Development of in-situ synthesized anion exchange membrane sensors with improved structural stability for robust nucleic acid detection

To improve the nucleic acid detection platform utilizing anion-exchange membrane (AEM) biosensors, an enhanced AEM was synthesized via one-pot photoinitiated polymerization from its molecular components, 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethyl acrylate (EGDMA), diallyldimethylammonium chloride (DDA), and the hydrophobic crosslinker bisphenol A-glycidyl methacrylate (bis-GMA). This membrane was designated HDAB (poly-HEMA-based AEM incorporating DDA and bis-GMA) membrane. FTIR analyses confirmed successful incorporation of bis-GMA in the HDAB membrane and scanning electron microscope imaging revealed its homogeneous structure. Compared to previous AEM formulations without bis-GMA, the HDAB membrane demonstrated significantly reduced water uptake (22 % vs 76 %) and reduced swelling (19 % vs 42 %), resulting in improved structural stability crucial for reproducible biosensor performance. Furthermore, the HDAB membrane can be in-situ synthesized in vat polymerization 3D-printed microfluidic chips to facilitate production and sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA) was identified as an efficient coupling agent to functionalize amino-modified DNA probes directly onto the HDAB membrane, significantly reducing its processing time. XPS analyses and fluorescence microscopy validated DNA probes functionalized by sulfo-SDA and subsequent target DNA hybridization. Electrokinetic measurements demonstrated HDAB membranes exhibited consistent limiting currents and long-term stability studies showed HDAB membranes maintained their detection performance for over 8 weeks, outperforming previous AEM formulations. Considering the mechanism of target DNA detection, easier differentiation of changes in measured I-V curves was achieved by monitoring static resistance shifts after converting the readout I-V curves to I-ΔR curves. Therefore, this improved HDAB membrane biosensor demonstrated enhanced reproducibility, shelf-life, and quantitative nucleic acid detection capabilities suitable for advancing this biosensing platform.