PAPER: Development of a lipase-based optical assay for detection of DNA

Pinijsuwan, S.; Shipovskov, S.; Surareungchai, W.; Ferapontova, E. E.; Gothelf, K. V.

Org. Biomol. Chem. 2011, 9, 6352–6356, doi: 10.1039/c0ob01165g

Center for DNA Nanotechnology, Department of Chemistry and iNANO, Aarhus University, Aarhus C, Denmark.

Abstract

A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate a detectable readout the captured lipase was applied to an optical assay that takes advantage of the enzymatic activity of lipase. The assay applies p-nitrophenol octanoate (NPO) as the substrate and in the presence of lipase the ester is hydrolyzed to p-nitrophenolate which has a strong absorbance at 405 nm. The method provides detection a detection limit of 200 fmol target DNA and it was able to distinguish single base mismatches from the fully complementary target.

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