Analytical methods founded upon whole cell-based assays are of importance in early stage drug development and in fundamental studies of biomolecular recognition. Here we have studied the binding of the monoclonal antibody trastuzumab to human epidermal growth factor receptor 2 (HER2) on human ovary adenocarcinoma epithelial cancer cells (SKOV3) using quartz crystal microbalance (QCM) technology. An optimized procedure for immobilizing the cells on the chip surface was established with respect to fixation procedure and seeding density. Trastuzumab binding to the cell decorated sensor surface was studied, revealing a mean dissociation constant, K-D, value of 7 +/- 1 nM (standard error of the mean). This study provides a new perspective on the affinity of the antibody-receptor complex presented a more natural context compared to purified receptors. These results demonstrate the potential for using whole cell-based QCM assay in drug development, the screening of HER2 selective antibody-based drug candidates, and for the study of biomolecular recognition. This real time, label free approach for studying interactions with target receptors present in their natural environment afforded sensitive and detailed kinetic information about the binding of the analyte to the target.

Phage display screening of a surface-immobilized adenine derivative led to the identification of a heptameric peptide with selectivity for adenine as demonstrated through quartz crystal microbalance (QCM) studies. The peptide demonstrated a concentration dependent affinity for an adeninyl moiety decorated surface (KD of 968 ± 53.3 μM), which highlights the power of piezoelectric sensing in the study of weak interactions. 

Background: The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements. Results: Biotin-selective thin (3-5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N, N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts. Conclusions: Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems.

The objective of this thesis was to explore novel approaches for studying molecular recognition at biological and biomimetic surfaces using the quartz crystal microbalance (QCM) biosensor technique. The first two papers focused on the synthesis and study of biotin selective polymer films prepared using the molecularly imprinted polymer (MIP) technique. Control over polymer structure is of importance for sensor reproducibility and sensitivity, and was addressed in Paper I where a simple strategy for fabricating uniform thin biotin imprinted polymer films was employed. In Paper II the binding of biotin moieties to thin (3-5 nm) biomimetic polymer films was examined and consequences for sensor performance discussed. The potential for using QCM as a tool for assessing the binding of small peptides derived from phage display screening was presented Paper III. Here, screening of a phage peptide library against immobilized adenine resulted in candidate peptides that were studied using this technique. In Paper IV a whole cell-based biosensor was developed for studying interactions with cell membrane-incorporated targets. Epithelial cancer cells, SKOV3, were attached to QCM sensor chips and the binding of the monoclonal antibody Herceptin^TM was studied. This approach demonstrates the potential of using QCM to study binding to membrane-incorporated targets, an alternative to assays based upon immobilized receptor structures lacking their natural context.

The expression of peptides on phage coat proteins via genetic engineering was first achieved in 1985 [1] and was followed by the construction of phage display combinatorial libraries in 1990 [2].  Phage display combinatorial libraries have been used not only to find peptides that interact with various biomolecules such as receptors, antibodies, enzyme active sites, and protein surfaces, but also to find peptides with affinity for metals and plastics and with function in non-biological environments [3].

In this study, we have challenged a cyclic heptamer phage display library with surfaces adenine functionalised surfaces with the aim of identifying peptide motifs selective for adenine. The synthesis and characterisation of surfaces, and results from phage display screening shall be presented.

[1] Smith, G.P. Science (1985) 228, 1315-1317.

[2] Scott, J.K.; Smith, G.P. Science (1990) 249, 386-390.

[3] Olofsson, L.; Söderberg, P.; Ankarloo, J.; Nicholls, I.A. J. Mol. Recognit. (2008) 19, 18-25.