No abstract

The behavior of proteins at biological and synthetic interfaces is often characterized by a strong history dependence caused by long relaxation times or irreversible transitions. In this work, we introduce the rate of adsorption as a means to systematically quantify the extent, and identify the underlying causes, of history dependence. We use multistep kinetic experiments in which the ith step is an exposure of a Si(Ti)O2 surface to a flowing fibronectin or cytochrome c solution of concentration ci for a time ti (ci ‫؍‬ 0 corresponds to a rinse) and measure the protein adsorption by optical waveguide light mode spectroscopy. The rate of adsorption is sensitive to the structure of the adsorbed layer, and we observe it to greatly increase, for a given adsorbed density, when going from a first to a subsequent adsorption step. This increase is most pronounced when the duration of the initial adsorption step is long. We attribute these observations to the gradual and irreversible formation of protein clusters or locally ordered structures and use them to explain previous underestimates of kinetic data by mesoscopic model descriptions. A thorough understanding of these complex postadsorption events, and their impact on history dependence, is essential for many physiological and biotechnological processes. Optical waveguide lightmode spectroscopy is a promising technique for their macroscopic quantification.optical waveguide lightmode spectroscopy ͉ interfacial kinetics ͉ surface diffusion ͉ surface aggregation

The controlled surface placement of protein molecules represents a crucial step toward many new biotechnological devices and processes. A promising means of directing the structure and formation rate of an adsorbed protein layer is through an applied electric potential difference. We present here a method for continuously measuring the protein adsorption under a direct current voltage using optical waveguide lightmode spectroscopy. An indium tin oxide-coated waveguiding sensor chip serves as the anode and adsorbing substrate, and a platinum counter electrode serves as the cathode in a parallel plate arrangement. For (negatively charged) human serum albumin in either pure water or N-[2-hydroxyethyl]piperazine-N‘-ethanesulfonic acid (HEPES) buffer, we find the transport-limited and initial surface-limited rates of adsorption to significantly increase with the applied potential. For (positively charged) horse heart cytochrome c, we observe no influence of the voltage on the transport-limited adsorption rate in either solvent and a decrease with the voltage in the initial surface-limited rate in a HEPES (but not a pure water) solvent. Interestingly, we find the rate of adsorption at moderate to high surface density to greatly increase with the voltage for both proteins; this effect is more pronounced in water than in HEPES. We attribute this enhanced adsorption to contact between electrode and protein patches of complementary charge, leading to more oriented and efficiently packed adsorbed molecules and, in the case of high voltage, to multilayer formation.

Adsorbed layers of proteins and other macromolecules often relax structurally more slowly than they form, rendering layer growth an out-of-equilibrium process. We show here how the interfacial cavity function, Phi (the average Boltzmann factor for a single probe molecule), may be determined, using kinetic data available from optical waveguide lightmode spectroscopy, and used as a continuous, in situ measure of history dependent adsorbed layer structure. The increase of Phi observed with residence time for fibronectin and lysozyme layers suggests post-adsorption clustering on a time scale longer than that predicted by a surface diffusion model.