Aptamer-based protein detection using a bioluminescent fusion protein

Abstract

An aptamer-based sandwich-type immunoassay is presented to detect human thrombin using a bioluminescent fusion protein, SSB–fLuc. Escherichia coli single-stranded DNA binding protein (SSB) is used as a linker between the aptamer and firefly luciferase (fLuc). For proof-of-principle, thrombin was used as the test analyte and thrombin aptamer as the sensing probe. In this fusion protein, both the SSB and the fLuc parts retained their biological activities after expression and purification. The SSB fragment of the fusion protein also had the thrombin aptamer binding ability either alone or in combination with thrombin as a triplex, which was confirmed by gel mobility shift assay using native polyacrylamide gels. The fusion protein can be used to detect thrombin in the nanomolar range. The present study thus demonstrates an aptamer-based bioluminescent assay that is simple and cost effective, and at the same time eliminates the need for labeling of either analytes or aptamers. This biomolecular detection scheme can be extended to the detection of a wide range of analytes.