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 mumol l-1. The %RSD was 7 at 5 mmol l-1 of cholic acid.

Viability of allergy (IgE) detection using an alternative aptamer receptor and electrochemical means

“…In particular their use has been largely noticed in quartz crystal sensing [28,31], microtitre-based assays [29,32,33], surface plasmon resonance [34] and fluorescence [35,36]. To this point, they have not been exploited in electrochemical methods to any great extent with the authors recognizing only the recent communication of Hianik et al [37].…”

Functional Nucleic Acid Sensors

“…A streptavidin functionalized alkaline phosphatase and its fluorogenic substrate were used for signal generation. Cholic acid was detected with a detection limit of 10 μM 429. Xu and co-workers functionalized AuNPs with the PDGF aptamer, while the free aptamer containing a biotin label 430.…”

Current research activity in biosensors

“…This methodology is called "in vitro selection". Since an aptamer consisting of DNA was designed for binding with protein in 1992 [34], various DNA aptamers have been designed to bind with thrombin [35], a transition state analog [36], bile acids, using enzyme-linked DNA aptamer [37], cholic acid [38], steroids using three-way DNA junctions [39], or rat brain tumor microvessels [40]. In addition, RNA aptamers can be adapted to biosensor applications by directly transduced molecular recognition of ATP, affording optical signals, although RNA is unstable compared with DNA as a recognition element [41].…”