Laura E. Valenti

Methylene blue dimerization does not interfere in surface-area measurements of kaolinite and soils

Methylene blue (MB) was adsorbed from aqueous solutions onto a kaolinite and four soil samples to determine the effects of MB dimerization on the measured surface area. Adsorption isotherms were prepared using four adsorbing solutions containing, respectively, 9, 46, 71, and 83% of MB molecules in the dimeric state. Langmuir-type isotherms were obtained in each case. The results indicate that equilibration occurs quickly. The aggregation state of MB molecules at the surface does not depend on the aggregation state in the initial adsorbing solutions, but on the final equilibrium concentration of MB. A comparison with the specific surface area measured by adsorption of ethylene glycol monoethyl ether indicates that MB adsorbs as a monomer, regardless of the aggregation number in solution. This result occurs owing to the strength of monomer-surface and monomer-monomer interactions. If monomer-surface interactions are favored, the MB dimer adsorbs in the monomeric form. If monomer-monomer interactions are favored, dimer adsorption may occur. The visible spectra of adsorbed molecules indicated that MB was present at the surface as a mixture of monomeric and dimeric species. These results suggest that dimers are formed in the contact region between two aggregating particles.

Infrared study of trifluoroacetic acid unpurified synthetic peptides in aqueous solution: trifluoroacetic acid removal and band assignment.

Synthetic peptide or protein samples are mostly unpurified with trifluoroacetic acid (TFA) used during the synthesis procedure, which strongly interferes with structure determination by infrared (IR) spectroscopy. The aim of this work was to propose a simple strategy to remove TFA contribution from attenuated total reflection (ATR)-IR spectra of the hexahistidine peptide (His6) in aqueous solution to study the conformation of this synthetic peptide without previous purification. Such a strategy is based on the subtraction mode widely employed to remove water contribution, and it is tested with TFA unpurified histidine as a model system. The subtraction is based on eliminating the strong TFA bands at 1147 and 1200cm(-1) by applying a scaling factor (as in buffer correction). The proposed modes represent excellent strategies that do not modify spectral features, and they provide reliable routines to obtain the synthetic peptide spectrum without TFA contribution. The conformational information from the corrected spectra at different pH values is deduced from semiempirical calculated IR spectra of different His6 conformers. The spectral features and the band positions of the corrected spectrum suggest that the peptide molecules mainly adopt an intermolecular β-sheet structure.

Unaffected features of BSA stabilized Ag nanoparticles after storage and reconstitution in biological relevant media.

Silver-coated orthopedic implants and silver composite materials have been proposed to produce local biocidal activity at low dose to reduce post-surgery infection that remains one of the major contributions to the patient morbidity. This work presents the synthesis combined with the characterization, colloidal stability in biological relevant media, antimicrobial activity and handling properties of silver nanoparticles (Ag-NP) before and after freeze dry and storage. The nanomaterial was synthesized in aqueous solution with simple, reproducible and low-cost strategies using bovine serum albumin (BSA) as the stabilizing agent. Ag-NP were characterized by means of the size distribution and morphology (UV-vis spectra, dynamic light scattering measurements and TEM images), charge as a function of the pH (zeta potential measurements) and colloidal stability in biological relevant media (UV-vis spectra and dynamic light scattering measurements). Further, the interactions between the protein and Ag-NP were evaluated by surface enhanced Raman spectroscopy (SERS) and the antimicrobial activity was tested with two bacteria strains (namely Staphylococcus aureus and Staphylococcus epidermidis) mainly present in the infections caused by implants and prosthesis in orthopedic surgery. Finally, the Ag-NP dispersed in aqueous solution were dried and stored as long-lasting powders that were easily reconstituted without losing their stability and antimicrobial properties. The proposed methods to stabilize Ag-NP not only produce stable dispersions in media of biological relevance but also long-lasting powders with optimal antimicrobial activity in the nanomolar range. This level is much lower than the cytotoxicity determined in vitro on osteoblasts, osteoclasts and osteoarthritic chondrocytes. The synthesized Ag-NP can be incorporated as additive of biomaterials or pharmaceutical products to confer antimicrobial activity in a powdered form in different formulations, dispersed in aqueous and non-aqueous solutions or coated on the surface of different materials.

Driving forces for the adsorption of a His-tag Chagas antigen. A rational approach to design bio-functional surfaces.

In order to rationally design a bio-functional surface based on the adsorption of a His-tag antigen, three requirements have to be considered: the bio-recognition element, the driving forces for the adsorption process and the detection mode of the bio-recognition event. This work is focused on the study of the adsorption mechanism of the His-tag H49 Chagas antigen on Ni(II) modified substrates. In order to construct the bio-functional surface, the gen of the H49 Chagas antigen was modified to incorporate His6 moiety at the N-terminal (His6-H49). Then, its physical adsorption and bio-affinity interaction with the solid substrate was studied by reflectometry. Besides His-Ni(II) bio-affinity interactions, His6-H49 was also physically adsorbed on Ni(II) modified substrates, leading to randomly oriented antigens. These loosely attached bio-molecules were partially removed using conditions of electrostatic repulsion. On the other hand, bio-affinity interactions, resulting in site-oriented molecules on the substrate, were only removable by specific competitors for Ni(II) surface sites. Finally, the surface bio-activity was determined from the peak separations of voltammetry waves due to the change of the electron transfer kinetics of a redox probe through the bio-functional surface (working electrode).

Stability of silver nanoparticles: agglomeration and oxidation in biological relevant conditions

Silver nanoparticles (Ag-NP) are the most used nanomaterial in consumer products due to the intrinsic antimicrobial capacity of silver. However, Ag-NP may be also harmful to algae, aquatic species, mammalian cells, and higher plants because both Ag+ and nanoparticles are responsible of cell damages. The oxidative dissolution of Ag-NP would proceed to completion under oxic conditions, but the rate and extent of the dissolution depend on several factors. This work correlates the effect of the capping agent (albumin and citrate) with the stability of Ag-NP towards agglomeration in simulated body fluid (SBF) and oxidation in the presence of ROS species (H2O2). Capping provides colloidal stability only through electrostatic means, whereas albumin acts as bulky ligands giving steric and electrostatic repulsion, inhibiting the agglomeration in SBF. However, citrate capping protects Ag-NP from dissolution to a major extent than albumin does because of its reducing power. Moreover, citrate in solution minimizes the oxidation of albumin-coated Ag-NP even after long incubation times. H2O2-induced dissolution proceeds to completion with Ag-NP incubated in SBF, while incubation in citrate leads to an incomplete oxidation. In short, albumin is an excellent capping agent to minimize Ag-NP agglomeration whereas citrate provides a mild-reductive medium that prevents dissolution in biological relevant media as well as in the presence of ROS species. These results provide insight into how the surface properties and media composition affect the release of Ag+ from Ag-NP, related to the cell toxicity and relevant to the storage and lifetime of silver-containing nanomaterials.

Driving Forces and Consequences of the Adsorption of Proteins to Carbon Nanotubes

Different strategies used to biofunctionalize CNTs with proteins, from direct physical adsorption on pristine CNTs to chemical treatments to achieve covalent interaction, are described. The discussion is focused on the consequences of the adsorption process on the structure and properties of both proteins and CNTs. On this base, recent developments in CNTs-proteins based biosensors (electrochemical and optical) and drug delivery systems are reviewed.

Albumin biofunctionalization to minimize the Staphylococcus aureus adhesion on solid substrates

Staphylococcus aureus has become the most common opportunistic microorganism related to nosocomial infections due to the bacteria capacity to form biofilms on biomedical devices and implants. Since bacterial adhesion is the first step in this pathogenesis, it is evident that inhibiting such a process will reduce the opportunity for bacterial colonization on the devices. This work is aimed at optimizing a surface biofunctionalization strategy to inhibit the adhesion of S. aureus on solid substrates. The first part of the work deals with the albumin adsorption-desorption process, studied by a factorial design of experiments to explore a wide range of experimental factors (protein concentration, pH, flow rate and adsorption time) and responses (initial adsorption rate, adsorbed amount, desorbed extent) for hydrophilic and hydrophobic substrates, with a reduced number of experiments. This approach allows the simultaneous evaluation of the factors affecting the albumin adsorption-desorption process to find a qualitative correlation with the amount of alive S. aureus adhered on albumin biofunctionalized substrates. The results of this work point to a relationship between bacterial adhesion and the degree of albumin relaxation on the solid substrate. In fact, the inhibition of bacterial adhesion on albumin biofunctionalized substrates is due to the surface perturbation on the native structure of the protein. On this base, a biofunctionalization strategy was designed using a solution of thermally treated albumin molecules (higher β-sheet or unordered secondary structure elements) to biofunctionalize solid substrates by dipping. With these albumin biofunctionalized substrates S. aureus adhesion was minimized.