There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Thrombin and its aptamers have been well studied and widely used as models in aptamer
based assays and sensors. Here we reported a thrombin-linked sandwich immunoassay
for proteins to demonstrate new applications of thrombin and the aptamers, converting
protein detection to analysis of thrombin label. In this assay, target protein was
sandwiched by the capture antibody on a microplate and the biotinylated detection
antibody. Thrombin bound to one biotinylated aptamer, and then the thrombin-labeled
aptamer was attached on the sandwich complex through streptavidin-biotin interaction
by using streptavidin as a linker. Thrombin catalyzed cleavage of fluorogenic peptide
substrates, generating fluorescence signals for target detection. Among a few different
anti-thrombin aptamers, the use of one nuclease resistant RNA aptamer having phosphorodithioate
(PS2) modification on a specific backbone position enabled higher assay sensitivity
due to its much higher affinity. This thrombin-linked sandwich immunoassay allowed
detection of prostate-specific antigen (PSA) at 2 pM, an important protein related
cancer disease, with high sensitivity and specificity. The strategy was general, and
also enabled sensitive detection of botulinum neurotoxin type A (BoNTA) light chain,
one toxin protein causing risk to human health. This assay combines advantages of
antibody recognition, aptamer affinity labeling, high affinity of aptamers, and enzyme
activity of thrombin. Labeling thrombin on the immunosandwich complex through simple
affinity binding overcomes limitations of covalent conjugating enzyme on antibody
in conventional immunoassay. This assay is promising in applications for protein detection.