High-density covalent immobilisation of DNA on plasma-activated surfaces for biosensing applications

Surface-immobilised 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 immobilisation density due electrostatic repulsion, and DNA-based biosensors typically require orientational control on the surface to be functional. Here, we improve DNA surface immobilisation by combining surfaces treated with a plasma-activated coating (PAC) and DNA probe strands with simple chemical modifications. It was found that DNA immobilisation 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 immobilised Gly-PEG-DNA are stable and can undergo multiple cycles of target hybridisation and dehybridisation, and bind a range of targets, including different length single-stranded DNA and DNA origami nanostructures. Finally, we demonstrate an application of a surface-immobilised 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 immobilising probe DNA on a range of surfaces for applications in biosensing and biophysics, enabling future templating of more complex multi-component sensors on surfaces using DNA origami nanostructures.