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.

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.

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.

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

Antithrombin-independent effects of heparins on fibrin clot nanostructure.

OBJECTIVE: Because of the widespread clinical use of heparins, their effects on the enzymatic cascade are very well known. In contrast, little is known about the direct effect of heparins on the nanostructure of fibrin fibers, even though this nanostructure plays a major role in the mechanical strength and lysis of clots. This lack of reliable data can be correlated with the lack of a nonintrusive, quantitative method to determine this structure. We recently developed such a method that allows the simultaneous determination of the average fiber radius and the protein content using spectrometric data. In this study, we assessed the nanostructure of fibrin in a system composed of human thrombin and fibrinogen. METHODS AND RESULTS: We provide quantitative evidence showing that both unfractionated heparin and low molecular weight heparin directly alter the nanostructure of fibrin fibers independent of their other actions on the coagulation cascade; as expected, the pentasaccharide fondaparinux has no effect. CONCLUSIONS: Our results show that in addition to the effect of heparin on the coagulation cascade, modifications of the fibrin nanostructure may also contribute to improved fibrinolysis.