Bioassay of bile acids using an enzyme-linked DNA aptamer.
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 mumol l-1. The %RSD was 7 at 5 mmol l-1 of cholic acid.
Kato
T
T
Research Center for Advanced Science and Technology, University of Tokyo, Japan.
Yano
K
K
Ikebukuro
K
K
Karube
I
I
D000818:Animals; D001647:Bile Acids and Salts / Q000032:analysis*; D001681:Biological Assay / Q000379:methods; D019826:Cholic Acid / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D008024:Ligands
D001647:Bile Acids and Salts; D008024:Ligands; D004247:DNA; D019826:Cholic Acid