Comparison of DNA immobilization efficiency on new and regenerated commercial amine-reactive polymer microarray surfaces

Abstract Reactive polymer-coated microarray substrates based on nucleophile-reactive N-hydroxysuccinimide (NHS) active ester chemistry lose their bio-immobilization reactivity to amine-terminated probe nucleophiles over time, both in print use and “dry” storage, due to their intrinsic hydrolytic instability. Poor or inconsistent DNA and protein probe immobilization efficiency is often observed with routine microarray printing conditions, with accompanying reliability and stability issues for assay. Because of surging popularity of microarraying applications, expectations for consistent assay results and the expense of losing microarray substrate immobilization performance, 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 freshly prepared slides in print-immobilization of oligonucleotide probes functionalized with primary amine reactive groups. DNA probe specific and non-specific surface binding due to physisorption versus nucleotide base amine covalent attachment was analyzed using both fresh and slides regenerated with NHS chemistry. Commercial reactive microarray substrates appear to retain DNA probes with both substantial covalent immobilization as well as some non-specific adsorption to the commercial arraying surface.

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