Mechanisms of Molecular Brush Inhibition of Protein Adsorption onto Stainless Steel Surface

摘要

Protein-resistant ("non-fouling") surfaces are particularly important in manyfields such as medical engineering, dentistry, pharmaceutical processes,bioprocessing, dairy and food manufacturing. Poly(ethylene glycol) (PEG)immobilized onto surfaces has been shown to confer high resistance to proteinadsorption. The reasons for variable performance and optimal protein repellency ofPEG layers have been the subject of much discussion; however there remains nogeneral consensus on the molecular mechanisms underlying the protein resistanceachieved with PEG coatings.The main objective of this study was to inhibit protein adsorption onto astainless steel surface. This objective requires an exploration of the mechanisms ofprotein adsorption on a stainless steel surface and how these mechanisms aremodified when a surface inhibits the adsorption of proteins. The stainless steelsurface has been chosen as a substrate as it is a commonly used material in manyrelevant applications such as in the dairy industry, in food processing and in clinicaluses.In order to elucidate the mechanisms of protein-PEG interactions theadsorption of lysozyme, β-casein, apo α-lactalbumin, holo α-lactalbumin and β-lactoglobulin onto various PEG-grafted surfaces was explored. The adsorption wasconducted at room temperature and at 40 C. The modification of bare SS surfacesand adsorption kinetics of proteins on unmodified and modified surfaces (i.e. barestainless steel and PEG surfaces) has been done in-situ and studied by means of aquartz crystal microbalance with dissipation sensing (QCM-D). The merit of themodification methods studied, compared to those of most published methods is thatthe process of modification is simple and easy, being done simply by passing asolution over the surface. The methods also do not involve any harmful or hazardouschemicals and thus are safe to be used even in food processing plants.The PEG coated surfaces prepared in this study were able to inhibit adsorptionof β-casein, α-lactalbumin (calcium enriched) and lysozyme proteins especially; thelowest adsorption of these achieved as a percentage of that on bare stainless steel, β-casein, 45 %, holo α-lactalbumin, 11 % and lysozyme, 1 %. By contrast, andunexpectedly, PEG molecules enhanced the adsorption of apo α-lactalbumin (theform without calcium). It is suggested that the PEG to apo α- lactalbuminhydrophobic interaction plays a dominant role which leads to protein aggregation atthe surface, for this latter observation. The results have shown that protein stability(i.e. whether it is a soft or a hard protein) greatly influenced the inhibitionperformance of PEG surfaces. It is apparently more difficult to prevent the adsorptionof soft proteins than hard proteins. This appears to be because soft proteins tend todenature regardless of the surface properties (i.e. hydrophilic or hydrophobic) andattach more effectively in their unfolded state. The results also indicated that higherPEG grafting density is not necessarily reflected in better protein inhibition.At the end of the project, a novel method of surface modification wasdeveloped. In this method, stainless steel surfaces were modified by coating thesurface with a protein layer (as a base) then followed by the attachment of PEGmolecules. Interestingly, the method developed showed an excellent potential forpreventing further protein adsorption at room and body temperatures. The adsorptionof β-casein, lysozyme, holo α-lactalbumin and β-lactoglobulin on the SS-lysozyme-PEG surfaces was down to about 3, 1, 4 and 0.4 %, respectively compared to that onthe bare surface. More interestingly and surprisingly also, there was almost zeroadsorption on those surfaces of mixed protein and single protein solutions at theconcentration found in milk. The method is believed to have the potential to beapplied in the pharmaceutical industry, in the biosensor field and in artificial medicalimplants with some modifications perhaps to suit the application.The modelling results demonstrated negative free energy changes onadsorption, consistent with the studied proteins being thermodynamically favoured toadsorb on bare SS. The adsorption of proteins was an endothermic process. Theproteins also showed large positive entropy changes on adsorption, indicatingadsorption-induced denaturation mechanisms (especially apo α-lactalbumin protein).At high temperatures and concentrations, the adsorption was governed first bydiffusion and later by surface kinetics, whereas under lower temperature (i.e. roomtemperature) and low concentration conditions (i.e. 0.1 g / L) the adsorption was ableto be described solely by surface-reactions.