How to Increase Adhesion Strength of Catechol Polymers to Wet Inorganic Surfaces.

Mussel wet adhesion is known for its outstanding strength on a variety of surfaces. On the basis of the hypothesis that 3,4-dihydroxyphenylalanine, a catecholic amino acid, governs mussel adhesion, chemists have put much effort into the design of adhesive synthetic polymers containing catechols. However, the exceptional properties exhibited by the native proteins were hardly captured. The attempts to make those polymers stick to wet inorganic surfaces resulted in low adhesive forces. Here we synthesized poly(dopamine acrylamide) and measured the interaction forces with various inorganic surfaces using atomic force microscopy-based single-molecule force spectroscopy. We show that hydroxylation of the surface plays a pivotal role on the formation of strong bonds. We demonstrate that depending on the conditions, the whole range of interactions, from weak interactions to covalent bonds, can come into play.

Mechanisms of Heparin-Induced Tau Aggregation Revealed by a Single Nanopore.

Protein aggregation is involved in many diseases, including Parkinson's and Alzheimer's. The latter is characterized by intraneuronal deposition of amyloid aggregates composed of the tau protein. Although large and insoluble aggregates are typically found in affected brains, intermediate soluble oligomers are thought to represent crucial species for toxicity and spreading. Nanopore sensors constitute an emerging technology that allows the detection of the size and populations of molecular assembly present in a sample. Here, we employed conical nanopores to obtain the particle distributions during tau aggregation. We identified three distinct populations, monomers, oligomers, and fibrils, which we could quantify along the aggregation process. By comparing tau wild type with a mutant carrying the disease-associated P301L mutation, we showed that the latter mutation promotes the formation of oligomers. We furthermore highlighted that the P301L mutation promotes fibril breakage. This work demonstrates that conical nanopore is a powerful tool to measure and quantify transient protein aggregate intermediates.

Amyloid Growth, Inhibition, and Real-Time Enzymatic Degradation Revealed with Single Conical Nanopore.

Amyloid fibrils are involved in several neurodegenerative diseases. However, because of their polymorphism and low concentration, they are challenging to assess in real-time with conventional techniques. Here, we present a new approach for the characterization of the intermediates: protofibrils and "end-off" aggregates which are produced during the amyloid formation. To do so, we have fashioned conical track-etched nanopores that are functionalized to prevent the fouling. Using these nanopores, we have followed the kinetic of amyloid growth to discriminate the different intermediates protofibrils and "end-off. Then, the nanopore was used to characterize the effect of promoter and inhibitor of the fibrillation process. Finally, we have followed in real-time the degradation of amyloid with peptase. Compare with the SiN nanopore, the track-etched one features exceptionally high success rate via functionalization and detection in "one-pot". Our results demonstrate the potential for a conical nanopore to be used as a routine technique for the characterization of the amyloid growth and/or degradation.

Unexpected Hard Protein Behavior of BSA on Gold Nanoparticle Caused by Resveratrol.

The understanding of the interactions between nanomaterials, biomolecules, and polyphenols is fundamental in food chemistry, toxicology, and new emerging fields, such as nanomedicine. Here, we investigated the effect of the resveratrol, a principal actor in drug-delivery application on the interaction between bovine serum albumin (BSA), employed as a vector for the delivery of polyphenol drugs, and gold nanoparticle (gNP), the most promising tool in theranostic applications. Through a combination of experimental techniques, which includes an initial evaluation by dynamic light scattering and surface plasmon resonance spectroscopy, we were able to evaluate the evolution of the gold nanoparticle aggregation with increasing ionic strength and the consequences of the BSA and resveratrol addition. To investigate the mechanisms of the interactions, we pursued at the single-molecule level using solid-state nanopore and fluorescence correlation spectroscopy. Our results show that without resveratrol, the BSA is adsorbed on the gNP in water or saline solution. In the presence of resveratrol, the BSA is normally absorbed on gNP in water, but the salt addition leads to its desorption. The resveratrol clearly plays a fundamental role, changing the protein behavior and making the BSA adsorption a reversible process in the presence of salt.

Orienting proteins by nanostructured surfaces: evidence of a curvature-driven geometrical resonance.

Experimental and theoretical reports have shown that nanostructured surfaces have a dramatic effect on the amount of protein adsorbed and the conformational state and, in turn, on the performances of the related devices in tissue engineering strategies. Here we report an innovative method to prepare silica-based nanostructured surfaces with a reproducible, well-defined local curvature, consisting of ordered hexagonally packed arrays of curved hemispheres, from nanoparticles of different diameters (respectively 147 nm, 235 nm and 403 nm). The nanostructured surfaces have been made chemically homogeneous by partially embedding silica nanoparticles in poly(hydroxymethylsiloxane) films, further modified by means of UV-O3 treatments. This paper has been focused on the experimental and theoretical study of laminin, taken as a model protein, to study the nanocurvature effects on the protein configuration at nanostructured surfaces. A simple model, based on the interplay of electrostatic interactions between the charged terminal domains of laminin and the nanocurved charged surfaces, closely reproduces the experimental findings. In particular, the model suggests that nanocurvature drives the orientation of rigid proteins by means of a "geometrical resonance" effect, involving the matching of dimensions, charge distribution and spatial arrangement of both adsorbed molecules and adsorbent nanostructures. Overall, the results pave the way to unravel the nanostructured surface effects on the intra- and inter-molecular organization processes of proteins.

Metal alloy solid-state nanopores for single nanoparticle detection.

Solid-state nanopore technology for nanoparticle sensing is considered for the development of analytical tools to characterise their size, shape or zeta potential. In this field, it is crucial to understand how the nanopore inner surface influences the dynamic of nanoparticle translocation. Here, three single nanopores directly drilled in metal alloys (titanium nitride, titanium-tantalum and tantalum) are considered. The translocation of polystyrene nanoparticles coated with ssDNA is investigated by the resistive pulse method at different concentrations and voltages. The results show that the nanoparticle energy barrier for entrance into the pore decreases for nanopores that exhibits a higher surface energy and hydrophilicity, while the dwell time is found to depend on the nanopore surface state. Overall, this study demonstrates that the control of nanopore surface state must be taken into account for the resistive pulse experiments for nanoparticle detection.

Probing the Cleaning of Polymeric Coatings by Nanostructured Fluids: A QCM-D Study.

Complex fluids composed of water, an organic solvent, and a surfactant have been recently employed as cleaning systems to remove hydrophobic materials, such as polymeric coatings, from solid surfaces. The simultaneous presence of surfactants and an organic solvent with good affinity for the polymer was proven necessary for the polymer's removal, but the comprehension of the cleaning mechanism is poorly understood. In this Article, we investigated the mechanism of removal, highlighting the specific role of each component in the interaction with the polymer film. In particular, the results from quartz crystal microbalance with dissipation monitoring (QCM-D) were compared with those obtained by using confocal microscopy to follow in situ the effect of a nanostructured fluid, i.e., a ternary formulation containing water, 2-butanone (MEK) as a good solvent for the polymer, and a nonionic surfactant (C9-11 ethoxylated alcohol, BR) on acrylic copolymer films (Paraloid B72). The results indicate a two-step process: (i) the penetration of the good solvent across the film causes the swelling of the polymer, the weakening of polymer-polymer interactions, and an increase of molecular mobility, followed by (ii) the slow adsorption of amphiphilic aggregates promoting the film detachment from the solid substrate. A different behavior is observed in the presence of similar formulations containing an anionic surfactant (sodium dodecyl sulfate, SDS), where the adsorption of SDS micelles on the surface of the polymeric film hinders solvent access into the polymer layer, rather than promoting its detachment from the solid substrate.

Kinetic discrimination of DNA single-base mutations by localized surface plasmon resonance.

Clinical application of DNA microarrays used for screening of single nucleotide polymorphisms (SNPs) are very important for diagnosis of diseases and appropriate treatment of patients. In this paper localized surface plasmon resonance (LSPR) technique has been used to study the DNA hybridization process for binary solutions of respectively perfectly matching (PM) and single base mismatching (MM) 93-mer ssDNA from KRAS codon 12. 5'-thiol modified 35-mer ssDNA has been linked to the Au nanodisks array as probe with a surface coverage of 2.8±0.1×1012/cm2. Probe's binding properties was investigated in details, obtaining a sensitivity down to 10nMand 13nM, respectively for PM and MM, showing that the hybridization process occurs at a lower rate for MM with respect to PM target. The competitive hybridization is accounted for by an inhibition model, where the non-complementary sequences kinetically hinder the hybridization of the perfect matching sequences, owing to their above mentioned affinity constant differences for the same probe. Accordingly, the single nucleotide polymorphisms can therefore be revealed in a single step and label free mode with high sensitivity and specificity by LSPR measurements.

Serum Protein-Resistant Behavior of Multisite-Bound Poly(ethylene glycol) Chains on Iron Oxide Surfaces.

Recent surveys have shown that the number of nanoparticle-based formulations actually used at a clinical level is significantly lower than that expected a decade ago. One reason for this is that the physicochemical properties of nanoparticles fall short for handling the complexity of biological environments and preventing nonspecific protein adsorption. In this study, we address the issue of the interactions of plasma proteins with polymer-coated surfaces. With this aim, we use a noncovalent grafting-to method to functionalize iron oxide sub-10 nm nanoparticles and iron oxide flat substrates and compare their protein responses. The functionalized copolymers consist of alternating poly(ethylene glycol) (PEG) chains and phosphonic acid grafted on the same backbone. Quartz crystal microbalance with dissipation was used to monitor polymer adsorption kinetics and evaluate the resistance to protein adsorption. On flat substrates, functionalized PEG copolymers adsorb and form a brush in moderate or highly stretched regimes, with densities between 0.15 and 1.5 nm-2. PEG layers using phosphonic acid as linkers exhibit excellent protein resistance. In contrast, layers prepared with carboxylic acid as the grafting agent exhibit mitigated protein responses and layer destructuration. The present study establishes a correlation between the long-term stability of PEG-coated particles in biofluids and the protein resistance of surfaces coated with the same polymers.

Chelating Surfaces for Native State Proteins Patterning: The Human Serum Albumin Case.

The paper reports a new "soft" surface functionalization strategy, based on a highly selective ion metal chelation process. The proposed stepwise methodology implies at first the construction of a monolayer of terpyridine-based thiol (Tpy), whose highly packed structuring has been followed in situ by using quartz crystal microbalance (QCM-D) measurements, showing that the monolayers consist of about 2.7 × 10(14) Tpy/cm(2). Then, the tridentate sites of the each Tpy moiety are employed to partially chelate divalent metal ions, providing an effective platform to anchoring proteins by completing the metal ion coordination with an available site on the protein of interest. We report the case study of the application of the process to the HSA immobilization onto various surfaces, including Tpy-Fe(II) and Tpy-Cu(II) complexes, as well as hydrophilic bare gold substrates and hydrophobic self-assembled Tpy-based monolayers. It is shown that the chelation interaction between Tpy-Cu(II) complexes and HSA produces the highest and most robust HSA immobilization, with an adsorbed mass at the steady state of ∼800 ng/cm(2), with respect to an average adsorption of ∼350 ng/cm(2) for the other surfaces. Furthermore, Cu(II)-chelated surfaces seem to promote a sort of protein "soft" landing, preventing the ubiquitous surface-induced major unfolding and transmitting an orientation information to the protein, owing to the highly specific symmetry coordination of the Tpy-Cu(II)-protein complex. Indeed, the interaction with a specific monoclonal antiboby (anti-HSA) indicated the lack of a significant protein denaturation, while a massive reorientation/denaturation process was found for all the remaining surfaces, including the Tpy-Fe(II) complex. Finally, the metal-ion-dependent HSA immobilization selectivity has been exploited to obtain micropatterned surfaces, based on the strikingly different strength of interaction and stability observed for Fe(II) and Cu(II) complexes.

Laminin adsorption on nanostructures: switching the molecular orientation by local curvature changes.

This work addresses the influence that the nanometric features of biologically relevant surfaces have on the conformation and properties of adsorbed laminin. It was observed that the adsorption kinetics and the nanomorphology of laminin were affected by the change in local curvature of chemically homogeneous nanostructured surfaces. The nanostructured surfaces were prepared by exploiting the self-assembly process of carboxylated polystyrene NPs, with diameters of 45, 109, and 209 nm, onto a polyelectrolyte multilayer formed by alternate deposition of poly(acrylic acid) and poly(allylamine hydrochloride) on gold. The anchored polymeric NPs were finally coated with a homogeneous layer of poly(allylamine hydrochloride), providing three surfaces with different nanometric local curvature. Atomic force microscopy was employed to characterize the relevant morphological parameters of the nanostructured surfaces. Quartz crystal microbalance with dissipation monitoring was employed to determine the adsorbed mass of laminin as well as its adsorption rate as a function of the local surface curvature. A model is proposed to explain the higher and faster laminin adsorption on surfaces with lower local curvature, where a switching of laminin anchoring orientation from a side-on to an end-on geometry can be predicted by a simple curvature-dependent parameter, γ, connecting the average nanostructure height h and the macromolecule radius of gyration R(g). The results provide a framework to understand the dependence of biomolecule orientation on local nanostructure.

Clay and DOPA containing polyelectrolyte multilayer film for imparting anticorrosion properties to galvanized steel.

A facile and green approach is developed to impart remarkable protection against corrosion to galvanized steel. A protecting multilayer film is formed by alternating the deposition of a polycation bearing catechol groups, used as corrosion inhibitors, with clay that induces barrier properties. This coating does not affect the esthetical aspect of the surface and does not release any toxic molecules in the environment.

Controlled density patterning of tolylterpyridine-tagged oligonucleotides.

An efficient surface anchoring strategy of tolylterpyridine-tagged DNA single strands (ssDNA-ttpy) synthesized on gold electrodes is reported. The method is based on exchange reactions between Fe(II)bis-terpyridine complexed SAMs and ssDNA-ttpy, and allows efficient hybrydization of the cDNA strands. Moreover, by using low-current focused ion beam lithography, micropatterned arrays are obtained, homogeneously covered with ssDNA-ttpy. The surface adsorption kinetics of ssDNA-ttpy, as well as its hybridization efficiency, was monitored by in situ quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The effective confinement of the ssDNA-ttpy at the micrometer level has been monitored by time of flight secondary ion mass spectrometry (ToF-SIMS) and ellipsometric surface imaging experiments, providing laterally resolved chemical and topographic mapping.

Fibronectin conformation switch induced by coadsorption with human serum albumin.

The dynamic adsorption of human serum albumin (HSA) and plasma fibronectin (Fn) onto hydrophobic poly(hydroxymethylsiloxane) (PHMS) and the structures of adsorbed protein layers from single and binary protein solutions were studied. Spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation monitoring (QCM-D) together with atomic force microscopy (AFM) were used to measure the effective mass, thickness, viscoelastic properties, and morphology of the adsorbed protein films. Adsorbed HSA formed a rigid, tightly bound monolayer of deformed protein, and Fn adsorption yielded a thick, very viscoelastic layer that was firmly bound to the substrate. The mixed protein layers obtained from the coadsorption of binary equimolecular HSA-Fn solutions were found to be almost exclusively dominated by Fn molecules. Further sequential adsorption experiments showed little evidence of HSA adsorbed onto the predeposited Fn layer (denoted as Fn ≫ HSA), and Fn was not adsorbed onto predeposited HSA (HSA ≫ Fn). The conformational arrangement of the adsorbed Fn was analyzed in terms of the relative availability of two Fn domains. In particular, (4)F(1)·(5)F(1) binding domains in the Hep I fragment, close to the amino terminal of Fn, were targeted using a polyclonal antifibronectin antibody (anti-Fn), and the RGD sequence in the 10th segment, in the central region of the molecule, was tested by cell culture experiments. The results suggested that coadsorption with HSA induced the Fn switch from an open conformation, with the amino terminal subunit oriented toward the solution, to a close conformation, with the Fn central region oriented toward the solution.

Patterning of lactoferrin using functional SAMs of iron complexes.

A new method has been developed that allows spatially resolved adsorption of lactoferrin on a surface, by means of specific non-covalent interaction between the native protein and a patterned self-assembled monolayer of an iron-containing terpyridine complex.