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.

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.

Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray.

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10-30% of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose-specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA-directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure-activity relationships between glycoclusters and lectins. High-throughput and multiplexed analysis of lectin-glycocluster interactions on a DDI microarray allows measurement of IC50 and dissociation constant (Kd ) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC50 and Kd values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC50 values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC50 values increase too. In contrast, the measured Kd values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single-molecule level (no surface cluster effect).

Effect of surface elasticity on the rheology of nanometric liquids.

The rheological properties of liquids confined to nanometer scales are important in many physical situations. In this Letter, we demonstrate that the long-range elastic deformation of the confining surfaces must be taken into account when considering the rheology of nanometric liquids. In the case of a squeeze-flow geometry, we show that below a critical distance D(c), the liquid is clamped by its viscosity and its intrinsic properties cannot be disentangled from the global system response. Using nanorheology experiments, we demonstrate that picometer elastic deflections of the rigid confining surfaces dominate the overall mechanical response of nanometric liquids confined between solid walls.

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.

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.

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.

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.

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.

Nanoparticles selectively immobilized onto large arrays of gold micro and nanostructures through surface chemical functionalizations.

Latex nanoparticles (100nm and 200nm diameter) were precisely located onto the gold regions of micro and nanopatterned gold/silica substrates through surface chemical functionalizations. The gold patterns were selectively functionalized with alkylthiols bearing biotin or amine headgroups. This selective functionalization allowed the trapping of streptavidin- or carboxy-functionalized latex nanoparticles onto the gold structures with very little non-specific adsorption onto the surrounding silica. Quantitative data of nanoparticle capture on gold and silica, obtained through SEM image analysis, showed a one to two order of magnitude increase on gold with a similar low coverage on silica (non-specific adsorption) thanks to chemical functionalizations. Single nanoparticles were captured at the gap of dimer gold nanostructures.

Orthogonal chemical functionalization of patterned gold on silica surfaces.

Single-step orthogonal chemical functionalization procedures have been developed with patterned gold on silica surfaces. Different combinations of a silane and a thiol were simultaneously deposited on a gold/silica heterogeneous substrate. The orthogonality of the functionalization (i.e., selective grafting of the thiol on the gold areas and the silane on the silica) was demonstrated by X-ray photoelectron spectroscopy (XPS) as well as time-of-flight secondary ion mass spectrometry (ToF-SIMS) mapping. The orthogonal functionalization was used to immobilize proteins onto gold nanostructures on a silica substrate, as demonstrated by atomic force microscopy (AFM). These results are especially promising in the development of future biosensors where the selective anchoring of target molecules onto nanostructured transducers (e.g., nanoplasmonic biosensors) is a major challenge.

DNA directed immobilization glycocluster array: applications and perspectives.

The present review concerns the recent advances in DNA directed immobilization (DDI) based glycocluster array. The impact of glycan immobilization on subsequent interactions with protein is discussed and the consequent pros and cons of DDI-based glycocluster array are reviewed. Finally, application in the discovery of anti-pathogen molecules is illustrated by screening for galactose or fucose glycoclusters targeting two Pseudomonas aeruginosa virulence factors (PA-IL and PA-IIL).

Capacitive detection of buried interfaces by a dynamic surface force apparatus.

We present here a new type of distance sensor mounted on a Surface Force Apparatus (SFA), able to detect the position of a buried interface and therefore the thickness of a thin solid or soft matter film coating the SFA surface(s). This sensor relies on the capacitance created by the two metallized surfaces of the SFA. An harmonic oscillation of these polarized surfaces creates a pico- to femto-amps current indicating their relative position. One of the specificities of this sensor is the relatively weak polarization used for the measurements, minimizing the electrical forces and their impact on other interactions, hydrodynamical and mechanical forces measured by the SFA. This original and simple design is of high interest for studying the viscoelastic properties of thin films, to detect adsorbed liquid layers or slippage at liquid-solid interfaces, or even to study complex fluids such as ionic liquids under polarization. We demonstrate the use of this sensor to study the flow boundary condition of silicon oil on a metal surface, and the elastic modulus of a thin elastomer layer.

AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL) filaments induced by multivalent glycoclusters.

Atomic force microscopy reveals that Pseudomonas aeruginosa LecA (PA-IL) and a tetra-galactosylated 1,3-alternate calix[4]arene-based glycocluster self-assemble according to an aggregative chelate binding mode to create monodimensional filaments. Lectin oligomers are identified along the filaments and defects in chelate binding generate branches and bifurcations. A molecular model with alternate 90° orientation of LecA tetramers is proposed to describe the organisation of lectins and glycoclusters in the filaments.