Chitosan has been proposed as a "natural" coagulating agent to solve wastewater problems. The main hindrance in this commercial chitosan application has been its low cost effectiveness. The hypothesis in our research is that chitosan complexes with natural polyanions is more effective than chitosan alone, particularly in recovering low concentration proteins from food processing wastewater. Chitosan (Chi) was reacted with alginate (Alg), pectin (Pec) and carrageenan (Car) ex-situ to obtain chitosan-polyanion complexes (Chi-Pol). Analysis by Fourier Transform Infrared (FTER) spectroscopy confirmed electrostatic interactions as the mechanism for complex formation. Scanning electron microscopy revealed a tight, non-porous structure except for the porous Chi-Car complex. Tests with a bovine serum albumin solution revealed low adsorption rates with slightly higher values for Chi-Car suggesting the need for an improved complexation method. Chi-Pol complexes prepared in-situ at different monomeric weight ratios (MR) were evaluated using pH 6 adjusted Cheddar cheese whey and surimi wastewater (SWW). Complexes used at 30 mg complex/L whey showed higher turbidity reductions than at 10 mg/L. MR had no significant effect on turbidity reduction except for Chi-Alg at 30 mg/L; the value (72 %) at MR = 0.2 was higher than for MR = 0.8. UV-Vis spectroscopy confirmed in-situ complex formation with a preference for the adsorption of specific whey protein fractions. Complexes formed at 0.2 and 0.8 MR were evaluated at two concentrations for the treatment of SWW. Tests at 50 mg/L showed a turbidity reduction of up to 97 % at 24 h with a 81-90 % recovery of SWW proteins. At 150 mg/L, similar efficacy was achieved in only 1 h with turbidity reductions ranging 94-99 % and 78-94 % protein recovery. FTIR analyses confirmed the adsorption of proteins as indicated by similarities in the three amide bands for Chi-Alg recovered solids and untreated SWW. Differential scanning calorimetry (DSC) was employed to study interactions of SWW proteins and Chi-Alg complexes. Untreated and complex bound SWW proteins revealed single exothermic peaks at 23.3 and 38.0°C, respectively. This suggested Chi-Alg and SWW protein interactions increased the thermal stability of SWW proteins. However, further thermal analysis studies are needed to confirm this finding. / Graduation date: 2001

Interactions between proteins and contact surfaces can have important implications in the food industry. Such interactions contribute to the course of fouling of membrane surfaces and they appear to mediate bacterial and spore adhesion to some degree as well. In addition to protein and solution properties, interfacial behavior is strongly influenced by contact surface properties. Among these, hydrophobicity and the potential to take part in acid-base interaction have received considerable attention, but in a quantitative sense we know very little about their respective influences on protein adsorption. It was the purpose of this research to quantify the equilibrium adsorptive behavior of the milk protein β-lactoglobulin as it is influenced by the presence of different contact surface functional groups. Monocrystalline and polished silicon surfaces were modified to be hydrophilic by oxidation and hydrophobic by silanization with dichlorodiethylsilane (DDES), dichlorodimethylsilane (DDMS), and dichlorodiphenylsilane (DDPS), each used at concentrations of 0.82, 3.3, and 82 mM. Surface hydrophobicities were evaluated with contact angle methods. Adsorption isotherms were constructed after allowing each modified silicon surface to independently contact β-lactoglobulin (0.01 M phosphate buffer, pH 7.0) at concentrations ranging between 200 and 2000 mg/L for eight h at room temperature. Surfaces were then rinsed and dried. Optical properties of the bare- and film-covered surfaces, necessary for calculation of adsorbed mass, were obtained by ellipsometry. Plots of adsorbed mass as a function of protein concentration exhibited attainment of plateau values beyond a protein concentration of about 200 mg/L. At high silane concentration, the plateau values associated with surfaces exhibiting ethyl groups were observed to be greatest followed by those exhibiting phenyl, methyl, then hydrophilic (OH) groups. At the low DDMS and DDES concentrations (0.82 and 3.3 mM), adsorbed mass did not increase beyond that value recorded for the hydrophilic surface. This is likely due to some critical spacing of methyl and ethyl groups being required to produce a favorable hydrophobic effect on adsorption. For surfaces treated with dichlorodiphenylsilane, adsorbed mass increased with silane concentration. Apparently, a favorable acid-base interaction effected by the hydrophilic surface is inhibited by the presence of small amounts of methyl and ethyl groups, but somewhat less inhibited by the presence of phenyl groups because the latter have the ability to undergo acid-base interaction. / Graduation date: 1993

Adsorption kinetic data recorded for α-lactalbumin, β-casein, β-lactoglobulin, bovine serum albumin and lysozyme at silianized silica surfaces of low and high hydrophobicity, along with a simple model for adsorption and surfactant-mediated elution of protein, were used to analyze the removal of each protein by sodium dodecylsulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) at each surface. The model relates resistance to surfactant elution to two rate constants: one governing conversion of removable protein to a nonremovable form (s₁), and one governing removal of protein by the surfactant (k [subscript s]). Elution of each protein from hydrophobic silica with SDS was interpreted as providing information relevant to protein-surface binding strength, or si; i.e., protein-specific differences in removal were a result of SDS adsorption to the surface and displacement of surface-bound protein, as opposed to solubilization driven by SDS binding to the protein. SDS-mediated removal of protein from surfaces of lower hydrophobicity were interpreted as generally proceeding according to a similar, displacement mechanism. The model indicated that data recorded for DTAB-mediated elution at each surface were generally less representative of protein-surface behavior, and more a function of k [subscript s], where differences in surfactant attachment to protein and solubilization appeared to play an important role in protein removal. Under controlled conditions use of the model would allow identification of cases where k [subscript s] in particularly protein specific, and illustrates the point that in such cases surfactant-mediated elution of a protein may reveal little about its surface behavior. / Graduation date: 1996

The effect of pH and ionic strength on the equilibrium adsorptive behavior of β-lactoglobulin onto hydrophobic and hydrophilic silicon surfaces was studied using ellipsometry. Plots of amount adsorbed (μg protein/cm²) as a function of protein concentration (mg/ml) exhibited attainment of plateau values beyond a protein concentration of 0.250 mg/ml. At a given pH and ionic strength, plateau values associated with hydrophobic surfaces were observed to be greater than those associated with hydrophilic surfaces. The Langmuir adsorption isotherm was chosen as the most appropriate model to represent the data and was used to compare results obtained under different experimental conditions. Effects of pH and ionic strength on protein adsorption at hydrophilic surfaces indicate that electrostatics played a major role, while pH and ionic strength effects on adsorption to hydrophobic surfaces reflect a greater importance of nonelectrostatic interactions. / Graduation date: 1991

It is widely accepted that the initial event that significantly influences biocompatibility is the nearly instantaneous adsorption of proteins from biological fluids onto the biomaterial surface. For blood-contacting devices, the complex layer of adsorbed plasma proteins is generally unfavourable and leads to major complications, including thrombus formation, inflammatory tissue responses, and microbial infections. Furthermore, protein interaction with passive films on metallic biomaterial surfaces may contribute to enhanced in vivo corrosion. To gain a better understanding of this phenomenon, the present thesis investigated the fundamental aspects of the interaction of the serum protein fibrinogen with a medical-grade stainless steel 316LVM surface using electrochemical and IR spectroscopy techniques. Aspects of this interaction included the thermodynamics and kinetics of fibrinogen adsorption, the effect of fibrinogen adsorption on the corrosion behavior of 316LVM stainless steel, and the conformational changes of fibrinogen upon its adsorption onto the stainless steel surface. / It was shown that fibrinogen readily adsorbs onto the 316LVM stainless steel surface. Increases in the bulk protein concentration resulted in a corresponding increase of the surface coverage, a dependence that was described by the Langmuir isotherm. Large, negative values of the calculated Gibbs energy of adsorption indicated a highly spontaneous and strong adsorption of fibrinogen onto the 316LVM stainless steel at all investigated temperatures. Although the adsorption process was shown to be endothermic under the applied experimental conditions, the primary driving force for the adsorption process was found to be the positive entropy gain that arises from structural loss and/or rearrangement of the protein upon adsorption, as well as dehydration of the protein and stainless steel surface during the adsorption process. Kinetic measurements indicated that fibrinogen adsorption occurs rapidly. / It was determined that for short contact times (1 hour), the addition of fibrinogen to the electrolyte enhanced the corrosion rate of the 316LVM stainless steel at the open circuit potential. For longer contact times (24 hours), an increase in the polarization resistance values was obtained, indicating an enhanced corrosion resistance of the material. It was postulated that the protein was not capable of complexing the well-stabilized passive film, and instead remained adsorbed to form a protective barrier to diffusion of oxygen-containing species from the electrolyte to the stainless steel surface. / The secondary structure of the surface-adsorbed fibrinogen molecules was investigated by modeling the experimental PM-IRRAS spectra. It was shown that the protein lost a certain extent of its secondary structure upon adsorption to the steel surface. Fibrinogen molecules adsorbed from more dilute solutions were also shown to possess a lower alpha-helical content than those adsorbed from more concentrated solutions, suggesting they laid on the stainless steel surface in a more linear configuration.

No description available.

In the past decade, single molecule imaging has advanced our understanding of processes at the molecular scale. Total internal reflection fluorescence (TIRF) microscopy is one implementation in particular that has been extensively applied in the study of protein adsorption to surfaces. The spatial and temporal resolution provided by TIRF has enabled dynamic measurements of individual proteins in solution, where previously only bulk measurements or static electron microscopy observations were possible. The ability to study individual proteins has revealed and sometimes clarified the complex interactions at their interfaces. Here, the utility of TIRF is expanded to introduce a new model of protein adsorption to the suface and to study the protein interface in contact with solution. Protein adsorption to surfaces has implications in surface biocompatibility, protein separation, and pharmaceutical nanoparticle development. For this reason, the phenomenon has been quantitatively by a variety of techniques, including single molecule imaging. The key data are the protein lifetimes on the surface, which have been shown to be broadly distributed and well-approximated by the sum of several exponential functions. The determined desorption rate constants are thought to reflect different interaction types between surface and protein, but the rates are not typically linked to a specific physical interaction. In the first part of this thesis, we establish appropriate imaging conditions and analysis methods for TIRF. A robust survival analysis technique is applied to capture the range of protein adsorption kinetics. In the second part, we utilize single molecule lifetime data from the adsorption of fibrinogen and bovine serum albumin (BSA) to glass surfaces and discover a heavy-tailed distribution: a very small fraction of proteins adsorbs effectively permanently, while the majority of proteins adsorb for a very short time. We then demonstrate that this characteristic power law behavior is well described by a model with a novel interpretation of the complex protein adsorption process. The second half of the thesis extends TIRF to study the solution-facing interface of the protein as opposed to the surface facing interface by establishing the parameters for a super-resolution imaging technique. Point accumulation for imaging nanoscale topography (PAINT) generates high-resolution images of the sample of interest through the positional tracking of many temporally-distinct instances of a fluorescent probe binding to the sample. Previously, this technique has been applied in the mapping of DNA nanostructures. Here, in the third part, we apply PAINT to the study of proteins. First, a workstream is established for a model system of Nile red and BSA. The kinetic parameters for the system are established to allow rational design of PAINT experiments with this system. The on-rate and off-rate for Nile red are determined. Additionally, the binding model between the two components is tested by studying how the presence of an inhibitor effects the parameters. In the final part, TIRF is used to study the protein-solution interface to examine the glycosylation of immunoglobulin A 1 (IgA1). Over 50% of eukaryotic proteins are glycosylated, and the glycan sequence is simultaneously difficult to study and crucial in the many functional roles proteins play. The glycosylation of IgA1, for example, plays a key role in the pathophysiology of IgA1 nephropathy. Lectins are proteins that bind to specifc glycan sequences and are often used to isolate glycosylated proteins. In this study, the appropriate surface conditions are established to allow specific binding between lectins and IgA1 glycans. The association and dissociation rate between lectins specific for the glycans on IgA1 are measured and affinity constants calculated. These efforts will help to rationally design experiments in the future to elucidate unknown glycan sequences on proteins.

Nonionic surfactants are commonly used to stabilize proteins during upstream and downstream processing and drug formulation. Surfactants stabilize the proteins through two major mechanisms: (i) their preferential location at nearby interfaces, in this way precluding protein adsorption; and/or (ii) their association with protein into "complexes" that prevent proteins from interacting with surfaces as well as each other. In general, both mechanisms must be at play for effective protein stabilization against aggregation and activity loss, but selection of surfactants for protein stabilization currently is not made with benefit of any quantitative, predictive information to ensure that this requirement is met. In certain circumstances the kinetics of surface tension depression (by surfactant) in protein-surfactant mixtures has been observed to be greater than that recorded for surfactant alone at the same concentration. We compared surface tension depression by poloxamer 188 (Pluronic�� F68), polysorbate 80 (PS 80), and polysorbate 20 (PS 20) in the presence and absence of lysozyme and recombinant protein, at different surfactant concentrations and temperatures. The kinetic results were interpreted with reference to a mechanism for surfactant adsorption governed by the formation of a rate-limiting structural intermediate (i.e., an "activated complex") comprised of surfactant aggregates and protein. The presence of lysozyme was seen to increase the rate of surfactant adsorption in relation to surfactant acting alone at the same concentrations for the polysorbates while less of an effect was seen for Pluronic�� F68. However, the addition of salt was observed to accelerate the surface tension depression of Pluronic�� F68 in the presence of lysozyme. The addition of a more hydrophobic, surface active protein (Amgen recombinant protein) in place of lysozyme resulted in greater enhancement of surfactant adsorption than that recorded in the presence of lysozyme. A simple thermodynamic analysis indicated the presence of protein caused a reduction in ���G for the surfactant adsorption process, with this reduction deriving entirely from a reduction in ���H. We suggest that protein accelerates the adsorption of these surfactants by disrupting their self associations, increasing the concentration of surfactant monomers near the interface. Based on these air-water tensiometry results, it is fair to expect that accelerated surfactant adsorption in the presence of protein (observed with PS 20 and PS 80) will occur with surfactants that stabilize protein mainly by their own adsorption at interfaces, and that the absence of accelerated surfactant adsorption (observed with F68) will be observed with surfactants that form stable surfactant-protein associations. Optical waveguide lightmode spectroscopy was used to test this expectation. Adsorption kinetics were recorded for surfactants (PS 20, PS 80, or F68) and protein (lysozyme or Amgen recombinant protein) at a hydrophilic solid (SiO���-TiO���) surface. Experiments were performed in sequential and competitive adsorption modes, enabling the adsorption kinetic patterns to be interpreted in a fashion revealing the dominant mode of surfactant-mediated stabilization of protein in each case. Kinetic results confirmed predictions based on our earlier quantitative analysis of protein effects on surface tension depression by surfactants. In particular, PS 20 and PS 80 are able to inhibit protein adsorption only by their preferential location at the interface, and not by formation of less surface active, protein-surfactant complexes. On the other hand, F68 is able to inhibit protein adsorption by formation of protein-surfactant complexes, and not by its preferential location at the interface. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Sept. 24, 2012 - Feb. 24, 2013.

Self-assembled monolayers have been proven to be well-ordered and to give stable ultrathin films. They show a remarkably high diversity with respect to their functionalisation giving rise to many possible applications. This thesis is focused on the potential use of these molecular thin films in life sciences. The reproduction of a membrane-like environment with these tightly packed and organized unimolecular layers has led to important breakthroughs in their nanotechnological application as biomaterials. Their straightforward modification allows the chemical and physical properties of biological interfaces to be altered. In particular, Oligo(ethylene glycol) based alkanethiol self-assembled monolayers were intensively studied as biointerfaces for their ability to resist the non specific adsorption of proteins. The electrostatic repulsion which originates from these monolayers was seen as one of the possible factors causing this protein repulsion. On the other hand proteins adsorb on alkanethiol self-assembled monolayers. This can be partially attributed to an attractive hydrophobic interaction between the biomolecules and the surface. As a result of the understanding of these two driving forces which are relevant for non-specific protein adsorption/repulsion, novel self-assembling molecules were tailored in an attempt to adjust the adsorption of proteins at the SAM-liquid interface. This was conceivable with these newly designed SAMs since they allow a combination of these forces. We have chosen the ionic strength of the liquid environment as the external parameter which could act on the amount of adsorbed proteins because the electrostatic force created by oligo(ethylene glycol) groups depends on it. In addition to the synthesis of six new molecules, the preparation and characterisation of the novel self-assembled monolayers are reported in this thesis. The density of the monolayers was estimated by X-ray photoelectron spectroscopy and ellipsometry, and the wettability properties were studied by measuring the contact angle. The total force acting on proteins from the SAMs was studied with an atomic force microscope, equipped with a tip mimicking proteins, by measuring force-distance curves. An in-situ technique was investigated in order to study the influence of the variation of this total force on the quantity of adsorbed proteins by varying the ionic strength.

The marked increase in surface-to-volume ratio associated with microscale devices for hemodialysis leads to problems with hemocompatibility and blood flow distribution that are more challenging to manage than those encountered at the conventional scale. In this work, stable surface modifications with pendant polyethylene oxide (PEO) chains were produced on polycarbonate microchannel and polyacrylonitrile membrane materials used in construction of microchannel hemodialyzer test articles. These coatings were evaluated in relation to protein repulsion, impact on urea permeability through the membrane, and impact on bubble retention through single-channel test articles. PEO layers were prepared by radiolytic grafting of PEO-PBD-PEO (PBD = polybutadiene) triblock copolymers to microchannel and membrane materials. Protein adsorption was detected by measurement of surface-bound enzyme activity following contact of uncoated and PEO-coated surfaces with ��-galactosidase. Protein adsorption was decreased on PEO-coated polycarbonate and polydimethyl siloxane (PDMS) materials by 80% when compared to the level recorded on uncoated materials. Protein adsorption on membrane materials was not decreased with PEO-PBD-PEO treatment; a PEI (polyethylene imide) layer exists on the AN69 ST membrane which is intended to trap heparin during membrane pre-treatment. It is still unclear how this PEI layer interacts with PEO-PBD-PEO. Neither the PEO-PBD-PEO triblocks nor the irradiation process was observed to have any effect on polyacrylonitrile membrane permeability to urea, nor did the presence of additional fibrinogen and bovine serum albumin (BSA) in the urea filtrate. The PEO-PBD-PEO treatment was not able to visibly reduce bubble retention during flow through single-channel polycarbonate test articles, however, the rough surfaces of the laser-etched polycarbonate microchannels may be causing this bubble retention. This surface treatment holds promise as a means for imparting safe, efficacious coatings to blood processing equipment that ensure good hemocompatibility and blood flow distribution, with no adverse effects on mass transfer. / Graduation date: 2013