Abstract
Herein, we report a new electrochemical assay for the endonuclease activity using a graphene monolayer. The approach involves a methylene blue (MB) tagged double-stranded (ds) DNA as an enzyme substrate with a single-stranded (ss) segment at the end for anchoring on graphene through π-π stacking. The nuclease enzyme mediates DNA hydrolysis following the release of dsDNA-MB from the graphene surface, which reduces the electrochemical current signal. The protocol was validated for the HaeIII and EcoRV restriction endonuclease enzyme. We also assayed the activity of methyltransferase enzyme and inhibition of restriction by HaeIII. The method allows real-time measurement and quantitative assay for endonuclease activities in a simple manner.
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