|Abstract: ||The physico-chemical phenomena occurring at charged interfaces are specifically affected by the type and the concentration of electrolytes. This has implications both in living and in inorganic systems. The discovery of the ‘ion specific effects’ dates back to Hofmeister (1888), who observed the specific effect of salts in promoting egg white proteins precipitation. Nowadays we are aware that ion specific effects are ubiquitous in all fields of science and technology where electrolytes play a role.
Until few years ago the occurrence of ion specific (Hofmeister) effects, although well recognized in a huge number of cases (i.e. protein precipitation and crystallization, viscosity of aqueous solutions, colloid stability, enzyme activities, etc.), was mainly related to ion-induced change of ‘water structure’. To depict these peculiar behaviors the words ‘kosmotropic’ (order maker) and ‘chaotropic’ (disorder maker) ion were coined. Recently, Ninham and co-workers have proposed an explanation of the Hofmeister phenomena based on the consideration that non- electrostatic ion-specific dispersion forces have to be evaluated at the same level of electrostatics in the Poisson-Boltzmann description of ion-ion and ion-surface interactions. The investigation of the effects that the type of salt and its concentration have on the properties (surface charge and potential, electrochemical behavior) of some proteins (BSA, cytochrome c, lysozyme) and of silica-based ordered mesoporours material (OMM) chosen as model systems, was the main purpose of this thesis. In particular, the present work is devoted to reach the following aims: I) To investigate the ion specific effects due to added electrolytes in different systems where soft or hard matter charged interfaces occur; II) To ascertain the interpretation of different experimental results through Ninham’s theory modeling; III) To show how the comprehension of Hofmeister effects may be used for technological purposes, for instance in the development of ‘controlled release systems’. This thesis can be outlined in two parts. The first part is dedicated to a description of the intermolecular forces in terms of the classical and new theories, which permits to understand ion specific effects at charged interfaces, and to the presentation of the experimental techniques that were used to investigate the various systems. In particular, Chapter 1 gives a brief summary of the classical theories of ‘electrical double layer’ and of ‘colloid stability’ used to describe the phenomena occurring at charged interfaces. Chapter 2 gives an overview of the most important ion-specific phenomena since the famous Hofmeister’s experiment, and of the most recent theoretical approaches currently used to explain ion specificity. Chapter 3 describes the experimental methods and the techniques that were used for the investigation of ion specific effects along the PhD work. In the second part of the thesis the papers where the properties of the charged interfaces were studied as a function of the ionic strength and the type of electrolyte are reported. Papers I and II deal with the model protein Bovine Serum Albumin (BSA) in aqueous solution where a liquid-liquid charged interface is formed.
In paper I the surface charge (Zp) and the Zeta potential (ζ) of BSA were determined by means of potentiometric titrations (PT) and electrophoretic light scattering (ELS), respectively. The measurements were carried on as a function of the pH and the ionic strength of the background electrolyte (NaCl) used in the measurements. The isoionic point (IIP) and the isoelectric point (IEP) of the protein were determined as the intercept of the surface charge and zeta potential curves with pH axis, respectively. The shift in the values of IIP (to higher pH) and of IEP (to lower pH), that occurs with increasing ionic strength, depends on the specific interaction of the ions of the background electrolyte with the surface of BSA. The experimental values of IIP and IEP were compared with the respective theoretical values calculated by means of the charge regulation model. In this model the H+ concentration at the protein surface (surface pH), is calculated introducing a term that takes into account the additional potential due to the dispersion forces proposed by Ninham. The same experimental techniques were used in paper II for the determination of the surface charge (Zp) and the electrophoretic mobility (μe) of BSA in the presence of different sodium salts at fixed ionic strength. In this case the values of electrophoretic mobility were used to calculate the effective charge (Zeff) of the protein by means of Henry’s equation. Zp is the net (proton) charge of the protein in the absence of other bound ions. Zeff, instead, is the charge of the protein including the ions (cations and anions) adsorbed on its surface. The difference between these two values (Zp-Zeff) calculated at a given pH, gives an estimation of ion-binding, that is the number of ions (cations and anions) bound to the protein surface. Paper III focuses on a silica-based Ordered Mesoporous Material (OMM), namely SBA-15, dispersed in an aqueous solution and thus forming a solid-liquid charged interface. Potentiometric titrations (PT) were used to estimate the surface charge density (σ) of silica surface of SBA-15 particles in water at different pH values. The surface charge density/pH curve was studied in the presence of salts with different cations but same anion. The experimental results were compared with the theoretical surface charge density (σ) vs pH curves calculated through Ninham’s theory. The theoretical curves for SBA-15 surface charge density were obtained using Ninham’s charge regulation model mentioned above for Paper I. Paper IV highlights the electrochemical behavior of cytochrome c (cyt c) as a function of salt type and concentration.
The electrochemical properties of cytochrome c, a redox protein, were investigated by means of differential pulse voltammetry (DPV) as a function of the ionic strength and the type of the supporting electrolyte used to guarantee the electrical conductivity of the solution. This work was carried out during my stay at the laboratory of Prof. Edmond Magner at the University of Limerick (Ireland). The redox potential (E°’) and the intensity of the current measured in the electrochemical experiments were firstly investigated as a function of the ionic strength using NaCl as the supporting electrolyte. The values of the redox potential were fitted using a model derived from the Debye-Hückel extended law. Then, the redox potential and the intensity of current were investigated in the presence of different electrolytes (cations and anions of the Hofmeister series) at a fixed ionic strength. The values of redox potential measured at different temperatures were used to obtain thermodynamic information (H°’ and S°’) about the redox process. Paper V reports a possible application of the electrolyte effects on charged interfaces for the development of a “controlled drug release system”. The adsorption and the release of Lysozyme (Lyz), a protein with antimicrobial activity, from two OMMs (SBA-15 and MSE) were investigated. The work focused on the effect of two weak electrolytes (buffers) used to fix the pH of the adsorbing solution. SBA-15 and MSE have both a hexagonal mesoporous structure, but different chemical composition and surface properties. In particular MSE is characterized by a higher “hydrophobic” character due to the presence of bridged ethylene groups linked to the silicon skeleton. The adsorption of the lysozyme on the two OMMs was carried out at two different pHs (pH 7.0 and 9.6) using two suitable buffers. Lysozyme release in ‘in-vitro’ physiological conditions was investigated. It was found that the Lysozyme loading changes very much, depending on the surface charge and on the type of surface-protein interactions. Non-electrostatic interactions, mainly involved in the adsorption/release process at the Lyz/MSE interface, seems to produce a stronger protein binding while electrostatic forces dominate at the Lyz/SBA-15 interface. Another very significant parameter that affects both adsorption and release is the pH of the adsorbing solution. The changing in the charge of the surfaces allows for a potential modulation of the global performance, both in terms of protein’s loaded amount, and in terms of release’s rate. The main conclusions of this work can be sketched by the following important remarks: I) All kinds of soft or hard matter charged interfaces (liquid-liquid and solid-liquid) are strongly affected by ion specific effects, in striking agreement with Hofmeister series (almost always). II) Theoretical modeling based on the modified Poisson-Boltzmann equation that includes ion dispersion potentials calculated introducing ab-initio ion polarizabilities of differently hydrated ions allowed to reproduce experimental electrochemical properties at a semi-quantitative level, thus validating further Ninham’s theoretical approach for intermolecular forces. III) In the case of OMMs used for immobilizing proteins, both adsorption and release can be modulated by the choice of different buffers (that modifies the pH). Ultimately, it can be asserted that the choice of background salt used in the adsorbing solutions may address different biotechnological purposes for instance a drug delivery system or a biocatalyst.|