Oriented antibodies as versatile detection element in biosensors

The aim of this thesis is to explore orientation of detection elements on biosensor surfaces. To this end, different strategies were combined such as surface chemistry and protein functionalization, with the aim to generate a platform for oriented immobilization of antibodies in biosensors. Chapter 2 investigates the formation of organic monolayers on oxide-free copper. Detailed studies were performed to characterize the monolayers and proof its quality. Apart from being the first oxide-free monolayers on copper reported thus far, further functionalization was successfully investigated. Chapter 3 gives an overview about approaches used to orient antibodies on surfaces. It also summarizes methods used to characterize the orientation of immobilized antibodies in a more direct manner. In chapter 4 a set of detection elements for tuberculosis bacteria is described. These are variable domains of llama heavy-chain antibodies, known as V_HH proteins. A number of V_HHs, selected by phage display, were expressed by <em>Escherichia coli</em> bacteria and characterized for binding towards <em>Mycobacterium tuberculosis</em> bacteria. Specificity of V_HHs was investigated and the antigen was identified. In chapter 5 the impact of orientation on the analyte binding capacity was studied by SPR as model biosensor. Established techniques (NH₂ coupling, biotyinylation) were used to immobilize V_HHs, and a comparison between oriented and random immobilized V_HHs was made. The effect of molecular weight, epitope number and affinity of the target analyte was investigated. In chapter 6, a novel coupling chemistry was used to immobilize V_HHs, and in this case the same chemistry could be used for oriented and random immobilization. V_HHs were engineered and functionalized with a non-natural amino acid to bear either one or five azide groups. Azide groups served as unique chemical handles on the V_HHs and were used to click proteins onto a cyclooctyne-modified surface in an oriented and random approach. Spectacular effects on biosensor sensitivity were observed when V_HHs were immobilized in an oriented manner. Finally, in chapter 7, the main results of this thesis are summarized and remaining problems as well as ideas for future research are discussed.