A New Model of Protein Adsorption Kinetics Derived from Simultaneous Measurement of Mass Loading and Changes in Surface Energy

Abstract

We describe a novel technology based on changes in the resonant frequency of an acoustically actuated surface and use it to measure temporal changes in the surface energy gamma (N m(-1)) of an elastomeric polymer membrane due to the adsorption of macromolecules from aqueous solution. The resonant elastomeric surface-tension (REST) sensor permits simultaneous determination of mass loading kinetics and gamma(t) for a given adsorption process, thereby providing a multivariable data set from which to build and test models of the kinetics of adsorption at solid-liquid interfaces. The technique is used to measure gamma(t) during the adsorption of either sodium dodecyl sulfate (SDS) or hen egg-white lysozyme (HEWL) onto an acrylic polymer membrane. The adsorption of SDS is reversible and is characterized by a decrease in gamma over a time period that coincides with that required for the mass loading of the membrane. For the adsorption of HEWL labeled with Alexa Fluor 532 dye, gamma continues to change long after the surface concentration of labeled HEWL, measured by using the elastomeric polymer membrane as an optical waveguide, reaches steady state. Gradual but significant changes in gamma(t) are observed as long as the concentration of protein in the bulk solution, c(b), remains nonzero. HEWL remains adsorbed to the membrane when c(b) = 0, but changes in gamma(t) are not observed under this condition, indicating that the interaction of bound protein molecules with those free in solution contribute to the prolonged change in the surface energy. This observation has been used to define a new model for the kinetics of globular protein adsorption to a solid-liquid interface that includes a mechanism by which the molecules in the bulk can facilitate the desorption of a sorbate molecule or change the energetic states of adsorbed molecules and, thus, the overall surface energy. The model is shown to capture the unique features of protein adsorption kinetics, including the relatively fast mass loading, the much more gradual change in surface energy that does not cease until the protein is removed from the bulk, the rapid desorption of an incubation-time-dependent fraction of bound protein when the protein is removed from the bulk, and the fixing of the residual surface concentration and surface energy at constant values once the removal of reversibly bound protein and free protein is complete.

Study of Adsorption of Globular Proteins on Hydrophobic Surfaces

“…Similar observations were reported by Clark et al, where the degree of reversibility of protein adsorption was found to decrease with increasing contact time [3]. The observed irreversibility of the desorption process has been attributed to interfacial conformational changes with time of the sorbate molecules resulting in strong interactions between the surface and the adsorbate [1].…”

Volumetric interpretation of protein adsorption: Kinetic consequences of a slowly-concentrating interphase

“…Understanding how protein arrives at, and adsorbs to, biomaterial surfaces is thus one of the most fundamental problems of biomaterials surface science. It is of practical interest therefore, to consider how this work might impact understanding of the fundamentals of biocompatibility.Based on the findings of this and recent work on protein adsorption [22][23][24]38], we conclude that immersion of a hydrophobic surface into a concentrated, multi-component protein solution such as blood [78,79] leads to virtually instantaneous accumulation of protein molecules from the proximal fluid phase (relative to the timescale of the observable acute biological responses to materials; see Appendix A). Given that (i) surface capacity for protein is actually quite small (in the range of 2-3 mg/m 2 or µmoles/m 2 for kDa-size proteins), (ii) the total blood-protein concentration is large (50-60 mg/mL, [78,79]), and that (iii) proteins exhibit surprisingly little difference in adsorption energetics across a broad range of blood protein types (3 decades in molecular weight, see refs.…”

“…Using a novel resonant elastomeric surface tension (REST) sensor, Clark et al recently measured rapid mass accumulation of hen egg-white lysozyme associated with much more gradual change in surface energy (tension of adsorbed protein) [38]. Clark et al thus capture the essence of observation (i); relatively fast mass adsorption that is not directly coupled to slow change in interfacial tension.…”

“…Partly because of this, we suspect, modern embellishments of RSA theory are forced to embrace an ever-increasing number of variables to explain all experimental outcomes (see, as a recent example, ref. [38]). We speculate (but do not show herein) that simpler protein-adsorption kinetic models can be assembled by linking diffusion-based theories (such as that of Varoqui and Pefferkorn [13]) with a 3D interphase model in a way that that fills an expanding interphase volume by diffusion at a rate and extent that is dependent on bulk-solution concentration, followed by relatively slow interphase concentration by the elimination of interphase water.…”

“…It is important to stress that this assumption is particular only to proteins at the relatively-high solution concentrations studied herein. It is known that mass-adsorption kinetics can be observed for proteins at low concentrations (≤ 1 mg/mL) by sensitive techniques with good time resolution [34,38].…”