Optimized Functional Covalent Immobilization of DNA on Plasma-Activated Surfaces for Biosensing Applications

Surface-immobilized DNA enables a broad range of biosensing technologies, from DNA aptamer sequences that directly bind targets to programmable tethers that bind other probe molecules such as antibodies and even to tether DNA nanostructures to achieve controlled nanoscale placement of sensing groups in complex patterns. However, chemical surface activation methods are often toxic and time consuming, the negative charge of DNA can limit surface immobilization density due electrostatic repulsion, and DNA-based biosensors typically require orientational control on the surface to be functional. Here, we improve DNA surface immobilization by combining surfaces treated with a plasma-activated coating (PAC) and DNA probe strands with simple chemical modifications. It was found that DNA immobilization on PAC-treated 96-well plates is improved 3-fold by high-yield click-chemistry modification of DNA probes with a glycine-polyethylene glycol (Gly-PEG) linker. We show that PAC-treated surfaces with immobilized Gly-PEG-DNA are stable and can undergo multiple cycles of target hybridization and dehybridization, and bind a range of targets, including different length single-stranded DNA and DNA origami nanostructures. Finally, we demonstrate an application of a surface-immobilized modular fluorescence biosensor. The orientational control provided by the Gly-PEG linker improves the sensor performance. Overall, we envision that these results will provide a simple and high-yield method for immobilizing probe DNA on a range of surfaces for applications in biosensing and biophysics, enabling future templating of more complex multicomponent sensors on surfaces using DNA origami nanostructures.