Thrombin-linked aptamer assay for sensitive detection of immunoglobulin E in sandwich format

Abstract

A thrombin-linked aptamer assay (TLAA) was developed with thrombin as an enzyme label for the detection of immunoglobulin E (IgE), an important biomarker for diseases. We constructed a sandwich assay for IgE using the anti-IgE antibody coated microplate and a DNA probe that contained the aptamer of IgE and the aptamer of thrombin. Taking advantage of aptamer affinity binding, the DNA probe could bind with IgE and thrombin, and the thrombin was labeled on the sandwich complex of IgE. As a protease, the thrombin catalyzed its fluorogenic peptide substrate to generate a fluorescent product. The assay showed a good linear relationship between the fluorescence signal and the IgE concentration ranging from 19 pM to 2.5 nM and a detection limit of 19 pM IgE. The detection limit further reached 0.625 pM when the TLAA was combined with rolling circle amplification (RCA) for multiple signal amplification. The TLAA exhibited high sensitivity and selectivity, and it allowed for determination of IgE in diluted human serum samples. This TLAA for IgE detection is promising for applications.