title: Temperature dependent activity and structure of adsorbed proteins on plasma polymerized N-isopropyl acrylamide.
abstract: Thorough studies of protein interactions with stimulus responsive polymers are necessary to provide a better understanding of their applications in biosensors and biomaterials. In this study, protein behavior on a thermoresponsive polymer surface, plasma polymerized N-isopropyl acrylamide (ppNIPAM), is investigated using multiple characterization techniques above and below its lower critical solution temperature (LCST). Protein adsorption and binding affinity are probed using radiolabeled proteins. Protein activity is estimated by measuring the immunological activity of an antibody adsorbed onto ppNIPAM using surface plasmon resonance. Conformation/orientation of the proteins is probed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and principal component analysis (PCA) of the TOF-SIMS data. In this work, we find that at low protein solution concentrations, ppNIPAM-treated surfaces are low fouling below the LCST, but protein retentive above it. The protein adsorption isotherms demonstrate that apparent affinity between soluble protein molecules and the ppNIPAM surface are an order of magnitude lower at room temperature than at 37 degrees C. Although direct protein desorption is not observed in our study when the surface temperature drops below the LCST, the binding affinity of surface adsorbed protein with ppNIPAM is reduced, as judged by a detergent elution test. Furthermore, we demonstrated that proteins adsorbed onto ppNIPAM are functionally active, but the activity is better preserved at room temperature than 37 degrees C. The temperature dependent difference in protein activity as well as TOF-SIMS and PCA study suggest that proteins take different conformations/orientations after adsorption on ppNIPAM above and below the LCST.
authors.affiliation: University of Washington Engineered Biomaterials, Department of Bioengineering, University of Washington, Seattle, Washington 98195-5061, USA.
authors.forename: Xuanhong
authors.initials: X
authors.lastname: Cheng
authors.forename: Heather E
authors.initials: HE
authors.lastname: Canavan
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Graham
authors.forename: David G
authors.initials: DG
authors.lastname: Castner
authors.forename: Buddy D
authors.initials: BD
authors.lastname: Ratner