Protein adsorption controlled by multivalent ions

Proteins are a substantial, and integral matter for life on earth since they are required, e.g. to transport nutrients, to regulate body functions, and to form the building blocks of muscles, skin, and hair. Depending on their structure and function, proteins can be divided into four groups: fibrous, globular, disordered, and membrane proteins. Globular proteins are water-soluble, spherical proteins and can show a rich phase behaviour featuring aggregation, liquid-liquid phase separation (LLPS), re-entrant condensation, and crystallisation in the presence of multivalent salts. Consequently, the dominant interactions are electrostatic forces. The knowledge about the driving force of the bulk phase behaviour is crucial to guide and manipulate the phase behaviour since certain aggregations are desired, whereas for example the formation of amyloid fibrils contribute to diseases such as Alzheimer. In this dissertation, the rich phase diagram of globular proteins with multivalent salt is utilised to investigate protein adsorption. Protein adsorption, meaning the accumulation or aggregation of proteins at a solid interface, occurs in numerous areas from medicine to food processing. Especially in the context of biomaterials, protein adsorption can be both an advantage and disadvantage since it facilitates biocompatibility, but can also induce a foreign body response, which leads to the rejection of an implant. While the general functions of proteins are well understood, less is known about underlying dominant molecular interactions which drive protein adsorption. The aim of this dissertation is to shed light on the dominant interactions driving and parameters influencing the adsorption behaviour such as protein type, cation and anion type, solvent, temperature, and surface properties. The main findings are summarised in the subsequent paragraphs. In the first results chapter, the correlation between bulk phase behaviour and protein adsorption at a net negatively charged surface as a function of the salt concentration is established. Re-entrant adsorption at the interface is observed, which reflects the bulk re-entrant condensation behaviour in an intriguing way measured by a reduced second virial coefficient (measure for dominant bulk interactions). The experimental findings can be described by the multivalent-ion-activated patchy particle model within the framework of classical density functional theory, which reduces the adsorption trend found to dominant electrostatic interactions between proteins and with the surface. In the second part, the effects of temperature and protein concentration on the protein adsorption are investigated, which induce simple adsorption or (diverging) wetting layer formation upon approaching bulk phase separation. Approaching LLPS increases attractive short-ranged interactions in the system facilitating the onset of a wetting transition. Through variations in protein concentration and temperature, LLPS formation can be promoted or prevented in the bulk solution. The wetting layer contains significantly more water trapped within the (protein) layer compared to a simple adsorption layer illustrating different layer morphologies between an adsorption and wetting layer. The experimental findings are in good agreement with the theoretical descriptions of ion-activated attractive patches experiencing an attractive wall potential within the framework of classical density functional theory. In the third part, the role of anions and cations on bovine serum albumin (BSA) in the bulk and at the interface are investigated. The bulk phase behaviour not only depends on the cation type used, but also on the anion type, which leads to different phase behaviours for different salts. Chloride is found to be ‘neutral’ in the sense that it neither affects the bulk nor interface behaviour of proteins, whereas for weakly hydrated anions (iodide salts), the established ion-activated patchy model is not valid since it neglects anion interactions. These anion interactions are assumed to consist of an interplay of electrostatic and hydrophobic interactions due to the different BSA binding sites for iodide. The phase transitions at the interface can also be induced by specific interface properties, whether these behaviours are reflected in bulk or not (i.e. bulk-independent adsorption), which emphasises the interplay of the substrate and bulk properties relating to protein adsorption. In the fourth part, the influence of anion type and solvents (includes different isotopes) on β-lactoglobulin (BLG) in the bulk and at the interface are investigated. The bulk and interface behaviour of BLG is not influenced by the anion used due to BLG’s weak affinity to anions and its preference for cation binding. In addition, BLG does not show an isotope effect. This could be explained with its predominant β-sheet structure, in which isotope substitution does not occur. At the interface, a much denser packing of the adsorption layer for BLG compared to BSA is found, which could explain why BLG crystallises and BSA does not. In the fifth part, preliminary results on surface modifications and their influence on protein adsorption are discussed. The parameters investigated include the surface charge, roughness (topography), and hydrophobicity, as well as substrates with implant coatings and protein-repellent properties and should be viewed as an outlook and starting point for future endeavours. A striking comparison between the bulk and interface behaviours of BLG and BSA is drawn in the conclusions summarising previous and current findings to explain the system behind protein adsorption and its underlying mechanisms. The knowledge of the mechanisms behind protein adsorption allows the targeted manipulation of parameters influencing protein adsorption such as temperature, salt type and concentration, and surface properties. This presents new prospects in the investigation of protein crystallisation through controlled nucleation at interfaces, which is particularly of relevance in the fields of pharmacology and biomaterials