Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate

The authors report on a microplate based thrombin assay by using an RNA aptamer (Toggle-25) as the affinity ligand. The aptamer is nuclease-resistant because it contains nucleobases modified with 2'-deoxyfluoro saccharides. The aptamer is coated on the surface of the wells of microplates where it captures thrombin from a sample. Following a washing step, a chromogenic or a fluorogenic peptide substrate is added which is cleaved by the captured thrombin to form a yellow product (with absorbance at 405 nm) or a blue fluorescent product (with an emission maximum at 444 nm) after incubation at 37 A degrees C for 2 h. The assay enables thrombin to be determined with a 10 fM detection limit when using the fluorogenic substrate. The assay is selective in that several other proteins do not interfere. The assay can be applied to the determination of thrombin in diluted human serum and plasma.