical response of treated samples. In addition, plasma-treated samples exhibited an unexpectedly higher resistance to aging. Finally, silane density was 35% lower than the one reported in literature for silica. However cells displayed a qualitatively higher spreading in opposition to the rounder appearance of cells on untreated zirconia. These results lay the foundations for the next generation of zirconia implants with biologically friendlier surfaces. STATEMENT OF SIGNIFICANCE: The use of zirconia-based ceramics in biomedical devices is broad and well accepted, especially in dental implants. However, they do not bond naturally to bone, therefore to ensure fixation surgeons typically rely on roughness at different scales, or on cements. Alternatively in this work we present a new perspective of surface modification through chemistry to enhance the interaction between surface and biological environment, without the downsides of roughness. This surface treatment is proposed for zirconia, which allowed a direct silanization of its surface and a higher cell attachment. The results of this research may open the possibility for the next generation of bioinert ceramic implants with more advanced tailored surfaces for increased osseointegration.

Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low-Nanomolar Multivalent Ligands.

Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low-nanomolar (Kd =19 nm, microarray) ligand with a tyrosine-based linker arm could be identified in a structure-activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.

pH driven addressing of silicon nanowires onto Si3N4/SiO2 micro-patterned surfaces.

pH was used as the main driving parameter for specifically immobilizing silicon nanowires onto Si3N4 microsquares at the surface of a SiO2 substrate. Different pH values of the coating aqueous solution enabled to experimentally distribute nanowires between silicon nitride and silicon dioxide: at pH 3 nanowires were mainly anchored on Si3N4; they were evenly distributed between SiO2 and Si3N4 at pH 2.8; and they were mainly anchored on SiO2 at pH 2. A theoretical model based on DLVO theory and surface protonation/deprotonation equilibria was used to study how, in adequate pH conditions, Si nanowires could be anchored onto specific regions of a patterned Si3N4/SiO2 surface. Instead of using capillary forces, or hydrophilic/hydrophobic contrast between the two types of materials, the specificity of immobilization could rely on surface electric charge contrasts between Si3N4 and SiO2. This simple and generic method could be used for addressing a large diversity of nano-objects onto patterned substrates.

Anti-heat shock protein autoantibody profiling in breast cancer using customized protein microarray.

Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%.