Proteins, e.g. antibodies or enzymes, are highly attractive molecules for both research and application due to their specific properties and functionalities. A vast majority of the applications involve immobilized molecules, i.e. proteins which are attached to a solid substrate. Ideally, an accurate control over the amount, position and orientation of the immobilized biomolecules is required, as the functionality of the proteins would be retained and they would hence remain available for the intended purpose. Unwanted side-effects from the immobilization such as protein agglomeration, denaturation or steric hindrance between neighbouring molecules could be minimized. However, despite its importance, a precise control (on the molecular level) of the immobilization process is scientifically and technologically still very challenging.
In this work we will present an elegant way of providing pre-defined protein binding-sites on solid surfaces by using vacuum-deposited metallic nanoparticles. These nanoparticles, with a size matching the size of the protein, could in principle allow for the immobilization of only one molecule per particle. Specific protein binding-sites on the surface are made redundant, however, if non-specific protein attachment is not prevented and proteins would cover the entire substrate surface. The avoidance of non-specific protein attachment is hence an important constraint in order to achieve localized binding sites. This combination of vacuum-deposited nanoparticles on a protein-repellent layer creates a truly bi-functional surface, i.e. consisting of protein-permissive and protein-repellent areas, allowing for a directed immobilization of proteins due to the chemical contrast between the two materials. In the scope of this thesis we will focus on tuning the amount and the position of immobilized proteins and we will investigate how the immobilization influences their biological functionality. The first part of this thesis is focussed on the preparation of clean and appropriately terminated metallic and semiconductor surfaces. It will be shown that a proper surface termination (e.g. an OH-termination) is a necessary precondition for the subsequent surface functionalization, e.g. with a protein-repellent PEG-silane layer. These PEG-silanized SiO2 surfaces reduced efficiently non-specific protein absorbtion, in contrast to unmodified SiO2 surfaces.
The second part reports on the deposition of nanoparticles onto the protein- repellent polymer layer. The nanoparticles with sizes below 5 nm, i.e. in the size range of proteins, are deposited from ultra-pure noble metals (Au and Pt) and do not require a stabilizing ligand layer. Nanoparticles used in this work are produced by two different vacuum-based methods, namely from atomic beams by molecular beam epitaxy and from pre-formed cluster beams from a laser vaporization cluster source. It will be investigated how these two methods yield different surface morphologies after nanoparticle deposition.
The main part of this thesis is focussing on the linker-molecule mediated immobilization of two structurally and functionally different types of proteins, namely antibodies (human- and anti-human- Immunoglobulin) and enzymes (Glucose oxidase), onto the deposited nanoparticles. We will link the number of immobilized proteins to the nanoparticle density by a combination of qualitative and quantitative analysis techniques, using various atomic force microscopy-, optical microscopy- and spectroscopy- techniques. We analyze the change in surface morphology after each individual surface preparation step, along with an in-situ detection of protein immobilization using a quartz crystal microbalance. This enables us to demonstrate that the amount of immobilized protein can be controlled by a variation of the nanoparticle coverage values.
This thesis also thrives to understand how protein immobilization on nanopar- ticles influences their biological functions. The biological activity of the two different protein types after immobilization will be compared with their activity in solution. Although a lower activity is observed for immobilized proteins, yet biological activity is retained and we can conclude that the concept of vacuum- deposited nanoparticles as protein binding sites on a protein-repellent layer is a valid approach.