Molecular-dynamics simulations of polymeric surfaces for biomolecular applications

In-vitro diagnostics plays a very important role in the present healthcare system. It consists of a large variety of medical devices designed to diagnose a medical condition by measuring a target molecule in a sample, such as blood or urine. In vitro is the latin term for in glass and refers here to the fact that the samples are investigated outside the living organism. The range of target molecules is very broad, spanning from salts to small molecules, proteins, nucleic acids and cells. An important segment in this range is the measurement of macromolecules, such as biomarker proteins or nucleic acids, in biological samples. Biosensors are compact systems for rapid detection of biological molecules. The first commercial biosensor was introduced in 1975 for glucose analysis by the Yellow Springs Instrument Company, based on the pioneering work of Clark and Lyons. Since that time, biosensors are becoming more integrated, more sensitive, smaller, faster and cheaper and are becoming available for more and more classes of biomarkers. The immunoassays can be performed nowadays in devices with very different formats, from the high-throughput parallel analysis on well-plates to integrated point-of-care biosensors using lab-on-a-chip technology. The solid phase in these devices is very often a polymeric glass. Polymeric glasses, such as polystyrene, are easy to process and can be produced at low costs, which makes them suitable for disposable cartridges in lab-on-a-chip devices. An important process in the immunoassays is the physisorption of the macromolecules to the polymeric solid phase, such as the non-specific binding of molecules from the biological sample onto the polymeric carrier. This process plays a very important role in the limit of detection, which is given by specific binding (signal) over non-specific binding (background). Therefore, the understanding of the non-specific binding of macromolecules to polymeric surfaces is crucial for the improvement of the sensitivity in these devices. The scope of this thesis is to gain fundamental knowledge on the physisorption of proteins onto polymeric surfaces and to understand how to model this process in atomistic details. The goal of this work is to model the interaction between myoglobin and polystyrene surfaces, within a clean buffer. Computer simulations provide detailed informations about the nature of the non-specific interactions between the biomolecule and the polymeric substrate, at molecular and atomic scales. The high level of detail obtained from simulations on a smaller scale is complementary to the experimental results obtained at a larger scale. The polymeric substrate is in our case modeled by an atactic amorphous polystyrene thin film. We would like to explore the possibility of changing the properties of the polymeric substrate, to prevent non-specific interaction, by chemical modification induced by oxidation. Pure polystyrene is a hydrophobic material. We tune the hydrophilicity of its surface by adding oxygen atoms to the phenyl rings of the polystyrene chain. This addition of oxygen is a way to mimic the oxidation of polystyrene surfaces that is performed in experiments. We represent the buffer solution in simulations by explicit water molecules described at atomistic level, to which we add Na+ and Cl- ions to reproduce the salt concentration in the experimentally used buffer solution. We chose myoglobin as model biomolecule because it is a relatively small globular protein, well studied in the past and represents a good candidate for practical applications. The first question we intend to answer is to which extent the model used to represent the polystyrene chains is important for the macroscopic properties of the polystyrene films and in particular to their interaction with the water molecules. To tackle this issue, we chose two representations of the polystyrene chains: the united atoms representations on one hand, in which only the heavy atoms are modeled explicitly and the hydrogen atoms are collapsed on the carbon atoms to which they are covalently bonded, and the dummy-hydrogens atoms representation on the other hand, in which the hydrogen atoms are modeled as interaction sites with no mass and with a positive partial electrical charge. The results of these simulations are presented in Chapter 3. We begin our systematic study on the interacting species in a biomedical device by characterizing the atactic amorphous polystyrene substrate. In Chapter 4 we present the results of molecular-dynamics simulations of polystyrene surfaces with controlled degree of oxidation. The variations in degree of oxidation at the surface, ranging from 0% to 24%, correspond to different degrees of hydrophilicity of the polystyrene surface, from hydrophobic to hydrophilic. We study the influence of the oxidation on the roughness of the film, both in vacuum and in water environment. We compare our results with experimental results from AFM measurements obtained by our collaborators. We also analyze the ordering of the molecular segments in non-oxidized and oxidized polystyrene at the interface with vacuum and with water. The structure of the water interface near polystyrene surfaces with different hydrophilicity is analyzed as well. Since the interaction between proteins and polymeric surfaces is a water-mediated process, it is very important to know how water behaves near these surfaces. In Chapter 5 we discuss the dynamics of water near non-oxidized (hydrophobic) and oxidized (hydrophilic) polystyrene surfaces, both in united-atoms and dummy-hydrogen atoms representations. We discuss the orientational dynamics of water molecules and its dependence on the distance from the interface. Furthermore, the translational diffusion of water molecules is briefly discussed. In Chapter 6 we study the nature of non-specific adsorption of myoglobin, as model protein, to hydrophobic and hydrophilic polystyrene surfaces. We investigate the importance of the orientation of the protein in the process of adsorption. We also discuss the influence of the hydrophilicity of the surface on the strength of adsorption of the protein. We conclude this thesis by Chapter 7, in which our main results are summarized. In addition, we give there an outlook on further interesting questions.