Neutron and X-ray reflectivity studies of human serum albumin adsorption onto functionalized surfaces of self-assembled monolayers.

Neutron and X-ray reflectivity (NR and XR) have been widely used for the investigation of the structure of thin organic films. Here we demonstrate how these sensitive techniques may be applied to the study of protein adsorption to well-characterized self-assembled monolayers (SAMs) with different chemical functionalities. NR can be used for in situ study, while XR provides complementary information on the initial surfaces and dried layers measured in air after protein has been adsorbed. In situ measurements of adsorption of human serum albumin onto a hydrophilic NH2-terminated monolayer clearly show the presence of a thin layer of adsorbed protein next to the SAM. Adsorption of albumin onto a hydrophobic, deuterated, CD3-terminated SAM causes even bigger changes in the NR. Upon replacing the protein solution with protein-free buffer solution, the reflectivities from both kinds of monolayers do not change, demonstrating that the albumin adsorption is irreversible after several hours of contact with the protein solution. X-ray reflectivity measurements of dried substrates performed ex situ in air provide a lower bound estimate of the amount of protein which must be at the interface in situ. This combination of techniques provides a uniquely sensitive approach for studying changes that occur upon protein adsorption at an interface.

Contact activation of the plasma coagulation cascade. I. Procoagulant surface chemistry and energy.

Contact activation of the intrinsic pathway of porcine blood plasma coagulation is shown to be a steep exponential-like function of procoagulant surface energy, with low activation observed for poorly water-wettable surfaces and very high activation for fully water-wettable surfaces. Test procoagulants studied were a system of oxidized polystyrene films with varying wettability (surface energy) and glass discs bearing close-packed self-assembled silane monolayers (SAMs) with well-defined chemistry consisting of 12 different terminating chemical functionalities. A monotonic trend of increasing coagulation activation with increasing water wettability was observed for the oxidized polystyrene system whereas results with SAM procoagulants suggested a level of chemical specificity over and above the surface energy trend. In particular, it was noted that coagulation activation by SAMs terminated with--CO2H was much higher than anticipated based on surface wettability whereas--NH3(+)-terminated SAMs exhibited very low procoagulant activity. SAMs terminated in--(CH2)2(CF2)7CF3 behaved as anticipated based on surface energy with very low procoagulant activity and did not exhibit special properties sometimes attributed to perfluorinated compounds. Quantitative ranking of the inherent coagulation activation properties of procoagulant surfaces was obtained by application of a straightforward phenomenological model expressed in a closed-form mathematical equation relating coagulation time to procoagulant surface area. Fit of the model with a single adjustable parameter to experimental measurements of porcine platelet-poor plasma coagulation time was very good, implying that assertions and simplifications of the model adequately simulated reality. Two important propositions of the model were that (1) the number of putative "activating sites" scaled linearly with procoagulant surface area, and (2) contact activation of the plasma coagulation cascade was catalytic in the sense that these activating sites were not consumed or "poisoned" by irreversible or slowly reversible protein adsorption during coagulation. An extension of the coagulation model proposed that procoagulant activation properties scale exponentially with the surface density of polar (acid-base) sites, which, in turn, was related to procoagulant wettability.

Contact activation of the plasma coagulation cascade. II. Protein adsorption to procoagulant surfaces.

A study of blood protein adsorption to procoagulant surfaces utilizing a coagulation time assay, contact angles, Wilhelmy balance tensiometry, and electron spectroscopy (ESCA) is presented. Using a new contact angle method of measuring protein adsorption termed "adsorption mapping" it was demonstrated that protein-adsorbent surfaces were inefficient activators of the intrinsic pathway of the plasma coagulation cascade whereas water-wettable, protein-repellent surfaces were efficient procoagulants. Repeated use of fully water-wettable (spreading) glass procoagulants in the coagulation time assay demonstrated that putative "activating sites" were not consumed in the coagulation of platelet-poor porcine plasma. Furthermore, these procoagulant surfaces retained water-wettable surface properties after incubation with blood proteins and saline rinse. The interpretation of these observations was that plasma and serum proteins were not adsorbed to water-wettable surfaces. However, ESCA of these same surfaces revealed the presence of a thin protein layer. Wilhelmy balance tensiometry resolved these seemingly divergent observations by demonstrating that protein was "associated" with a bound hydration layer, but not formally adsorbed through a surface dehydration or ionic interaction mechanism.