Enzyme-assisted mineralization of calcium phosphate: exploring confinement for the design of highly crystalline nano-objects.

In hard tissues of vertebrates, calcium phosphate (CaP) biomineralization is a fascinating process that combines specific physicochemical and biochemical reactions, resulting in the formation of extracellular matrices with elegant nanoarchitectures. Although several "biomimetic" strategies have been developed for the design of mineralized nanostructured biointerfaces, the control of the crystallization process remains complex. Herein, we report an innovative approach to overcome this challenge by generating, in situ, CaP precursors in a confined medium. For this purpose, we explore a combination of (i) the layer-by-layer assembly, (ii) the template-based method and (iii) the heterogeneous enzymatic catalysis. We show the possibility of embedding active alkaline phosphatase in a nanostructured multilayered film and inducing the nucleation and growth of CaP compounds under different conditions. Importantly, we demonstrate that the modulation of the crystal phase from spheroid-shaped amorphous CaP to crystalline platelet-shaped hydroxyapatite depends on the degree of confinement of active enzymes. This leads to the synthesis of highly anisotropic mineralized nanostructures that are mechanically stable and with controlled dimensions, composition and crystal phase. The present study provides a straightforward, yet powerful, way to design anisotropic nanostructured materials, including a self-supported framework, which may be used in broad biomedical applications.

Calcium phosphate mineralization through homogenous enzymatic catalysis: Investigation of the early stages.

HYPOTHESIS: The crystallization of calcium phosphate (CaP) is a ubiquitous process that occurs in several mineralized tissues and involves a variety of biochemical and chemical reactions. This issue has been hitherto continuously studied from supersaturated solutions (chemical procedure), i.e. by adding calcium and orthophosphate ions in a homogenous phase. Yet, both in vivo and in vitro investigations have clearly shown the implication of enzymes, namely alkaline phosphatase (ALP), to initiate the mineralization process by generating orthophosphate ions. EXPERIMENTS: We report a thorough investigation on the mechanism of enzyme-induced mineralization in homogenous phase (enzymatic procedure). For this purpose, ALP is introduced in Ca2+/Mg2+-containing solution (pH = 7.4; 37 °C), and its activity modulated by the concentration of its substrate. FINDINGS: Results show that after 24 h of mineralization both chemical and enzymatic procedures lead to the formation of well-crystalline hydroxyapatite nano-objects, however with noticeable impact on their shape and dimensions. Remarkably enough, by combining in situ monitoring and ex situ characterizations, we identify several intermediate phases, including amorphous phase, dicalcium phosphate dehydrate phase (DCPD or brushite) and Whitlockite (WH). Besides, mineralized nano-objects with a core-shell structure is observed, and hydroxyapatite platelets are shown to grow on the surface of their shell.

Dual stimuli-responsive coating designed through layer-by-layer assembly of PAA-b-PNIPAM block copolymers for the control of protein adsorption.

In this paper, we describe the successful construction, characteristics and interaction with proteins of stimuli-responsive thin nanostructured films prepared by layer-by-layer (LbL) sequential assembly of PNIPAM-containing polyelectrolytes and PAH. PAA-b-PNIPAM block copolymers were synthesized in order to benefit from (i) the ionizable properties of PAA, to be involved in the LbL assembly, and (ii) the sensitivity of PNIPAM to temperature stimulus. The impact of parameters related to the structure and size of the macromolecules (their molecular weight and the relative degree of polymerization of PAA and PNIPAM), and the interaction with proteins under physico-chemical stimuli, such as pH and temperature, are carefully investigated. The incorporation of PAA-b-PNIPAM into multilayered films is shown to be successful whatever the block copolymer used, resulting in slightly thicker films than the corresponding (PAA/PAH)n film. Importantly, the protein adsorption studies demonstrate that it is possible to alter the adsorption behavior of proteins on (PAA-b-PNIPAM/PAH)n surfaces by varying the temperature and/or the pH of the medium, which seems to be intimately related to two key factors: (i) the ability of PNIPAM units to undergo conformational changes and (ii) the structural changes of the film made of weak polyelectrolytes. The simplicity of construction of these PNIPAM block copolymer-based LbL coatings on a large range of substrates, combined with their highly tunable features, make them ideal candidates to be employed for various biomedical applications requiring the control of protein adsorption.

Direct AFM force mapping of surface nanoscale organization and protein adsorption on an aluminum substrate.

We investigate the nanoscale organization of a superficially hydroxylated Al substrate and its effect on subsequent protein adsorption using atomic force microscopy (AFM). For this purpose we used a mode which allows a direct mapping of a variety of surface properties (adhesion, elasticity, dissipation, etc.) to be probed simultaneously with topographical images. The hydroxylation treatment leads to a drastic modification of the surface morphology, owing to the formation of AlOOH compounds. In air, AFM images revealed the formation of regular nanorod-like structures randomly distributed, inducing the appearance of nanoporous domains on the surface. In buffer solution, prior to the adsorption of proteins, the surface nanoscale organization is preserved, mainly due to the chemical stability of AlOOH compounds under these conditions. The adsorption of proteins on the obtained nanostructured surface was performed using either a globular (ß-lactoglobulin) or a fibrillar (collagen) protein and by modulating the adsorbed amount through the incubation time or the concentration of proteins in solution. At low amounts, collagen adsorbs on the whole surface without preferential localization. The surface topography remains similar to the bare surface, while significant changes were evidenced on adhesion and elasticity maps. This is due to the fact that the surface became adhesive and less stiff, owing to the presence of a soft and hydrated protein layer. By contrast, ß-lactoglobulin tends to diffuse into the nanoporous domains, leading to their filling up, and the surface is blurred with a thick and dense protein layer upon increasing the amount of adsorbed molecules. Our findings demonstrate the interest in using AFM for surface mapping to investigate the mechanism of protein adsorption at the nanoscale on materials with high surface roughness.

Design of mixed PEO/PAA brushes with switchable properties toward protein adsorption.

Adsorption of proteins at interfaces is an ubiquitous phenomenon of prime importance. Layers of poly(ethylene oxide) (PEO) are widely used to repel proteins. Conversely, proteins were shown to adsorb deeply into brushes of poly(acrylic acid) (PAA), and their subsequent partial release could be triggered by a change of pH and/or ionic strength (I). Mixed brushes of these polymers are thus promising candidates to tune protein adsorption onto new smart surfaces. In this work, the synthesis of such mixed brushes was performed based on a "grafting to" approach, the two polymers being either grafted sequentially or simultaneously. Detailed characterization of the obtained brushes using static water contact angle measurements, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and polarization-modulation reflection-absorption infrared spectroscopy is presented. While sequential grafting of the two polymers for different reactions times did not give rise to a broad range of composition of mixed brushes, simultaneous grafting of the polymers from solutions with different compositions allows for the synthesis of a range of mixed brushes (mass fraction of PEO in the mixed brushes from 0.35 to 0.65). A key example is then chosen to illustrate the switchable behavior of a selected mixed PEO/PAA brush toward albumin adsorption. The adsorption behavior was monitored with a quartz crystal microbalance. The mixed brush could adsorb high amounts of albumin, but 86% of the adsorbed protein could then be desorbed upon pH and I change. The obtained properties are thus a combination of the ones of PEO and PAA, and a highly switchable behavior is observed toward protein adsorption.

Review--Interactions between diatoms and stainless steel: focus on biofouling and biocorrosion.

There is a considerable body of information regarding bacterially enhanced corrosion, however, this review focuses on diatoms (unicellular algae) whose contribution to biocorrosion is less well studied. The reasons why diatoms have been neglected in studies of biocorrosion in natural waters are discussed and the question whether diatoms should be considered as inert with respect of electrochemical processes is considered. A particular focus is given to the case of stainless steels (SS), which are widely used in variety of applications in natural waters. Basic information on the cell biology of diatoms is included in the review, particularly with respect to their ability to 'sense' and adhere to surfaces. Investigations at the nanoscale are reviewed as these studies provide information about the behavior of cells at interfaces. Recent advances include the use of atomic force microscopy (AFM), although only a few studies have been applied to diatoms. Regarding the electrochemical behavior of SS, the mechanisms by which diatoms influence the potential ennoblement process is discussed. Such studies reveal the association of diatoms, in addition to bacteria, with biocorrosion processes.

nPEG-TiO2 nanoparticles: a facile route to elaborate nanostructured surfaces for biological applications.

We report the synthesis of diacid-terminated PEG-functionalized cubic TiO(2) nanocrystals by a simple one-step solvothermal method, and their further use to form nanostructured surfaces for protein immobilization. The relevance and major interest of the so-obtained nanocrystals are the presence of terminal carboxylic acid groups at their surface, as confirmed by infrared analyses, in addition to the surrounding PEG chains, essential to avoid non specific interactions. These functional chemical groups were used to (i) immobilize the synthesized nanocubes on a cysteamine-modified Au surface, and to (ii) attach proteins via a presumable covalent link. AFM images show that the shapes and the narrow size distribution of the nanocubes, observed by TEM, were preserved after their immobilization on the modified Au surface. Moreover, the efficiency and specificity of antigen recognition were demonstrated using spectroscopic analyses. Our successful approach provides a versatile and facile way to elaborate specific and sensitive nanostructured surfaces for biosensors.

Growth mechanism of confined polyelectrolyte multilayers in nanoporous templates.

We investigate the mechanism of polyelectrolyte multilayer (PEM) assembly in nanoporous templates with a view to synthesizing nanotubes or nanowires under optimal conditions. For this purpose, we focus on the effect of parameters related to the geometrical constraints (pore diameter), the size of the macromolecules (their molar mass and the ionic strength), and the interaction between the pore walls and the adsorbed chains (modulated by the ionic strength). Our results reveal the existence of two regimes in the mechanism of PEM growth: (i) the first regime is comparable to that observed on flat substrates, including the influence of ionic strength and (ii) the second regime, which is slower in terms of kinetics, results from the interconnection established between polyelectrolyte chains across the pores and leads to the formation of a dense gel. As a consequence, the diffusion of polyelectrolytes in nanopores becomes the controlling factor of PEM growth in this second regime. The dense gel, owing to its peculiar structure, enhances the formation of nanowires or of partially occluded nanotubes in some cases, depending on initial pore dimensions.

Enzymatic approach in microbial-influenced corrosion: a review based on stainless steels in natural waters.

The electrochemical behavior of stainless steels (SS) in natural waters is characterized by the ennoblement of their free corrosion potential (E(corr)). This phenomenon depends strongly on the settlement of biofilms on SS surfaces. Many hypotheses have been proposed to explain the biofilm action, in particular the enzymatic catalysis plays an important role by shifting the cathodic and/or anodic processes. However, there are still only few studies relating the use of purified enzymes. In contrast with bacteria-associated corrosion, the direct influence of enzymes is still poorly documented. The aim of this review is to show the benefits of the enzymatic approach in the study of biocorrosion. Indeed, enzymatic systems may constitute convenient models to mimic microbial influenced corrosion and to evaluate the behavior of metallic materials in natural waters.

Ennoblement of stainless steel in the presence of glucose oxidase: nature and role of interfacial processes.

The ennoblement of the free corrosion potential (E(corr)) of AISI 316L stainless steel which did not occur in synthetic fresh water (SFW), was observed after introduction of glucose oxidase (Gox) and glucose, or of hydrogen peroxide (H(2)O(2)). The composition of the surface was monitored using AFM and XPS, a detailed XPS analysis being based on the discrimination between oxygen of organic and inorganic nature proposed in a previous study. In H(2)O(2) medium, the main changes regarding the inorganic phase were the increase of the oxygen concentration in the passive film, the increase of the molar concentration ratio of oxidized species Fe(ox)/Cr(ox) and the growth of nanoparticles, presumably made of ferric oxide/hydroxide. In Gox medium, no significant changes were observed in both oxygen concentration and Fe(ox)/Cr(ox) ratio, but the density of colloidal particles decreased, indicating a dissolution of Fe oxide/hydroxide under the influence of gluconate. In contrast with H(2)O(2), in SFW and Gox the amount of organic compounds increased due to the accumulation of polysaccharides and proteins. The influence of glucose oxidase on the ennoblement of stainless steel is not due to indirect effects of H(2)O(2) through the change of surface composition. The E(corr) ennoblement seems to be directly due to the presence of H(2)O(2) and to the electrochemical behavior of H(2)O(2) and related oxygen species. This consideration is important for understanding and controlling microbial influenced corrosion.

Evolution of the passive film and organic constituents at the surface of stainless steel immersed in fresh water.

The evolution of the surface of a conventional stainless steel (AISI 316L) immersed in aqueous medium simulating fresh water (pH approximately 8) was studied using XPS and AFM. A detailed analysis of XPS spectra allowed a distinction to be made between oxygen of organic and inorganic nature. During the first 48 h, the main changes concern the inorganic phase: the oxygen concentration in the passive layer increases, owing both to oxidation of metal elements, including conversion of Fe(II) to Fe(III), and to hydration; the molar ratio of oxidized species Fe(ox)/Cr(ox) decreases slightly; the formation of colloidal particles, presumably made of ferric hydroxide, is observed by AFM. After longer periods of immersion, the Fe(ox)/Cr(ox) is higher, while the coating of colloidal particles reaches a full surface coverage. The amount of organic compounds increases further and the XPS spectra reveal the accumulation of polysaccharides and proteins, which indicate that these organic compounds are of biological origin.