CuAAC-mediated Protein Immobilization towards Next Generation Biosensing Devices

Abstract

Miniaturization of microarrays is one of the key challenges in the development of advanced biosensing devices. Downscaled biosensors require smaller amounts of expensive biological receptor material, such as antibodies, enzymes and cells. This lowers their costs tremendously and makes them superior to existing devices. To achieve this size reduction, revision and optimization of currently used techniques is required and new promising routes should be explored. The immobilization process of the bioreceptors to the transducer is one of the key features needing further optimization. Commonly used covalent immobilization techniques utilize multiple functional groups of the protein’s autogenic amino acids, leading to random oriented layers. However to achieve better performance, it can be envisioned that a different approach should be followed using a site-specific and bio-orthogonal group present at the outside of the bioreceptor. This is a result of the fact that a stable and unique oriented coupling of the receptor to the surface will lead to a homogeneous surface coverage, which is a prerequisite for device miniaturization. ‘Click’ chemistry, e.g. CuAAC, provides opportunities towards this controlled coupling. This is exemplified by the presented research, in which non site-specifically functionalized Staphylococcus aureus protein A (SpA) is used as a model bioreceptor to optimize the CuAAC immobilization conditions. Furthermore, a comparison with existing immobilization procedures, e.g. physical adsorption and covalent EDC/NHS coupling, has been performed, demonstrating that ‘click’ strategy can be a viable alternative. Finally, experiments with C-terminal alkyne-functionalized Maltose Binding Protein (MBP) demonstrate the potential improvements of site-specific immobilization to complementary functionalized surfaces. Ellipsometry, a technique with nanogram sensitivity, is used for the online quantification of the immobilized surface mass.