Toward Distinguishing between the Superchaotropic and Hydrophobic Characters of Nanometric-Sized Ions in Interaction with PEGylated Surfaces.

In this study, we explore the superchaotropic effect of various polyoxometalate or boron cluster nano-ions on hydrophilic neutral surfaces. Nano-ions, characterized by low charge densities, exhibit strong adsorption on non-ionic hydrophilic surfaces like PEGylated micelles. This adsorption phenomenon was attributed to the enthalpically favorable dehydration of nano-ions, the so-called superchaotropic effect. Here, we investigate the adsorption of three nano-ions, α-SiW12O404-, α-PW12O403-, and B12I122-, with decreasing charge density or increasing superchaotropicity (or hydrophobicity), on hydrophilic solid surfaces, PEGylated gold nanoparticles, and PEGylated gold-coated quartz crystal. Solid surfaces are devoid of hydrophobic regions, enabling the study of the subtle nuance between hydrophobic and superchaotropic effects. Unlike adsorption on PEGylated micelles, the adsorption constant decreases with a reduced charge density, aligning with the well-established principle that hydrophobic ions do not adsorb on hydrophilic surfaces. This research improves our understanding of the subtle difference between superchaotropic and hydrophobic effects in nano-ion adsorption phenomena.

Dynamic Instability of Individual Carbon Nanotube Growth Revealed by In Situ Homodyne Polarization Microscopy.

Understanding the kinetic selectivity of carbon nanotube growth at the scale of individual nanotubes is essential for the development of high chiral selectivity growth methods. Here we demonstrate that homodyne polarization microscopy can be used for high-throughput imaging of long individual carbon nanotubes under real growth conditions (at ambient pressure, on a substrate) and with subsecond time resolution. Our in situ observations on hundreds of individual nanotubes reveal that about half of them grow at a constant rate all along their lifetime while the other half exhibits stochastic changes in growth rates and/or switches between growth, pause, and shrinkage. Statistical analysis shows that the growth rate of a given nanotube essentially varies between two values, with a similar average ratio (∼1.7) regardless of whether the rate change is accompanied by a change in chirality. These switches indicate that the nanotube edge or the catalyst nanoparticle fluctuates between different configurations during growth.

Probing the local structure of nanoscale actinide oxides: a comparison between PuO2 and ThO2 nanoparticles rules out PuO2+x hypothesis.

Actinide research at the nanoscale is gaining fundamental interest due to environmental and industrial issues. The knowledge of the local structure and speciation of actinide nanoparticles, which possibly exhibit specific physico-chemical properties in comparison to bulk materials, would help in a better and reliable description of their behaviour and reactivity. Herein, the synthesis and relevant characterization of PuO2 and ThO2 nanoparticles displayed as dispersed colloids, nanopowders, or nanostructured oxide powders allow to establish a clear relationship between the size of the nanocrystals constituting these oxides and their corresponding An(iv) local structure investigated by EXAFS spectroscopy. Particularly, the first oxygen shell of the probed An(iv) evidences an analogous behaviour for both Pu and Th oxides. This observation suggests that the often observed and controversial splitting of the Pu-O shell on the Fourier transformed EXAFS signal of the PuO2 samples is attributed to a local structural disorder driven by a nanoparticle surface effect rather than to the presence of PuO2+x species.

Surface Properties of Alkoxysilane Layers Grafted in Supercritical Carbon Dioxide.

Silicon oxide surface properties can be easily modified by grafting alkoxysilane molecules. Here, we studied the structure and the morphology of ultrathin layers prepared by the grafting of alkoxysilanes having different head groups (thiol, amine, and iodo) in supercritical carbon dioxide (CO2) on model plane silicon oxide surfaces. Several characterization techniques (X-ray reflectivity, water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy (AFM)) were used to determine the physicochemical properties of the layers prepared at different temperatures. Moreover, for the first time, AFM peak force measurements were used to delve deeper into the determination of the structure of these ultrathin alkoxysilane layers. The results show that the grafting temperature and the nature of the head group strongly affect the morphology and structure of the grafted layers. Dense monolayers are obtained with 3-(mercaptopropyl)trimethoxysilane at 60 °C, polycondensed layers are always prepared with [3-(aminoethylamino)propyl]trimethoxysilane, and a dense bilayer is synthesized with 3-(iodopropyl)triethoxysilane at 120 °C.

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed.

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

On the Operational Aspects of Measuring Nanoparticle Sizes.

Nanoparticles are defined as elementary particles with a size between 1 and 100 nm for at least 50% (in number). They can be made from natural materials, or manufactured. Due to their small sizes, novel toxicological issues are raised and thus determining the accurate size of these nanoparticles is a major challenge. In this study, we performed an intercomparison experiment with the goal to measure sizes of several nanoparticles, in a first step, calibrated beads and monodispersed SiO2 Ludox®, and, in a second step, nanoparticles (NPs) of toxicological interest, such as Silver NM-300 K and PVP-coated Ag NPs, Titanium dioxide A12, P25(Degussa), and E171(A), using commonly available laboratory techniques such as transmission electron microscopy, scanning electron microscopy, small-angle X-ray scattering, dynamic light scattering, wet scanning transmission electron microscopy (and its dry state, STEM) and atomic force microscopy. With monomodal distributed NPs (polystyrene beads and SiO2 Ludox®), all tested techniques provide a global size value amplitude within 25% from each other, whereas on multimodal distributed NPs (Ag and TiO2) the inter-technique variation in size values reaches 300%. Our results highlight several pitfalls of NP size measurements such as operational aspects, which are unexpected consequences in the choice of experimental protocols. It reinforces the idea that averaging the NP size from different biophysical techniques (and experimental protocols) is more robust than focusing on repetitions of a single technique. Besides, when characterizing a heterogeneous NP in size, a size distribution is more informative than a simple average value. This work emphasizes the need for nanotoxicologists (and regulatory agencies) to test a large panel of different techniques before making a choice for the most appropriate technique(s)/protocol(s) to characterize a peculiar NP.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Conditions to minimize soft single biomolecule deformation when imaging with atomic force microscopy.

A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers.

The timeline of corona formation around silica nanocarriers highlights the role of the protein interactome.

Magnetic mesoporous silica nanoparticles (M-MSNs) represent promising targeting tools for theranostics. Engineering the interaction of nanoparticles (NPs) with biological systems requires an understanding of protein corona formation around the nanoparticles as this drives the biological fate of nanocarriers. We investigated the behavior of proteins in contact with M-MSNs by high-throughput comparative proteomics, using human and bovine sera as biological fluids, in order to assess the adsorption dynamics of proteins in these media. Using system biology tools, and especially protein-protein interaction databases, we demonstrated how the protein network builds up within the corona over the course of the experiment. Based on these results, we introduce and discuss the role of the "corona interactome" as an important factor influencing protein corona evolution. The concept of the "corona interactome" is an original methodology which could be generalized to all NP candidates. Based on this, pre-coating nanocarriers with specific proteins presenting minimal interactions with opsonins might provide them with properties such as stealth.

High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field.

BACKGROUND: The toxicity of manufactured fumed silica nanoparticles (NPs) remains poorly investigated compared to that of crystalline silica NPs, which have been associated with lung diseases after inhalation. Amorphous silica NPs are a raw material for manufactured nanocomposites, such as cosmetics, foods, and drugs, raising concerns about their potential toxicity. RESULTS: The size of the NPs was determined by dynamic light scattering and their shape was visualized by atomic force microscopy (10 ± 4 nm). The pertinent toxicological concentration and dynamic ranges were determined using viability tests and cellular impedance. We combined transcriptomics and proteomics to assess the cellular and molecular effects of fumed silica in A549 human alveolar epithelial cells. The "no observed transcriptomic adverse effect level" (NOTEL) was set to 1.0 µg/cm(2), and the "lowest observed adverse transcriptional effect level" (LOTEL) was set at 1.5 µg/cm(2). We carried out genome-wide expression profiles with microarrays and identified, by shotgun proteomics, the exoproteome changes in lung cells after exposure to NP doses (0.1, 1.0, 1.5, 3.0, and 6.0 µg/cm(2)) at two time points (24 h and 72 h). The data revealed a hierarchical, dose-dependent cellular response to silica NPs. At 1.5 µg/cm(2), the Rho signaling cascade, actin cytoskeleton remodeling, and clathrin-mediated endocytosis were induced. At 3.0 µg/cm(2), many inflammatory mediators were upregulated and the coagulation system pathway was triggered. Lastly, at 6.0 µg/cm(2), oxidative stress was initiated. The proteins identified in the extracellular compartment were consistent with these findings. CONCLUSIONS: The alliance of two high-throughput technologies allowed the quantitative assessment of the cellular effects and molecular consequences of exposure of lung cells to low doses of NPs. These results were obtained using a pathway-driven analysis instead of isolated genes. As in photography, toxicogenomics allows, at the same time, the visualization of a wide spectrum of biological responses and a "zoom in" to the details with a great depth of field. This study illustrates how such an approach based on human cell culture models is a valuable predictive screening tool to evaluate the toxicity of many potentially harmful emerging substances, alone or in mixtures, in the framework of future regulatory reinforcements.

Conformational dynamics of individual antibodies using computational docking and AFM.

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen-binding fragments and one crystallizable fragment using a surface-constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X-ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X-ray structure consequence of the antibody flexibility. We conclude that high-resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica.

Nanoscale structural features determined by AFM for single virus particles.

In this work, we propose "single-image analysis", as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis.

Subcellular localization and interaction network of the mRNA decay activator Pat1 upon UV stress.

To identify nucleo-cytoplasmic shuttle proteins that relocate to the nucleus upon UV stress, we selected 18 targets on the basis of their conservation amongst eukaryotes and their relatively poor functional description. Their relocation was assayed using quantitative nuclear relocation assay (QNR). We focused on Pat1, a component of the cytoplasmic foci called processing bodies (p-bodies), because it had the strongest response to the stress. We verified that Pat1 accumulates in the nucleus after GFP tagging and fluorescence microscopy. Using tandem affinity purification coupled to a mass spectrometry shotgun detection and quantitation approach, we explored the dynamics of Pat1 protein-protein interaction network after UV stress. We have shown that Pat1 co-purifies with Dhh1 specifically upon UV stress. We observed that the nuclear accumulation of Pat1 upon UV stress is abolished in a dhh1∆ strain. These data provide the first evidence that Dhh1 is required for Pat1 nuclear relocation after UV stress.

Computational reconstruction of multidomain proteins using atomic force microscopy data.

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.

Pilot in vivo toxicological investigation of boron nitride nanotubes.

Boron nitride nanotubes (BNNTs) have attracted huge attention in many different research fields thanks to their outstanding chemical and physical properties. During recent years, our group has pioneered the use of BNNTs for biomedical applications, first of all assessing their in vitro cytocompatibility on many different cell lines. At this point, in vivo investigations are necessary before proceeding toward realistic developments of the proposed applications. In this communication, we report a pilot toxicological study of BNNTs in rabbits. Animals were injected with a 1 mg/kg BNNT solution and blood tests were performed up to 72 hours after injection. The analyses aimed at evaluating any acute alteration of hematic parameters that could represent evidence of functional impairment in blood, liver, and kidneys. Even if preliminary, the data are highly promising, as they showed no adverse effects on all the evaluated parameters, and therefore suggest the possibility of the realistic application of BNNTs in the biomedical field.

Single and multiple bonds in (strept)avidin-biotin interactions.

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

Tobacco mosaic virus as an AFM tip calibrator.

The study of high-resolution topographic surfaces of isolated single molecules is one of the applications of atomic force microscopy (AFM). Since tip-induced distortions are significant in topographic images the exact AFM tip shape must be known in order to correct dilated AFM height images using mathematical morphology operators. In this work, we present a protocol to estimate the AFM tip apex radius using tobacco mosaic virus (TMV) particles. Among the many advantages of TMV, are its non-abrasivity, thermal stability, bio-compatibility with other isolated single molecules and stability when deposited on divalent ion pretreated mica. Compared to previous calibration systems, the advantage of using TMV resides in our detailed knowledge of the atomic structure of the entire rod-shaped particle. This property makes it possible to interpret AFM height images in term of the three-dimensional structure of TMV. Results obtained in this study show that when a low imaging force is used, the tip is sensing viral protein loops whereas at higher imaging force the tip is sensing the TMV particle core. The known size of the TMV particle allowed us to develop a tip-size estimation protocol which permits the successful erosion of tip-convoluted AFM height images. Our data shows that the TMV particle is a well-adapted calibrator for AFM tips for imaging single isolated biomolecules. The procedure developed in this study is easily applicable to any other spherical viral particles.

Deciphering the energy landscape of the interaction uranyl-DCP with antibodies using dynamic force spectroscopy.

Previous studies on molecular recognition of uranyl-DCP (dicarboxy-phenanthroline chelator) compound by two distinct monoclonal antibodies (Mabs U04S and U08S) clearly showed the presence of a biphasic shape in Bell-Evans' plots and an accentuated difference in slopes at the high loading rates. To further explore the basis in the slope difference, we have performed complementary experiments using antibody PHE03S, raised against uranyl-DCP but, presenting a strong cross-reactivity toward the DCP chelator. This work allowed us to obtain a reallocation of the respective contributions of the metal ion itself and that of the chelator. Results led us to propose a 2D schematic model representing two energy barriers observed in the systems Mabs U04S- and U08S-[UO(2)-DCP] where the outer barrier characterizes the interaction between UO(2) and Mab whereas the inner barrier characterizes the interaction between DCP and Mab. Using dynamic force spectroscopy, it is thus possible to dissect molecular interactions during the unbinding between proteins and ligands.

On molecular recognition of an uranyl chelate by monoclonal antibodies.

The energy landscape of the uranyl (UO2) chelate dissociated from a monoclonal antibody U08S was investigated using dynamic force spectroscopy (DFS). The uranyl ion (UO2(2+)) is chelated with the ligand dicarboxy-phenanthroline (DCP). The monoclonal antibody U08S was raised against UO2-DCP and does not cross-react with DCP alone. The results of plotting the most probable force against the logarithm of the loading rate show two distinguished values of slopes of multiple fitting lines, as observed in our previous study on that system with monoclonal antibody U04S (Odorico et al., 2007a. Biophys. J. 93: 645-654.). It indicates an unbinding process undergoing at least two activation states. We have generated the histogram of unbinding events with respect to the composite stiffness of the complex between the protein and the uranyl compound. Combining the model of Bell and Evans with that of Williams, we have estimated the number of parallel bonds involved in the unbinding process and determined the value of stiffness for individual bonds. We propose that the uranyl compound binds to the two antibodies U04S and U0c at structurally equivalent locations and forms the interaction with similar coordination modes. In addition, the unbinding process goes through two steps; the first weakens the bonding of the central metal with AspL50 of the antibody and the second breaks other non-bonded interactions of the compound with the antibody.

Surface response methodology for the study of supported membrane formation.

We report on the investigations of the formation of the tethered lipid bilayer by vesicle deposition on amine-functionalized surfaces. The tethered bilayer was created by the deposition of egg-PC vesicles containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide as anchoring molecules on an amine-coated surface. This approach is an easy route for the formation of a biomimetic-supported membrane. A Doelhert experimental design was applied to determine the conditions leading to the formation of a continuous and defect-free tethered bilayer on different surfaces (gold and glass). Doehlert designs allow modeling of the experimental responses by second-order polynomial equations as a function of experimental factors. Four factors expected to influence bilayer formation were studied: the lipid concentration in the vesicle suspension, the mass percentage of anchoring molecules in the vesicles, the contact time between the vesicles and the surface, and the resting time of the membrane after buffer rinse. The optimization of the membrane preparation parameters was achieved by monitoring lipid assembly formation using surface plasmon resonance spectroscopy on gold and by fluorescence recovery after photobleaching on glass. Three characteristic responses were systematically measured: the bilayer thickness, the lipid diffusion coefficient, and the lipid mobile fraction. The simultaneous inspection of the three characteristics revealed that a restricted experimental domain leads to properties that are in accordance with a bilayer presence. The factors of this domain are a lipid concentration from 0.1 to 1 mg/mL, 4-8% of anchoring molecules in the vesicles, 1-4 h of contact time between vesicles and surface, and 21-24 h of resting time after buffer rinse. Under these conditions, a membrane having a lipid mass per surface between 545 +/- 5 and 590 +/- 10 ng/cm2, a diffusion coefficient of between 2.5 +/- 0.3 x 10(-8) and 3.60 +/- 0.5 x 10(-8) cm2/s, and a mobile fraction between 94 +/- 2 and 99 +/- 1% was formed. These findings were confirmed by atomic force microscopy observations, which showed the presence of a continuous and homogeneous bilayer in the determined experimental domain. This formation procedure presents many advantages; it provides an easily obtainable biomimetic membrane model for proteins studies and offers a versatile tethered bilayer because it can be adapted easily to various types of supports.