Abstract
We compare immobilization methods for oligonucleotides on carboxylic acid surfaces with the goal of improving hybridization efficiency for single-stranded DNA (ssDNA) bioassays. When immobilized via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), ssDNA (either modified with a terminal amine or not) specifically binds to the surface; without EDC, binding is minimal. EDC-activated probes can, however, bind covalently to the surface through nucleobase as well as terminal amino groups. Unmatched base pairs from the former are detected via melting curve analysis: the target begins to unwind at∼40°C below the double-strand melting temperature of∼64°C. To eliminate such backbone binding, we immobilized azide-functionalized DNA via click chemistry, resulting in hybridization efficiencies 5 times higher than with EDC. This improvement, and the room-temperature hybridization of the click chemistry process, make it an important alternative to EDC for reliable DNA assays with maximum specificity and sensitivity.
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