Bioassay of bile acids using an enzyme-linked DNA aptamer

Abstract

A new analytical method for the detection of bile acids has been developed by adopting an alkaline phosphatase-linked DNA oligomer that binds to bile acids. A 5′-biotin-labeled DNA oligomer with a 40-nucleotide length that is defined by the in vitro selection method was connected with alkaline phosphatase through an avidin–biotin linkage and applied to an enzyme immunoassay format. Sample solutions were incubated with small aliquots for a cholic acid-immobilized agarose matrix, on which the alkaline phosphatase-linked DNA oligomer had been bound prior to carrying out the assay. The amount of the alkaline phosphatase-linked DNA oligomer dissociated from the cholic acid-immobilized agarose matrix, which was detected using a fluorogenic substrate for alkaline phosphatase, indicated the amount of bile acids in the samples. The results suggest that the DNA aptamer directly linked with the reporter enzyme is applicable as a detector ligand for the immunoassay format. A linear calibration range was obtained for cholic acid between 0.1 to 5 mmol l⁻¹ with a limit of detection of 10 μmol l⁻¹. The %RSD was 7 at 5 mmol l⁻¹ of cholic acid.