Polydimethylacrylamide surface functionalization for the optimization of enzymatic assays in technical and diagnostic applications

Abstract

Enzymes are biochemical reaction catalysts that are of crucial importance for everyday life. Beside playing an essential role in accelerating and regulating biochemical reactions in living organisms, enzymes contribute significantly to both technical and medical applications. The ability to regulate the rate and specificity of reactions gives enzymes their importance. In technical applications, enzymes have already led to significant changes as they offer a sustainable and environmentally friendly alternative to conventional chemical processes. For healthcare, enzymes are valuable diagnostic and therapeutic targets as they are associated with a wide range of diseases. The objective of this work was to address current limitations and drawbacks of enzymatic applications, while also optimizing their applicability and expanding their scope. The first aim was to develop a method to enhance existing enzymatic processes, thus providing an essential tool on the way to greener and more sustainable chemistry. In the second part of the project, this method was to be adapted for the use in a diagnostic assay to detect bacterial enzymes. In order to achieve these goals, different strategies involving polydimethylacrylamide surface functionalization were implemented for enzymatic applications in technical and biomedical contexts. In order to enhance enzymatic assays for more sustainable reactions, different polymer surfaces were functionalized with a polydimethylacrylamide hydrogel in a simple one-step method. PDMA was crosslinked as a copolymer with methylacryloylbenzo-phenone by UV-induced C,H-insertion reactions and thus immobilized on the surface. The surface functionalization aimed to increase the hydrophilicity of the polymer surfaces, leading to less non-specific adsorption of enzymes. Various enzymatic assays were used to test the effect of the surface functionalization on enzyme performances. The integration of PDMA led to decreased non-specific adsorption of the enzymes to the surfaces, resulting in enhanced enzymatic performance in all assays. The study revealed significant increases in enzymatic substrate conversion, and both initial and maximum velocities. In course of this research, the one-step UV-crosslinking process for PDMA surface functionalization was further modified by adding β-lactam antibiotics and β-lactamase inhibitors. The incorporation of these molecules aimed to provide additional functio-nality to the PDMA-functionalized surfaces, with the intention of optimizing β-lactamase assays and potentially improving diagnostic applications as well as antibiotic susceptibility testing for bacterial infections. Representatives of different classes of β-lactam antibiotics and β-lactamase inhibitors were successfully immobilized on the surface and the interaction with various β-lactamases was tested in a chromogenic β-lactamase assay. Results proved that sufficient amounts of antibiotics and inhibitors were immobilized using this method and that they are not losing their accessibility for β-lactamase bindings. The assays comprising various antibiotics and inhibitors revealed that the developed surface functionalization enabled successful susceptibility testing of β-lactamases from different classes. Antibiotic susceptibility testing was also proved possible for β-lactamases isolated from bacterial cultures, which mimicked clinical samples. During this study it was further accomplished to transfer the method of antibiotic immobilization to 3D printed microfluidic devices, paving the way for the development of point-of-care tests.