In situ mapping of biomineral skeletal proteins by molecular recognition imaging with antibody-functionalized AFM tips.

Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.

One-step electrochemical elaboration of SnO2 modified electrode for lead ion trace detection in drinking water using SWASV.

A facile method was proposed for the elaboration of an electrochemical sensor for heavy metal's trace detection by using square wave anodic stripping voltammetry (SWASV); this method is based on a simple anodic conversion of tin electrode into Sn/SnO2 modified electrode. Both electrochemical and physico-chemical techniques were used to confirm the modification process and better understand the electrode's behavior. Then, depending on the operating conditions, the response signal was studied and adjusted in order to obtain optimal sensor performance. When optimized, the proposed method reached a lowest detection limit (LOD) of 2.15 µg L-1 (0.0104 µM), and quantification limit (LOQ) of 5.36 µg L-1 (0.0259 µM), in linearity range between from 6.2 and 20.7 µg L-1. Additionally, after having used the elaborated electrode for ten successive measurements, the repeatability remains very high with an RSD of approximately 5.3%; furthermore, ten other species appear to have very slight effect on Pb(II) detection. Finally, for the method validation, the proposed electrode was able to sense different lead concentration integrated in a local bottled spring water by showing recovery levels ranging from 103.8 to 108.4%.

New insight on spatial localization and microstructures of calcite-aragonite interfaces in adult shell of Haliotis tuberculata: Investigations of wild and farmed abalones by FTIR and Raman mapping.

In the present study, we investigated the shell microstructures of the gastropod European abalone Haliotis tuberculata in order to clarify the complex spatial distribution of the different mineral phases. Our studies were carried out with a standardized methodology on thirty adult European abalone H. tuberculata (5-6 cm long) composed of 15 wild individuals and 15 individuals taken from the France Haliotis hatchery. The macroscopic (binocular) and microscopic observations coupled with Fourier Transform Infrared Spectroscopy (FTIR) and Raman vibrational analysis allowed to unambiguously detect, identify and localize calcite and aragonite. For the first time it has been shown that calcite is present in 100% of farmed and wild adult shell. The microstructural details of the calcite-aragonite interfaces were revealed by using both confocal micro-Raman mapping and Scanning Electron Microscopy (SEM) observations. Calcite zones are systematically found in the spherulitic layer without direct contact with the nacreous layer. The calcite area - nacreous layer interface is made of a thin spherulitic layer with variable thickness from a few micrometers to several millimeters. In order to contribute to a better understanding of the biomineralization process, a model explaining the hierarchical arrangement of the different phases of calcium carbonate is presented and discussed. Finally, it has been shown that these calcitic zones can be connected to each other within the shells and that their spatial distributions correspond to streaks perpendicular to the direction of length growth.

Mud-Based Construction Material: Promising Properties of French Gravel Wash Mud Mixed with Byproducts, Seashells and Fly Ash as a Binder.

The French gravel industry produces approximatively 6.5 million tons of gravel wash mud each year. This material offers very promising properties which require an in-depth characterization study before its use as a construction material, otherwise it is removed from value cycles by disposal in landfills. We examined the suitability of gravel wash mud and seashells, with fly ash as a binder, as an unfired earth construction material. Thermal and mechanical characterizations of the smart mixture composed of gravel wash mud, Crepidula fornicata shells and fly ash are performed. The new specimens exhibit high compressive strengths compared to usual earth construction materials, which appears as a good opportunity for a reduction in the thickness of walls. The use of fly ash and Crepidula shells in addition to gravel wash mud provides high silica and calcium contents, which both react with clay, leading to the formation of tobermorite and Al-tobermorite as a result of a pozzolanic reaction. Considering the reduction in porosity and improvements in strength, these new materials are good candidates to contribute significantly to the Sustainable Development Goals (SDGs) and reduce carbon emissions.

Haslea silbo, A Novel Cosmopolitan Species of Blue Diatoms.

Specimens of a new species of blue diatoms from the genus Haslea Simonsen were discovered in geographically distant sampling sites, first in the Canary Archipelago, then North Carolina, Gulf of Naples, the Croatian South Adriatic Sea, and Turkish coast of the Eastern Mediterranean Sea. An exhaustive characterization of these specimens, using a combined morphological and genomic approach led to the conclusion that they belong to a single new to science cosmopolitan species, Haslea silbo sp. nov. A preliminary characterization of its blue pigment shows similarities to marennine produced by Haslea ostrearia, as evidenced by UV-visible spectrophotometry and Raman spectrometry. Life cycle stages including auxosporulation were also observed, providing data on the cardinal points of this species. For the two most geographically distant populations (North Carolina and East Mediterranean), complete mitochondrial and plastid genomes were sequenced. The mitogenomes of both strains share a rare atp6 pseudogene, but the number, nature, and positions of the group II introns inside its cox1 gene differ between the two populations. There are also two pairs of genes fused in single ORFs. The plastid genomes are characterized by large regions of recombination with plasmid DNA, which are in both cases located between the ycf35 and psbA genes, but whose content differs between the strains. The two sequenced strains hosts three plasmids coding for putative serine recombinase protein whose sequences are compared, and four out of six of these plasmids were highly conserved.

Electrochromic Properties and Electrochemical Behavior of Marennine, a Bioactive Blue-Green Pigment Produced by the Marine Diatom Haslea ostrearia.

Marennine has long been known as the unique peculiar pigment responsible for the natural greening of oysters. It is specifically produced by the marine diatom Haslea ostrearia and it is a natural blue molecule indeed promising for food industry because of the rarity of such non-toxic, blue-colored pigments. In the search for its still not defined molecular structure, investigation of the color changes with the redox state has been carried out combining different approaches. Reducing and oxidizing chemicals have been added to purified marennine solutions and a stable blue-green color has been confirmed for the oxidized state, while a yellow color corresponded to the reduced unstable state. Raman spectroscopy has been used to monitor changes in the Raman spectra corresponding to the different colored states, and cyclic voltammetry has allowed the detection of a redox system in which protons and electrons are exchanged. These findings show that marennine is a suitable stable blue pigment for use in food applications and help in the elucidation of the chromophore structure.

Organic-inorganic interactions revealed by Raman spectroscopy during reversible phase transitions in semiconducting [(C2H5)4N]FeCl4.

The alkylammonium halogenoferrate families are subjected to diverse studies according to their wide field application. However, these compounds show various transitions depending on the preparation process. In this paper, the [(C2H5)4N]FeCl4 compound was successfully synthesized using a slow evaporation solution growth method at room temperature. An optical absorption measurement confirms the semiconductor nature with a band gap around 2.95 eV. The X-ray powder diffraction (XRPD) data confirmed the formation of a single-phase with hexagonal-type structure. The differential scanning calorimetry (DSC) indicated that the [(C2H5)4N]FeCl4 compound undergoes eight reversible phase transitions between 193 and 443 K. At high temperature (T > 423 K) the plastic nature of the crystals was confirmed. Temperature-controlled X-ray diffraction reveals that the thermal expansion of the crystal structure is non homothetic in the (a,b) plane and along the c axis. The temperature dependence of the Raman spectra up to 443 K revealed specific reorientations and molecular displacements of the organic and inorganic components associated with the phase transitions. We aim to thermally stabilize the [(C2H5)4N]FeCl4 compound which has a band gap suitable for photocatalytic processes.

Insight into the structural, elastic and electronic properties of a new orthorhombic 6O-SiC polytype.

Different polytypes of SiC are described and predicted in literature. Here, we report the first occurrence of an orthorhombic 6O-SiC polytype as rock-forming mineral in the nickel laterite mine of Tiebaghi (New Caledonia). This new class of SiC crystallizes in the space group Cmc21 with 12 atoms per unit cell [a = 3.0778(6) Å, b = 5.335(2) Å, c = 15.1219(6) Å, α = 90°, ß = 90°, γ = 120°]. The density of 6O-SiC is about 3.22 g/cm3 and the calculated indirect bandgap at room temperature of 3.56 eV is identical to 6H-SiC. Our results suggest that 6O-SiC is the intermediate state in the wurtzite to rocksalt transformation of 6H-SiC.

Raman Open Database: first interconnected Raman-X-ray diffraction open-access resource for material identification.

Detailed crystallographic information provided by X-ray diffraction (XRD) is complementary to molecular information provided by Raman spectroscopy. Accordingly, the combined use of these techniques allows the identification of an unknown compound without ambiguity. However, a full combination of Raman and XRD results requires an appropriate and reliable reference database with complete information. This is already available for XRD. The main objective of this paper is to introduce and describe the recently developed Raman Open Database (ROD, http://solsa.crystallography.net/rod). It comprises a collection of high-quality uncorrected Raman spectra. The novelty of this database is its interconnectedness with other open databases like the Crystallography Open Database (http://www.crystallography.net/cod and Theoretical Crystallography Open Database (http://www.crystallography.net/tcod/). The syntax adopted to format entries in the ROD is based on the worldwide recognized and used CIF format, which offers a simple way for data exchange, writing and description. ROD also uses JCAMP-DX files as an alternative format for submitted spectra. JCAMP-DX files are compatible to varying degrees with most commercial Raman software and can be read and edited using standard text editors.

Antibiofilm Activity of Polyamide 11 Modified with Thermally Stable Polymeric Biocide Polyhexamethylene Guanidine 2-Naphtalenesulfonate.

The choice of efficient antimicrobial additives for polyamide resins is very difficult because of their high processing temperatures of up to 300 °C. In this study, a new, thermally stable polymeric biocide, polyhexamethylene guanidine 2-naphtalenesulfonate (PHMG-NS), was synthesised. According to thermogravimetric analysis, PHMG-NS has a thermal degradation point of 357 °C, confirming its potential use in joint melt processing with polyamide resins. Polyamide 11 (PA-11) films containing 5, 7 and 10 wt% of PHMG-NS were prepared by compression molding and subsequently characterised by FTIR spectroscopy. The surface properties were evaluated both by contact angle, and contactless induction. The incorporation of 10 wt% of PHMG-NS into PA-11 films was found to increase the positive surface charge density by almost two orders of magnitude. PA-11/PHMG-NS composites were found to have a thermal decomposition point at about 400 °C. Mechanical testing showed no change of the tensile strength of polyamide films containing PHMG-NS up to 7 wt%. Antibiofilm activity against the opportunistic bacteria Staphylococcus aureus and Escherichia coli was demonstrated for films containing 7 or 10 wt% of PHMG-NS, through a local biocide effect possibly based on an influence on the bacterial eDNA. The biocide hardly leached from the PA-11 matrix into water, at a rate of less than 1% from its total content for 21 days.

Promising silicones modified with cationic biocides for the development of antimicrobial medical devices.

We have tested silicones containing 2% or 5% of the cationic biocides polyhexamethylene guanidine dodecylbenzenesulfonate (PHMG-DBS), 1-octyl-3-methylimidazolium tetrafluoroborate (OMIM-BF4) or 1-dodecyl-3-methylimidazolium tetrafluoroborate (DMIM-BF4) against the major relevant bacterial and yeast species in health care-associated infections (HCAI). Study conducted according to the international standard ISO 22196 revealed that silicones containing 2% or 5% DMIM-BF4 or 5% PHMG-DBS presented the highest antimicrobial activity, leading to a logarithmic growth reduction of 3.03 to 6.46 and 3.65 to 4.85 depending on the bacterial or fungal species. Heat-pretreated silicones containing 2% DMIM-BF4 kept a high activity, with at least a 3-log reduction in bacterial growth, except against P. aeruginosa where there was only a 1.1-log reduction. After 33days, the release ratio of cationic biocide from silicone films containing 5% of DMIM-BF4 was found to be 5.6% in pure water and 1.9% in physiological saline solution, respectively. No leaching of PHMG-DBS polymeric biocide was detected under the same conditions. These results demonstrate unambiguously that silicones containing 2% DMIM-BF4 or 5% PHMG-DBS present high antimicrobial activity, as well as high leaching resistance and therefore may be good candidates for the development of safer medical devices.

New promising antifouling agent based on polymeric biocide polyhexamethylene guanidine molybdate.

A new polymeric biocide polyhexamethylene guanidine (PHMG) molybdate has been synthesized. The obtained cationic polymer has limited water solubility of 0.015 g/100 mL and is insoluble in paint solvents. The results of acute toxicity studies indicate moderate toxicity of PHMG molybdate, which has a median lethal dose at 48 h of 0.7 mg/L for Daphnia magna and at 96 h of 17 mg/L for Danio rerio (zebrafish) freshwater model organisms. Commercial ship paint was then modified by the addition of a low concentration of polymeric biocide 5% (w/w). The painted steel panels were kept in Dnipro River water for the evaluation of the dynamics of fouling biomass. After 129-d exposure, Bryozoa dominated in biofouling of tested substrates, forming 86% (649 g/m2 ) of the total biomass on control panel surfaces. However, considerably lower Bryozoa fouling biomass (15 g/m2 ) was detected for coatings containing PHMG molybdate. Dreissenidae mollusks were found to form 88% (2182 g/m2 ) of the fouling biomass on the control substrates after 228 d of exposure, whereas coatings containing PHMG molybdate showed a much lower biomass value of 23.6 g/m2 . The leaching rate of PHMG molybdate in water was found to be similar to rates for conventional booster biocides ranging from 5.7 µg/cm2 /d at the initial stage to 2.2 µg/cm2 /d at steady state. Environ Toxicol Chem 2017;36:2543-2551. © 2017 SETAC.

Structural behavior of laser-irradiated γ-Fe2O3 nanocrystals dispersed in porous silica matrix : γ-Fe2O3 to α-Fe2O3 phase transition and formation of ε-Fe2O3.

The effects of laser irradiation on γ-Fe2O3 4 ± 1 nm diameter maghemite nanocrystals synthesized by co-precipitation and dispersed into an amorphous silica matrix by sol-gel methods have been investigated as function of iron oxide mass fraction. The structural properties of γ-Fe2O3 phase were carefully examined by X-ray diffraction and transmission electron microscopy. It has been shown that γ-Fe2O3 nanocrystals are isolated from each other and uniformly dispersed in silica matrix. The phase stability of maghemite nanocrystals was examined in situ under laser irradiation by Raman spectroscopy and compared with that resulting from heat treatment by X-ray diffraction. It was concluded that ε-Fe2O3 is an intermediate phase between γ-Fe2O3 and α-Fe2O3 and a series of distinct Raman vibrational bands were identified with the ε-Fe2O3 phase. The structural transformation of γ-Fe2O3 into α-Fe2O3 occurs either directly or via ε-Fe2O3, depending on the rate of nanocrystal agglomeration, the concentration of iron oxide in the nanocomposite and the properties of silica matrix. A phase diagram is established as a function of laser power density and concentration.

Experimental evidence of ultrathin polymer film stratification by AFM force spectroscopy.

By performing Atomic Force Microscopy measurements of pull-off force as a function of the temperature, we were able to probe the dynamic of supported thin polystyrene (PS) films. Thermal transitions induce modifications in the surface energy, roughness and surface modulus that are clearly detected by AFM and related to PS chain relaxation mechanisms. We demonstrated the existence of three transition temperatures that can be associated to the relaxation of polymer chains located at different depth regions within the polymer film. Independently of the film thickness, we have confirmed the presence of a region of high mobility for the polymer chains at the free interface. The thickness of this region is estimated to be above 7nm. The detection of a transition only present for film thicker than the gyration radius Rg is linked to the dynamics of polymer chains in a bulk conformation (i.e. not in contact with the free interface). We claim here that our results demonstrate, in agreement with other techniques, the stratification of thin polymer film depth profile in terms of relaxation behavior.

Density functional theory calculations on azobenzene derivatives: a comparative study of functional group effect.

Density functional theory (DFT) calculations have been used to investigate the structural properties, dipole moments, polarizabilities, Gibbs energies, hardness, electronegativity, HOMO/LUMO energies, and chemical potentials of trans and cis configurations of eight para-substituted azobenzene derivatives. All properties have been obtained using the B3LYP functional and 6-31++G(d,p) basis set. The planar structures have been obtained for all optimized trans configurations. The energy difference between trans and cis configurations for considered derivatives was found to be between 64.2-73.1 kJ/mole. It has been obtained that the p-aminodiazo-benzene (ADAB) has the difference in the dipole moments between trans and cis forms higher than for trans and cis azobenzene.

Quantification of the dissolved inorganic carbon species and of the pH of alkaline solutions exposed to CO2 under pressure: a novel approach by Raman scattering.

Dissolved inorganic carbon (DIC) content of aqueous systems is a key function of the pH, of the total alkanility (TA), and of the partial pressure of CO2. However, common analytical techniques used to determine the DIC content in water are unable to operate under high CO2 pressure. Here, we propose to use Raman spectroscopy as a novel alternative to discriminate and quantitatively monitor the three dissolved inorganic carbon species CO2(aq), HCO3(-), and CO3(2-) of alkaline solutions under high CO2 pressure (from P = 0 to 250 bar at T = 40 °C). In addition, we demonstrate that the pH values can be extracted from the molalities of CO2(aq) and HCO3(-). The results are in very good agreement with those obtained from direct spectrophotometric measurements using colored indicators. This novel method presents the great advantage over high pressure conventional techniques of not using breakable electrodes or reference additives and appears of great interest especially in marine biogeochemistry, in carbon capture and storage and in material engineering under high CO2 pressure.

Marennine, promising blue pigments from a widespread Haslea diatom species complex.

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.

Preparation techniques alter the mineral and organic fractions of fish otoliths: insights using Raman micro-spectrometry.

The high spatial resolution analysis of the mineral and organic composition of otoliths using Raman micro-spectrometry involves rigorous protocols for sample preparation previously established for microchemistry and trace elements analyses. These protocols often include otolith embedding in chemically neutral resin (i.e., resins which do not contain, in detectable concentration, elements usually sought in the otoliths). Such embedding may however induce organic contamination. In this paper, Raman micro-spectrometry reveals the presence of organic contamination onto the surface obtained from the use of epoxy resin, specifically Araldite. This contamination level varies depending on otolith structures. Core and checks, known as structural discontinuities, exhibit the most important level of contaminations. Our results suggest that otolith embedding with resin affects the organic matrix of the otolith, probably through an infiltration of the resin in the crystalline structure. The interpretation of chemical otolith signatures, especially Raman otolith signatures, and stable isotope analyses should then be revised in light of these results. In this respect, we propose a method for the correction of Raman otolith signatures for contamination effects.

Patterning surface by site selective capture of biopolymer hydrogel beads.

This communication describes the fabrication of microstructured biopolymer surfaces by the site-selective capture of pectin hydrogel beads. A positively charged surface consisting of poly-L-lysine (PLL) was subjected to site-selective enzymatic degradation using patterned polydimethylsiloxane (PDMS) stamps covalently modified with trypsin, according to the recently described method. The patterned surface was used to capture ionically cross-linked pectin beads. The desired patterning of the hydrogel surfaces was generated by site-selective immobilization of these pectin beads. The ability of the hydrogels to be dried and swollen in water was assessed.

Site-selective surface modification using enzymatic soft lithography.

Surface modification with functional polymers or molecules offers great promise for the development of smart materials and applications. Here, we describe a versatile and easy-to-use method of site-selective surface modification based on the ease of microcontact printing and the exquisite selectivity of enzymatic degradation. A micropatterned poly-L-lysine (PLL) layer on solid substrates was prepared by enzymatic degradation using trypsin enzyme immobilized on a prestructured poly(dimethlylsiloxane) (PDMS) stamp. After the enzymatic degradation of PLL and the removal of the degradation products, very well defined patterning was revealed over a large scale by fluorescence microscopy and atomic force microscopy (AFM). We investigate the advantage of our method by comparison with traditional microcontact printing and found that lateral diffusion was reduced, yielding a more accurate reproduction of the master. We also demonstrate that the stamp can be reused without reinking. The patterned surface was used for site-selective modification. The strategy was applied to two applications: the first is dedicated to the creation of amino-silane patterned surfaces, and the second illustrates the possibility of patterning polyelectrolyte multilayered thin films.