Analysis of regenerated amine-reactive polymer microarray slides

Reactive polymer-coated microarray substrates based on N-hydroxyl succinimide (NHS) chemistry lose their bio-immobilization reactivity to probe nucleophiles over time, both in use and in storage, due to their intrinsic hydrolytic instability. Poor DNA and protein probe immobilization efficiency is often observed with routine microarray printing conditions, with accompanying reliability and stability issues for assay. We report a one-step reaction to regenerate NHS-reactive chemistry in situ on these microarray polymer surfaces with simple, straightforward reaction chemistry. Surfaces regenerated with this method perform equal to or better than freshly prepared slides in print-immobilization of oligonucleotide probes functionalized with primary amine reactive groups. DNA probe specific and non-specific surface binding as well as target hybridization were analyzed using both fresh and regenerated slides. Commercial microarray substrates appear to retain DNA probes with both substantial covalent immobilization and some non-specific adsorption to the surface.