Quantification and control of the surface density of azide groups presented by coating a functional polymer Hikaru Amo, Yusuke Kanki, Kenta Morita, Tatsuo Maruyama Graduate School of Engineering, Kobe University Strain-promoted azide-alkyne cycloaddition (SPAAC) is a type of Huisgen 1,3-dipolar cycloaddition reaction, also known as a copper-free click reaction. SPAAC is widely used for the modification of biomolecules such as proteins, DNA and cells. It is a bioorthogonal reaction that does not affect various functional groups in living organisms and it does not use cytotoxic copper catalysts. In our previous study, cyclic alkynes were presented on a plastic substrate by coating a functional polymer, and DNA molecules were successfully immobilised via SPAAC [1] . In this study, azide groups were presented on a surface of a plastic substrate by coating a functional polymer. Then, we quantified the density of azide groups presented on the substrate surface using cyclic alkyne- modified fluorescein, and controlled the density of azide groups by blending polymers for coating. In addition, a surface patterned with azide groups was prepared on a substrate surface by microcontact printing (μCP). Poly(MMA- r -HEMA) (PME) was synthesized via free-radical polymerization of methyl methacrylate and 2-hydroxyethyl methacrylate. Five different kinds of functional polymers were then synthesised by conjugating various azide molecules to the hydroxy groups of the side chains of PME. A 1 wt% functional polymer solution (solvent: ethyl acetate) was coated on an acrylic substrate to present azide groups on the substrate surface. The surface of the coated substrate was measured by X-ray photoelectron spectroscopy, and an N1s peak derived from the azide groups was observed, indicating that the azide groups were successfully presented on the surface. The azide group–presenting substrate was immersed in a solution containing fluorescent molecules modified with cyclic alkynes (Nflu-DACN) to conjugate the fluorophores with azide groups presented on the substrate surface via SPAAC. Subsequently, the side chains of functional polymers were hydrolyzed to release the fluorophores. The fluorescence intensity in a solution was then measured to quantify the surface density of azide groups. As a result, the surface density of azide groups was successfully quantified for the substrates coated with five different kinds of functional polymers. We found that the density of azide groups varied depending on the structures of the side chains. When a mixture solution of PME and a functional polymer was coated on a substrate surface, the azide group density increased as the ratio of the functional polymer in the mixture increased. Thus, the azide group density was successfully controlled. The surface patterned with azide groups was prepared using the μCP method. Ink of a Nflu-DACN solution was applied to a PDMS stamp. The stamp was brought into contact with a substrate surface presenting azide groups. The observation using a confocal laser scanning microscope demonstrated that the stamped surface had the surface morphology that matched the shape of the stamp, indicating the successful surface patterning. References 1. Miyahara et al. , Colloid Polym. Sci. , 2019, 297 , 927
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