An easily transferable protocol for in-situ quasi-non-invasive analysis of protein binders in works of art.

Proteomic approaches based on mass spectrometry have become increasingly popular for protein binder's identification in works of art. The identification of the binder employed may offer key information on paintings and other polychrome objects and contribute to assess their historical and technical context, also providing useful hints for a proper restoration and/or conservation treatment. Usually, the protocols employed to this purpose are invasive and at least micro sampling is required. Here, we present a simple transferable method for a quasi-non-invasive analysis of binders in artworks based on the use of a very small poly (2-hydroxyethyl methacrylate)/poly (vinylpyrrolidone) hydrogel (3 mm × 3 mm) previously loaded with trypsin for the in-situ digestion of proteins and applied onto the objects' surface. Upon extraction of digested peptides from the hydrogel, they were examined by MALDI-TOF-MS and/or LC-ESI-MS/MS. The method was validated on fresh and aged model pictorial layers; optical microscope images, and spectrophotocolorimetry confirmed that neither damage nor color alteration of the painting layer occurred, and no hydrogel residue was left. X-ray photoelectron spectroscopy carried out on paint models confirmed that the treatment with trypsin-loaded gels did not modify the pigment composition, even on aged samples. The protocol was successfully applied to a painting on wood mockup aged thirty years, a statue dated XV century exposed in San Lorenzo church (Bisceglie, Bari, Apulia), and a liturgical scroll Benedictio ignis et fontis (Benedizionale) of the Museo Diocesano of Bari dated eleventh century; in all these objects the proteinaceous binder was readily and successfully identified.

A quasi non-destructive approach for amber geological provenance assessment based on head space solid-phase microextraction gas chromatography-mass spectrometry.

Head space (HS) solid-phase micro-extraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) was used to analyze the volatile fraction of ambers of different geological origin. In particular, Romanian (romanite) and Baltic (succinite) amber samples were studied. Both types of amber have nearly similar bulk chemical compositions and could probably reflect only some differences of paleobiological and/or diagenetic origin. The present study shows that amber head space fingerprint, obtained by SPME/GC-MS, can provide a simple and quasi non-destructive method capable of romanite/succinite differentiation. Among the numerous compounds present in the head space, a number of few informative variables could be selected that were able to differentiate the ambers as demonstrated by Principal Component and Cluster Analysis.

An innovative, easily fabricated, silver nanoparticle-based titanium implant coating: development and analytical characterization.

Microbial colonization and biofilm formation on implanted devices represent an important complication in orthopaedic and dental surgery and may result in implant failure. Controlled release of antibacterial agents directly at the implant site may represent an effective approach to treat these chronic complications. Resistance to conventional antibiotics by pathogenic bacteria has emerged in recent years as a major problem of public health. In order to overcome this problem, non-conventional antimicrobial agents have been under investigation. In this study, polyacrylate-based hydrogel thin coatings have been electrosynthesised on titanium substrates starting from poly(ethylene glycol diacrylate)-co-acrylic acid. Silver nanoparticles (AgNPs) with a narrow size distribution have been synthesized using a "green" procedure and immobilized on Ti implant surfaces exploiting hydrogel coatings' swelling capabilities. The coatings have been characterized by XPS and SEM/EDX, while their silver release performances have been monitored by ICP-MS. The antibacterial activity of these AgNP-modified hydrogel coatings was tested evaluating in vitro inhibition growth of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, among the most common pathogens in orthopaedic infections. Moreover, a preliminary investigation of the biocompatibility of silver-loaded coatings versus MG63 human osteoblast-like cells has been performed. An important point of strength of this paper, in fact, is the concern about the effect of silver species on the surrounding cell system in implanted medical devices. Silver ion release has been properly tuned in order to assure antibacterial activity while preserving osteoblasts' response at the implant interface.

Analytical characterization of laser-generated copper nanoparticles for antibacterial composite food packaging.

A new type of nanomaterial has been developed as antibacterial additive for food packaging applications. This nanocomposite is composed of copper nanoparticles embedded in polylactic acid, combining the antibacterial properties of copper nanoparticles with the biodegradability of the polymer matrix. Metal nanoparticles have been synthesised by means of laser ablation, a rising and easy route to prepare nanostructures without any capping agent in a liquid environment. As prepared, nanoparticle suspensions have been easily mixed to a polymer solution. The resulting hybrid solutions have been deposited by drop casting, thus obtaining self-standing antibacterial packages. All samples have been characterized by UV-Vis spectroscopy, X-ray photoelectron spectroscopy and electro-thermal atomic absorption spectroscopy. Ion release data have been matched with bioactivity tests performed by Japanese Industrial Standard (JIS) method (JIS Z 2801:2000) against Pseudomonas spp., a very common Gram-negative microbial group able to proliferate in processed food.

Fingerprinting of egg and oil binders in painted artworks by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of lipid oxidation by-products.

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based approach was applied for the detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products in extracts of small (50-100 µg) samples obtained from painted artworks. Ageing of test specimens under various conditions, including the presence of different pigments, was preliminarily investigated. During ageing, the TAGs and PLs content decreased, whereas the amount of diglycerides, short-chain oxidative products arising from TAGs and PLs, and oxidized TAGs and PLs components increased. The examination of a series of model paint samples gave a clear indication that specific ions produced by oxidative cleavage of PLs and/or TAGs may be used as markers for egg and drying oil-based binders. Their elemental composition and hypothetical structure are also tentatively proposed. Moreover, the simultaneous presence of egg and oil binders can be easily and unambiguously ascertained through the simultaneous occurrence of the relevant specific markers. The potential of the proposed approach was demonstrated for the first time by the analysis of real samples from a polyptych of Bartolomeo Vivarini (fifteenth century) and a "French school" canvas painting (seventeenth century).

Ciprofloxacin-modified electrosynthesized hydrogel coatings to prevent titanium-implant-associated infections.

New promising and versatile materials for the development of in situ sustained release systems consisting of thin films of either poly(2-hydroxyethyl methacrylate) or a copolymer based on poly(ethylene-glycol diacrylate) and acrylic acid were investigated. These polymers were electrosynthesized directly on titanium substrates and loaded with ciprofloxacin (CIP) either during or after the synthesis step. X-ray photoelectron spectroscopy was used to check the CIP entrapment efficiency as well as its surface availability in the hydrogel films, while high-performance liquid chromatography was employed to assess the release property of the films and to quantify the amount of CIP released by the coatings. These systems were then tested to evaluate the in vitro inhibition of methicillin-resistant Staphylococcus aureus (MRSA) growth. Moreover, a model equation is proposed which can easily correlate the diameter of the inhibition haloes with the amount of antibiotic released. Finally, MG63 human osteoblast-like cells were employed to assess the biocompatibility of CIP-modified hydrogel coatings.

Electrosynthesis of hydrogel films on metal substrates for the development of coatings with tunable drug delivery performances.

Novel polyacrylates-based hydrogel thin films were prepared by electrochemical polymerization, a new method to obtain hydrogels directly onto metal substrates. 2-Hydroxy-ethyl-methacrylate (HEMA), a macromer poly (ethylene-glycol diacrylate) (PEGDA) and PEGDA copolymerized with acrylic acid (AA) were used to obtain hydrogels. The electrosynthesized coatings were characterized by X-ray photoelectron spectroscopy, to assess their surface chemical composition, and by water content determination measurements, to characterize the swelling behavior. In particular, quartz crystal microbalance with dissipation monitoring was used to evaluate the pH-dependency of the swelling for AA-containing hydrogels. Moreover, a model protein (bovine serum albumin) and a model drug (caffeine) were entrapped within the hydrogel coatings during electrosynthesis, to examine the release performances and mechanisms of the electrosynthesized hydrogels. It was observed that all the examined polymers showed significant release properties and, in particular, AA-containing hydrogel films confirmed a strong pH-dependence as expected. These coatings seem to be promising in orthopedic field for in situ drug delivery applications.

Analytical characterization of chitosan nanoparticles for peptide drug delivery applications.

Chitosan-cyclodextrin hybrid nanoparticles (NPs) were obtained by the ionic gelation process in the presence of glutathione (GSH), chosen as a model drug. NPs were characterized by means of transmission electron microscopy and zeta-potential measurements. Furthermore, a detailed X-ray photoelectron spectroscopy study was carried out in both conventional and depth-profile modes. The combination of controlled ion-erosion experiments and a scrupulous curve-fitting approach allowed for the first time the quantitative study of the GSH in-depth distribution in the NPs. NPs were proven to efficiently encapsulate GSH in their inner cores, thus showing promising perspectives as drug carriers.

Analytical investigations of poly(acrylic acid) coatings electrodeposited on titanium-based implants: a versatile approach to biocompatibility enhancement.

A polyacrylic acid film was synthesized on titanium substrates from aqueous solutions via an electroreductive process for the first time. This work was done in order to develop a versatile coating for titanium-based orthopaedic implants that acts as both an effective bioactive surface and an effective anti-corrosion barrier. The chemical structure of the PAA coating was investigated by X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was employed to evaluate the effect of annealing treatment on the morphology of the coatings in terms of their uniformity and porosity. Inductively coupled plasma mass spectrometry was used to measure ion concentrations in ion release tests performed on Ti-6Al-4V sheets modified with PAA coatings (annealed and unannealed). Results indicate that the annealing process produces coatings that possess considerable anti-corrosion performance. Moreover, the availability and the reactivity of the surface carboxylic groups were exploited in order to graft biological molecules onto the PAA-modified titanium implants. The feasibility of the grafting reaction was tested using a single aminoacid residue. A fluorinated aminoacid was selected, and the grafting reaction was monitored both by XPS, using fluorine as a marker element, and via quartz crystal microbalance (QCM) measurements. The success of the grafting reaction opens the door to the synthesis of a wide variety of PAA-based coatings that are functionalized with selected bioactive molecules and promote positive reactions with the biological system interfacing the implant while considerably reducing ion release into surrounding tissues.

Synthesis, analytical characterization and bioactivity of Ag and Cu nanoparticles embedded in poly-vinyl-methyl-ketone films.

The electrosynthesis of copper and silver core-shell nanoparticles (NPs) by the sacrificial anode technique, employing tetraoctylammonium (TOA) salts as base electrolyte for the first time, is described. These surfactants were selected because they combine high NP stabilizing power with useful disinfecting properties. The resulting colloids were mixed with a solution of an inert dispersing polymer and used to prepare nanostructured composite thin films. The morphologies and chemical compositions of the nanomaterials were characterized by Transmission Electron Microscopy (TEM) and X-ray Photoelectron Spectroscopy (XPS). The TEM reveals that the average core diameter of the metal NPs ranges between 1.7 and 6.3 nm, as a function of the nature of the metal and of the electrosynthesis conditions, and does not change significantly upon inclusion in the polymer matrix. An appreciable concentration of the metal is detected on the nanoparticle surface by XPS. High-resolution XP spectra indicate that both copper and silver are present at zero oxidation state in all of the materials (colloids and composite films). This demonstrates the high efficiency of the surfactant at controlling the morphology and the chemical composition of the nanodispersed metal in both the as-synthesized colloid and in the polymeric dispersion. The nanocoatings are shown to exert a marked inhibitory effect on the growth of eukaryote and prokaryote target microrganisms, and experimental evidence of a synergic disinfecting effect due to the surfactant and the nanodispersed metal is provided. On the basis of these stability and bioactivity results, it is clear that Cu-NPs and Ag-NPs are suitable for application in disinfecting or antifouling paint and coating formulations.

Electrosynthesis and analytical characterization of PMMA coatings on titanium substrates as barriers against ion release.

The performance of polyacrylic coatings as barrier films against corrosion of titanium-based orthopaedic implants was investigated. In particular, poly(methyl methacrylate) (PMMA) was electrosynthesized on titanium substrates by electro-reductive processes from aqueous monomer solutions. The obtained PMMA coatings were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The effect of an annealing treatment on the morphology of coatings with respect to uniformity and porosity of films was assessed by scanning electron microscopy (SEM). An inductively coupled plasma-mass spectrometry (ICP-MS) technique was used for ion concentration measurements in ion release tests performed on TiAlV sheets modified with PMMA coatings (annealed and unannealed). Results indicated that the annealing process produces coatings with considerable anticorrosion performances.

Analytical characterization of bioactive fluoropolymer ultra-thin coatings modified by copper nanoparticles.

Copper-fluoropolymer (Cu-CFx) nano-composite films are deposited by dual ion-beam sputtering. The extensive analytical characterization of these layers reveals that inorganic nanoparticles composed of Cu(II) species are evenly dispersed in a branched fluoropolymer matrix. In particular, X-ray photoelectron spectroscopy has been employed to study the surface chemical composition of the material and to assess how it changes on increasing the copper loading in the composite. Transmission electron microscopy reveals that the copper nanoclusters have a mean diameter of 2-3 nm and are homogeneously in-plane distributed in the composite films. Electrothermal atomic absorption spectroscopy has been used to study the kinetics of copper release in the solutions employed for the biological tests. The Cu-CFx layers are employed as bioactive coatings capable of inhibiting the growth of target microorganisms such as Saccharomyces cerevisiae, Escherichia coli, Staphylococcus aureus, and Lysteria. The results of the analytical characterization enable a strict correlation to be established among the chemical composition of the material surface, the concentration of copper dissolved in the microorganisms broths, and the bioactivity of the nano-structured layer.

Analytical characterization of collagen- and/or hydroxyapatite-modified polypyrrole films electrosynthesized on Ti-substrates for the development of new bioactive surfaces.

The design and development of new bioactive surfaces on titanium-based materials employed in orthopedic implants is described. The new biosurfaces consist of thin polypyrrole films, directly grown on implant materials and modified by the inclusion of hydroxyapatite and/or collagen during the polymer electrodeposition step. The experimental procedure has been optimized in terms of loading and distribution of the bioactive components. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) investigations have been performed in order to control the effectiveness of film modifications. In particular, XPS has been used to check the presence of biocompounds in the surface and sub-surface region of the polymer film, which is a critical requisite for a positive interface interaction between the biomaterial and the surrounding tissue.

A combined XPS-SEM/EDX investigation on explanted UHMW polyethylene acetabular cups: possible role of silicon traces in the wear debris.

An investigation was started aimed at a better understanding of the complex phenomena leading to chemical degradation and morphological deterioration of UHMW polyethylene cups in total hip prostheses. Analysis was performed on retrieved implants which needed revision due to inflammation and pain problems. Preliminary results obtained by parallel XPS and SEM/EDX experiments gave evidence, for the first time, that silicon traces are involved in the process of particle formation and segregation onto the surface of the cups. The extent of modification of the surface chemical composition of cups and the process of particle segregation seem to be correlated to both the implant time and to some particular features of patient (age, activity, style of life, etc.). Investigation on a large number of samples is in progress in order to test this hypothesis. The results obtained so far confirmed the potential of surface spectroscopies (XPS) in biomaterial investigations.

Synthesis, analytical characterization, and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates.

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin (RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.

Development and analytical characterization of cysteine-grafted polypyrrole films electrosynthesized on Pt- and Ti-substrates as precursors of bioactive interfaces.

The grafting of cysteine to polypyrrole(PPY)-coated platinum and titanium substrates has been investigated with the aim of developing innovative bioactive materials of interest for bone implants. Polypyrrole has been chosen as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates, of any shape and dimension, leading to remarkably adherent overlayers. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS) which showed the presence of aminoacid residues onto the polymer surface. Information obtained by an accurate curve fitting of significant regions in the spectra (C1s, N1s, and O1s signals) and by a cross-check of peak area ratios, before and after the grafting process, gave evidence that cysteine forms covalent bonds to pyrrole rings, preferentially in beta-positions, via the sulfydryl group, leaving both amino and carboxylic functionalities available for further chemistry. The surface density of cysteine residues was evaluated by microgravimetric measurements performed by the electrochemical quartz crystal microbalance and was found suitable for the exploitation of these modified surfaces as bioactive systems. Some preliminary results are reported on the adhesion of neonatal rat calvarial osteoblasts onto titanium substrates after coating by a PPY film modified by a polypeptide having cysteine as a terminal residue and containing the Arg-Gly-Asp aminoacid adhesive sequence.

Effect of antidenaturant drugs on lysozyme deposit formation on soft contact lenses by liquid chromatography--electrochemical detection.

The effect of some antidenaturant drugs on the formation of lysozyme deposits on soft contact lenses was investigated by in vitro experiments. Results were obtained which encourage the use of this strategy to reduce lens spoilage. At the same time emphasis is given to the possibility that some functionalities (i.e. lysine groups) present in drug molecules could promote protein adsorption. In order to evaluate at a quantitative level the effectiveness of the drugs used to minimize the formation of deposits on soft contact lenses, a selective method for the determination of adsorbed lysozyme was developed. It is based on the hydrolysis of the 'lens/adsorbed lysozyme' system, followed by high performance liquid chromatography coupled to electrochemical detection of the released tryptophan residues. The lysozyme calibration curve was linear up to 5 mg ml-1 and the detection limit was 5 ng injected.

Two methods of examining protein deposits on hydrophilic contact lenses.

Our aim in these studies has been to develop spectroscopic techniques for quantitative analysis of the early phases of protein deposition on soft contact lenses. We produced protein deposition in vitro by soaking lenses in a 0.025% chloral hydrate lysozyme solution at 37 degrees C. Two groups of lenses were so treated: one for 15 days and the other for 6 months. The study of in vivo deposits was carried out on lenses used by healthy wearers. From the same six patients we obtained two consecutive groups of 12 lenses: one group worn for 8 hours and the other group worn for 7 days. We tested the lenses by an indirect (destructive) method (based on a colorimetric assay of amino acid complexes obtained by basic hydrolysis) and by a direct (non-destructive) method consisting of spectrophotometry performed on intact lenses. Using the indirect method we found that the lenses absorbed in vitro for 6 months had lysozyme deposits of 5 +/- 1 micrograms; while the lenses adsorbed for 15 days supplied an average of 0.8 +/- 0.3 micrograms of lysozyme. Lenses worn by healthy volunteers show protein deposition equivalent to a lysozyme quantity ranging from 0.8 to 1.6 micrograms. We found a lower limit of detection with the direct method of approximately 5 micrograms, limiting its applicability.