Flow Microcalorimetry Research Articles

The conformational stability of ovalbumin and lysozyme in the aqueous solutions of various cosolvents

A thermodynamic method is reported to monitor the conformational stability of lysozyme and ovalbumin in the presence of various cosolvents. Heats of dilution of the proteins in concentrated aqueous solutions of urea, ethanol or glucose have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the proteins in the different solvent media are derived: They allow to gain information about the influence of the cosolvents on the interactions between two interacting hydrated molecules of a protein, hence on its conformational stability. The two proteins behave very differently in the various cosolvents. In glucose, the coefficients for ovalbumin are positive up to 3 mol kg−1 of cosolvent and then negative, while those for lysozyme are negative up to 6 mol kg−1. In urea, coefficients for ovalbumin are positive, while negative for lysozyme up to 7 mol kg1. In ethanol, coefficients for ovalbumin are almost invariant, even at the highest concentrations of cosolvent, underlining that the hydration shell of the protein is such to maintain essentially unaltered the native conformation. For lysozyme, coefficients are negative and almost invariant up to 20 mol kg−1 ethanol: Then, a jump occurs toward much more large and negative values. The observed behaviors are rationalized also on the basis of the results previously obtained for small model molecules in concentrated solutions of urea, ethanol or glucose. The differences between the two proteins are explained in terms of the effects of the cosolvents on hydrophilic and hydrophobic interactions and account for the structural characteristics of each protein. In fact, notwithstanding both are globular proteins, they are differently packed and that could make them to react differently toward the action of a given cosolvent.

Competitive adsorption of lysozyme and myoglobin on mesostructured cellular foam silica

A model of multicomponent multilayer adsorption has been developed to describe the simultaneous adsorption of lysozyme (14.3 kDa, pI = 11.35) and myoglobin (17.6 kDa, pI = 7.2) on mesostructured cellular foam (MCF) silica in 0.02 M potassium phosphate buffer at pH 7 and 25 °C. The mechanism of adsorption was proposed based on batch adsorption isotherms and flow microcalorimetry (FMC) traces for the enthalpy of adsorption. Thermodynamic derivations of Guggenheim–Anderson–deBoer (GAB) and Langmuir models were used to develop the model based on the proposed mechanism. Regression analysis demonstrated the effect of each of the proposed mechanistic equations.

Development of a flow system for studying biofilm formation on medical devices with microcalorimetry

Isothermal microcalorimetry (IMC) is particularly suited to the study of microbiological samples in complex or heterogeneous environments because it does not require optical clarity of the sample and can detect metabolic activity from as few as 104CFU/mL cells. While the use of IMC for studying planktonic cultures is well established, in the clinical environment bacteria are most likely to be present as biofilms. Biofilm prevention and eradication present a number of challenges to designers and users of medical devices and implants, since bacteria in biofilm colonies are usually more resistant to antimicrobial agents. Analytical tools that facilitate investigation of biofilm formation are therefore extremely useful. While it is possible to study pre-prepared biofilms in closed ampoules, better correlation with in vivo behaviour can be achieved using a system in which the bacterial suspension is flowing. Here, we discuss the potential of flow microcalorimetry for studying biofilms and report the development of a simple flow system that can be housed in a microcalorimeter. The use of the flow system is demonstrated with biofilms of Staphylococcus aureus.

Thermodynamic study of the interaction between linear plasmid deoxyribonucleic acid and an anion exchange support under linear and overloaded conditions

Anion-exchange chromatography has been successfully used in plasmid DNA (pDNA) purification. However, pDNA adsorption mechanism using this method is still not completely understood, and the prediction of the separation behavior is generally unreliable. Flow microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces and mechanisms involved in the adsorption process of biomolecules onto several chromatographic systems. Thus, using FMC, this study aims to understand the adsorption mechanism of linear pDNA (pVAX1-LacZ) onto the anion-exchange support Fast Flow (FF) Q-Sepharose. Static binding capacity studies have shown that the mechanism of pDNA adsorption onto Q-Sepharose follows a Langmuir isotherm. FMC experiments resulted in thermograms that comprised endothermic and exothermic heats. Endothermic heat major contributor was suggested to be the desolvation process. Exothermic heats were related to the interaction between pDNA and Q-Sepharose primary and secondary adsorption. Furthermore, FMC revealed that the overall adsorption process is exothermic, as expected for an anion-exchange interaction. Nevertheless, there are evidences of the presence of nonspecific effects, such as reorientation and electrostatic repulsive forces.

Functional group effects on the enthalpy of adsorption for self-assembly at the solution/graphite interface.

The thermodynamics of self-assembly have long been explored by either experimental or theoretical investigations which are often unable to account for all the factors influencing the assembly process. This work interrogates the thermodynamics of self-assembly at a liquid/solid interface by measuring the enthalpy of adsorption encompassing analyte-analyte, analyte-solvent, analyte-substrate, and solvent-substrate interactions. Comparison of the experimental data with computed lattice energies for the relevant monolayers across a series of aliphatic analytes reveals similar ordering within the series, with the exceptions of the fatty acid and bromoalkane adsorbates. Such a discrepancy could arise when the lattice energies do not account for important interactions, such as analyte-analyte interactions in solution. Flow microcalorimetry provides a uniquely inclusive view of the thermodynamic events relevant to self-assembly at the liquid/solid interface.

Flow Micro‐Calorimetry and FTIR Spectroscopy Study of Interfacial Interactions in Uncoated and Coated Calcium Carbonate Filled Polyurethane Adhesives

SummaryThermoplastic polyurethane (TPU) ‐ calcium carbonate interactions were studied using flow micro‐calorimetry (FMC) and diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS). FMC enabled the determination of adsorption and desorption energies; in this study a model compound approach was used to acquire insight in to the effect of calcium carbonate type and presence of stearate coating on polymer – filler interactions. It was anticipated that this data will assist in the understanding of differing responses obtained from parallel plate rheometry and viscoelastic measurements of the filled polyurethanes. Three calcium carbonates (coated and uncoated precipitated calcium carbonate, and natural ultramicronized uncoated calcium carbonate) were used. A stronger TPU‐filler interaction was shown in the uncoated precipitated calcium carbonate due to the fact that more of the surface was available for interaction.

Interactions between biomolecules and an iron-silica surface

An iron-silica adsorbent (1:1 ratio by mass) with a pore cell size of 11.4nm and BET surface area of 610m2g−1 was synthesized and compared to a mesocellular foam (MCF) silica and iron oxide for amino acid (alanine, aspartic acid, lysine and histidine) and lysozyme adsorption. Amino acid batch adsorption loadings did not exceed estimated monolayer coverage and were generally quantitatively similar on iron-silica and MCF silica; the observed loadings followed the trend alanine>lysine/aspartic acid≫histidine. Multilayers were observed on the strongly positive iron oxide surface. The dynamic heat of biomolecule adsorption was directly measured with flow microcalorimetry (FMC); the enthalpies of adsorption of alanine, aspartic acid, lysine, and histidine on iron-silica were −0.046, −0.73, −5.3 and −14.3kJmol−1, respectively. Alanine adsorption was mediated by the surrounding water, aspartic acid adsorbed through weak electrostatic attractions, and lysine and histidine formed bonds during adsorption. Lysozyme monolayer loading was higher on iron-silica than on MCF silica, calorimetric studies indicate a different lysozyme adsorption mechanism on iron-silica and MCF silica.

Physisorption of Three Calix[4]arenes on Alkyl-Functionalized Mesoporous Silica for Biomimetic Ligand Development

Calixarene physisorption onto alkyl-functionalized silica support is presented as an alternative to tethering to reduce synthetic complexity. Three versions of calix[4]arene were synthesized with differing upper rim functional groups: aromatic amine, tert-butyl, and unfunctionalized. Calixarene molecules physisorbed throughout the alkyl layer on the silica following a linear isotherm with a maximum observed loading of 300 μmol g–1. Large exothermic adsorption enthalpies (−60 to −260 kJ mol–1), attributed to van der Waals interactions, were measured for the calixarene physisorption using flow microcalorimetry; large negative entropies were calculated (−140 to −780 J mol–1 K–1). Amine-calix[4]arene was physisorbed onto C18-silica (NCS) through solvent evaporation. The myoglobin uptake on NCS (47 μmol g–1) was twice that on C18-silica; additionally, a greater heat of adsorption (−16.9 ± 2.9 kJ mol–1) was observed on NCS than on C18-support (−12.7 ± 2.2 kJ mol–1).

Effect of fly ash washing conditions on the properties of coupling agent modified polypropylene/fly ash composites

Two fly ash samples (one from UK, UKFA, and another from South Africa, SAFA) were washed with water and 1 M hydrochloric acid (followed by water) prior to incorporation (at 65% wt) into polypropylene homopolymer, containing an unsaturated carboxylic acid coupling agent (Lubrizol Solplus® C800) with dicumyl peroxide free radical initiator. Melt blending was achieved using a Haake Rheomix 600 mixing chamber, and composite test plaques were compression moulded. Flexural and impact testing was then carried out. Unwashed and water washed fly ash–based composites responded well to C800 modification, and flow microcalorimetry (FMC) and diffuse reflectance Fourier transform infrared spectroscopy studies confirmed strong interaction between C800 and fly ash. However, washing the fly ash with HCl led to a reduction in composite flexural and impact properties. Scanning electron microscopy imaging of the latter composite fracture surfaces revealed poor filler‐matrix adhesion, which was thought to be due to reduced interaction between C800 and HCl washed fly ash. The latter was confirmed using FMC. Reduction of C800/fly ash interaction led to a reduction in the nucleation of polypropylene (PP) crystallization and a decrease in melt flow rate. The latter may be due to a shift in locus of PP–C800 addition reactions from the interfacial region to the bulk matrix. POLYM. COMPOS., 35:698–707, 2014. © 2013 Society of Plastics Engineers

Flow micro-calorimetry and diffuse reflectance Fourier transform infrared spectroscopy studies in filled polyurethane adhesives by using dimethyl adipate as a model compound

Interactions between nano-scale filler particles (precipitated calcium carbonate, carbon black and fumed silica) and model compounds (dimethyl adipate and butan-2-one) are quantified using flow micro-calorimetry (FMC) and diffuse reflectance Fourier transform infrared spectroscopy (DRFTIRS). Carbonyl groups of dimethyl adipate interact strongly with silanol groups on the fumed silica surface but weakly with the uncoated precipitated calcium carbonate. In general, higher surface area loading imparts high level of adsorption because of the nanofiller has more adsorption sites. Carbon black is an exception likely due to the less accessible surface groups and the presence of relatively important amount of micropores.

Synthesis of glycylglycine-imprinted silica microspheres through different water-in-oil emulsion techniques

Sol–gel molecularly imprinted materials (MIMs) are traditionally obtained by grinding and sieving of a monolith formed by bulk polymerization. However, this process has several drawbacks that can be overcome if these materials are synthesized directly in the spherical format. This work aimed at the development of two efficient methods to prepare spherical glycylglycine-templated silica (“whole-imprinted” and surface-imprinted) through a combination of sol–gel and emulsion techniques. The synthesis of the microspheres was optimized regarding emulsion and sol–gel parameters. Imprinting efficiency of the prepared materials was studied by solid phase extraction and flow microcalorimetry. The particles prepared with glycylglycine and functional monomer, in basic medium (using cyclohexane as non-polar continuous medium) presented the highest imprinting factor – 2.5 – and the respective surface-imprinted material presented an imprinting factor of 1.5. The results of flow microcalorimetry confirmed the action of different mechanisms of glycylglycine adsorption: entropically-controlled interactions were present for the “whole-imprinted” material, indicating adsorption inside small imprinted pores; enthalpically-controlled interactions were observed for the surface-imprinted material, a behaviour more compatible with a template/surface-only interaction. Globally, the two approaches allowed for a successful imprinting effect which was more extensive for the “whole-imprinted” material, whereas the surface-imprinting feature confers to the surface-imprinted xerogel advantages regarding mass transfer kinetics. Overall, the spherical particles obtained by both approaches presented characteristics, such as sphericity, mesoporosity, easy/fast accessibility to imprinted sites, important indicators that these materials may be candidates for stationary phases for efficient, selective chromatographic separation.

Energetics of biomolecule adsorption on mesostructured cellular foam silica

Heats of adsorption measured by flow microcalorimetry (FMC) were used to understand the energetics of biomolecule adsorption on mesostructured cellular foam (MCF) silica. Tryptophan (Trp), lysozyme (LYS), and bovine serum albumin (BSA) were used as probe molecules. The FMC results confirmed that attractive interactions (both electrostatic and van der Walls interactions) between the biomolecules and acid-washed MCF silica were the driving force for adsorption, even when the protein (BSA) and the surface were both negatively charged and repulsion interactions might be expected. Multiple exothermic events occurred, possibly because of multipoint interactions between biomolecules and MCF silica resulting from multiple binding sites on the biomolecule and a curved pore structure. Interestingly, the magnitude of the enthalpy of adsorption (ΔHTotal) increased with increasing biomolecule size at pH 5.2; the number of exothermic peaks corresponded to the number of binding regions on the biomolecule. In addition, standard Gibbs energies of adsorption (ΔG°) and entropy of adsorption (ΔS°) were calculated from batch adsorption isotherms and the measured enthalpy of adsorption. BSA adsorption energetics were significantly affected by changing the pH from 4 to 5.2. This effect was attributed to a dramatic conformational change of BSA as a function of pH. The energetics of adsorption provide invaluable insight into the mechanism biomolecule adsorption.

Influence of Morphology and Crystallinity on Surface Reactivity of Nanosized Anatase TiO2 Studied by Adsorption Techniques. 1. The Use of Gaseous Molecular Probes

Various titanium dioxide nanoparticles were prepared by sol–gel method in order to obtain samples showing different sizes and morphologies. An original approach based on the adsorption of gaseous molecules from the gas phase was proposed to gain information about surface energy of nanosized TiO2 anatase in terms of interfacial reactivity and heterogeneity. Argon, nitrogen, and ammonia were selected as such surface molecular probes. The mainly observed crystallographic faces of anatase particles were the {101} and {001} surfaces together with the {100} one. Their abundance was correlated with the energy distribution inferred from the local isotherms of argon adsorption in the low-pressure range. The acid character of the anatase surface was probed by nitrogen molecules, and, consequently, the location of polar sites on the particle surface could be determined in correlation with the argon adsorption domains. Moreover, the number and the strength of surface acid sites were evaluated with the aid of two-cycle adsorption of gaseous ammonia supplemented by appropriate flow microcalorimetry measurements. This molecular probe revealed significant differences among the samples depending on their crystal shape or face distribution.

Effects of silver nanoparticles on thermal properties of DBSA-doped polyaniline/PVC blends

Dodecylbenzenesulfonic acid (DBSA)-doped polyaniline (PAND) has been synthesized by redoping (PANDR) and aqueous polymerization (PANDA) methods. Silver nanoparticles were incorporated into the PANDR/tetrahydrofuran solution (PANDS) and then mixed with poly (vinyl chloride) (PVC) solution to prepare PANDS/PVC nanocomposites. In the present study, effects of silver nanoparticles on thermal properties of PAND/PVC blends have been investigated by employing thermal gravimetric analysis and heat flow microcalorimetry techniques. From these results it has been observed that the thermal stability of blends have increased by increasing the concentration of PAND in blends and nanocomposites. Addition of silver nanoparticles has suppressed the dehydrochlorination process and evolution/degradation of DBSA in PANDS/PVC nanocomposites. Presence of silver nanoparticles in PAND/PVC nanocomposites has reduced the mobility of PANI chains which in turn inhibited the transfer of free radicals formed during degradation of PAND and PVC through inter-chain reactions; hence, degradation process has been slowed down and thermal stability has been improved. Embedment of silver nanoparticles has reduced thermal weight loss corresponding to polymer degradation step and attains lower heat flow level in inert atmosphere for nanocomposites in contrast to those with no nanoparticles, thereby further improving thermal stability of nanocomposites. The heats of oxidation measured for blends and nanocomposites were independent of PAND/PVC blends composition.

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg−1, the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules.The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on the possible mode of action of the two cosolvents on the stability of proteins in solution.

Effect of sodium chloride concentration on the interaction of N-(N-glycylglycyl)glycine with xylitol and D-mannitol in aqueous solutions

Abstract Enthalpies of mixing of N -( N -glycylglycyl)glycine with xylitol and D-mannitol and their respective enthalpies of dilution in aqueous sodium chloride solutions have been determined at 298.15 K using flow microcalorimetry. The experimental results obtained have been analyzed to give the heterotactic enthalpic interaction coefficients ( h xy , h xxy , h xyy ) of the solutes in terms of the McMillan–Mayer model. It has been found that the heterotactic enthalpic pairwise interaction coefficients h xy between N -( N -glycylglycyl)glycine and xylitol or D-mannitol in aqueous sodium chloride solutions are negative and become less negative with the increase of the concentration of sodium chloride. The h xy values for the interaction between N -( N -glycylglycyl) glycine and D-mannitol are less than that between N -( N -glycylglycyl) glycine and xylitol. The results are discussed in terms of solute–solute and solute–solvent interactions.

Effect of Nanoscale Pore Space Confinement on Cadmium Adsorption from Aqueous Solution onto Ordered Mesoporous Silica: A Combined Adsorption and Flow Calorimetry Study

The competitive adsorption of Cd(II) ions has been studied from aqueous solutions of cadmium nitrate in the presence of 0.1 M sodium nitrate at 298 K onto nonporous Spherosil XO75LS and ordered porous silica of the SBA-15 type with the purpose of tracking the effect of confinement in pores. Four SBA-15 mesoporous samples, prepared using pluronic P103 and P123 as templates at two temperatures of 303 and 373 K and differing in both the pore size (from 3.8 to 6.4 nm) and the areal density of acid sites reactive toward gaseous ammonia, were used. The equilibrium adsorption isotherms and the enthalpy curves for the related displacement process were determined by means of solution depletion method and liquid flow microcalorimetry, respectively. The experimental adsorption curves were transformed into normalized plots showing the ratio between the amount of cadmium retained at the solid–liquid interface and the number of surface silanol groups. Estimates of the standard molar Gibbs free energy and entropy of adsorption were made in the pseudoplateau adsorption region. It was postulated that the adsorbing Cd2+ cations ion exchanged with the initially adsorbed Na+ ions, the overall displacement process being endothermic and entropy-driven in the whole concentration range studied, mostly due to small modifications of the hydration shells of adsorbing and desorbing species. The areal density of surface silanols, influenced by the surface curvature within pore space, appeared to be the major factor that induced significant changes in the molar enthalpy of displacement.

Energetics of protein adsorption on amine-functionalized mesostructured cellular foam silica

The energetics of lysozyme adsorption on aminopropyl-grafted MCF silica (MCF-NH2) are compared to the trends observed during lysozyme adsorption on native MCF silica using flow microcalorimetry (FMC). Surface modification on MCF silica affects adsorption energetics significantly. All thermograms consist of two initial exothermic peaks and one later endothermic peak, but the heat signal trends of MCF-NH2 are opposite from those observed for adsorption onto native MCF silica in salt solutions of sodium acetate and sodium sulfate. At low ionic strength (0.01 M), LYS adsorption onto MCF-NH2 was accompanied by a large exotherm followed by a desorption endotherm. With increasing ionic strength (0.1 and 3.01 M), the magnitude of the thermal signal decreased and the total process became less exothermic. Also a higher protein loading of 14 μmol g −1 was obtained at low ionic strength in batch adsorption isotherm measurements. Taken together, the FMC thermograms and batch adsorption isotherms reveal that MCF-NH2 has the nature of an ion exchange adsorbent, even though lysozyme and the aminopropyl ligands have like net charges at the adsorption pH. Reduced electrostatic interaction, reduced Debye length, and increased adsorption-site competition attenuate exothermicity at higher ionic strengths. Thermograms from flow microcalorimetry (FMC) give rich insight into the mechanisms of protein adsorption. A two-step adsorption mechanism is proposed in which negatively charged surface amino acid side chains on the lysozyme surface make an initial attachment to surface aminopropyl ligands by electrostatic interaction (low ionic strength) or van der Waals interaction (high ionic strength). Secondary attachments take place between protruding amino acid side chains and silanol groups on the silica surface. The reduced secondary adsorption heat is attributed to the inhibitory effect of the enhanced steric barrier of aminopropyl group on MCF silica.

Energetics of lysozyme adsorption on mesostructured cellular foam silica: Effect of salt concentration

The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.

Microcalorimetric Study for the Binding of Ionic Drugs to Human Erythrocytes and the Ghost Membranes

The binding of phenothiazine derivatives (as cationic drugs) and non-steroidal anti-inflammatory drugs (as anionic drugs) to human erythrocytes and ghost membranes has been compared with respect to their thermodynamic characteristics, by flow microcalorimetry at pH 7.4 and 37 C. From enthalpyentropy correlation, it was shown that anionic and cationic drugs are bound to different binding sites on the membranes. Phenothiazines bind to a single common site of the erythrocyte membranes with relatively high binding affinities (K = 10(4)-10(5) M-1). The binding is entropy-driven and characterized by a small negative enthalpy (delta H) and a positive entropy change (delta S), reflecting hydrophobic interactions. However, the binding reaction for the intact erythrocytes shows large negative values for both delta H and delta S. The values of K for the membranes and delta H for the intact erythrocytes increased with the increase of the hydrophobic character of the substituent group at the C-2 position of the phenothiazine nucleus (H less than Cl less than CF3). The results indicate that phenothiazines bind and or penetrate to the inner membranes of the erythrocytes and react with intracellular components such as haemoglobin, while anti-inflammatory drugs bind to the surface protein on the membranes with a lower affinity (K = 10(3) M-1) than phenothiazines, reflecting the small negative delta H and positive delta S for the interaction with intact erythrocytes.