Quantification of Protein Adhesion Strength to Surface Attached Poly (N- isopropylacrylamide) Networks by Hydrodynamic Detachment Shear Stresses
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Chemical and Biomedical Engineering
Ryan Toomey, Ph.D.
Aydin Sunol, Ph.D.
Nathan Gallant, Ph.D.
William Lee, Ph.D.
Sameer Varma, Ph.D.
IgG Adsorption, Peptide-Polymer Conjugate, Polystyrene Microsphere, Spinning Disk, Thermoresponsive Hydrogel
Stimuli responsive coatings offer a versatile method by which to manipulate interfacial interactions of proteins in a desired way. However, there exists little guidance as to how the structure of a responsive polymer coating influences adsorption of proteins. In this dissertation, the adsorption behavior of immuglobulin G (IgG) on poly (N-isopropylacryamide) (PNIPAAm) hydrogel coatings was investigated as a function of film thickness. PNIPAAm exhibits a hydrophilic to hydrophobic transition above a critical temperature of ~32°C in aqueous solutions. In this research, through the use of quartz crystal microbalance with dissipation (QCM-D) it was observed that the adsorption was thickness dependent and became non-reversible as the temperature was decreased. Interestingly, QCM-D results also suggested a similar amount of protein adsorption on both hydrated and dehydrated PNIPAAm surfaces. A rigid film analysis using Sauerbrey equation revealed a multi-layer formation on the collapsed PNIPAAm coatings. Although it is allegedly reported that PNIPAAm favors adsorption above the critical temperature due to hydrophobic interactions, there have been several studies that reported adsorption of proteins below the critical temperature. To better understand the QCM-D results, hydrodynamic shear force assays in a spinning disk configuration were performed in order to quickly measure and quantify adhesion of polystyrene (PS) probe spheres (10μm) to the PNIPAAm coatings in both the solvated (hydrophilic) and collapsed (hydrophobic) state. The influence of polymer coating thickness, polymer chain cross-link density, microsphere concentration and adsorption time on the adhesion characteristics of the coatings was investigated in relation with volume phase transition of the polymer coatings.
A series of experiments on quantification of the temperature dependent adhesion of proteins adsorbed on surface attached PNIPAAm coatings of thicknesses was performed as the surface chemistry was switched from hydrophilic to hydrophobic. First, adhesion of polystyrene (PS) microspheres on PNIPAAm coatings was quantified in order to have a guideline for temperature dependent adhesion performance of these coatings. PS particles were subjected to a range of detachment shear stresses through hydrodynamic flow in a spinning disk configuration. These experiments provide an indirect method to determine the force of adhesion since it is proportional to the hydrodynamic force. Model protein, IgG, was then linked to PS microspheres and the mean adhesion strength of the IgG coated PS microspheres were determined through the detachment shear stresses. The influence of PS deposition time, PS bead concentration, PNIPAAm coating thickness and PNIPAAm cross-link density on the adhesion strength were addressed. The results indicated that in the collapsed state, the adhesion of bare hydrophobic PS microspheres depends strongly on coating thickness. For hydrophilic charged PS microspheres the adhesion was always higher on the hydrated PNIPAAm surfaces and appeared not to be strongly affected by the increase in PNIPAAm coating thickness. The adhesion of IgG was higher on the collapsed PNIPAAm surfaces and the adhesion trend did not significantly change as the PNIPAAm film thickness was increased. For PNIPAAm coatings with the cross-link density reduced by factor of 10, the adhesion was again higher on the collapsed PNIPAAm surface and scaled linearly with thickness. Moreover, the influence of thickness became prominent at the higher thickness values (165 nm-185 nm). In addition, the adhesion of carboxylated microspheres on PNIPAAm did not reach equilibrium and increased linearly with microsphere deposition time.
A study on the sensing characteristics of PNIPAAm coatings in response to heavy metal ions was also conducted in this dissertation. The temperature-dependent swelling behavior of poly(N-isopropylacrylamide) and tripeptide Gly-Gly-His/poly(NIPAAm) conjugate hydrogel coatings were investigated using a quartz crystal microbalance with dissipation (QCM-D) while in contact with NaCl, ZnCl2, NiCl2, and CuCl2 solutions. To fabricate the tripeptide conjugated gels, precursor gels of poly(NIPAAm-co-3-aminopropylmethacrylamide[3.5 mole%]) were synthesized via free radical polymerization. The metal binding tripeptide, Gly-Gly-His, was subsequently synthesized in the gel via a Merrifield solid phase peptide synthesis (SPPS) technique, in which the amino group of the copolymer gel provided a functional site to support peptide synthesis. It was found that the logarithm of the transition temperature of the tripeptide Gly-Gly-His/poly(NIPAAm) conjugate hydrogel was proportional to the ionic strength, showing two distinct regions at low and high ionic strengths for the divalent ions. In the low ionic strength regime, the salting out constants were 0.08 M-1, 0.07 M-1, and 0.06 M-1 for Cu2+, Ni2+, and Zn2+, respectively, which follows the known trend for binding of the ions to Gly-Gly-His. In the high ionic strength region, when the metal-ion binding sites in the tripeptide conjugate hydrogel were saturated, the salting out constants were similar to the salting out constants associated with pure poly(NIPAAm).
Scholar Commons Citation
Sanden, Gulnur, "Quantification of Protein Adhesion Strength to Surface Attached Poly (N- isopropylacrylamide) Networks by Hydrodynamic Detachment Shear Stresses" (2014). USF Tampa Graduate Theses and Dissertations.