Fluorescent aptamer-based assay for thrombin with large signal amplification using peroxidase mimetics

Abstract

This article describes an aptamer-based thrombin assay using a hemin-based peroxidase mimetic for signal amplification. Thrombin is recognized by an immobilized primary aptamer (G1-quadruplex). The G1-quadruplex/hemin complex formed quenches the fluorescence of CdTe quantum dots (QDs) due to photoinduced electron transfer (PET). In the next step, thrombin is associated with a secondary aptamer (G2-quadruplex) consisting of Pt nanoparticles (NPs), G2-quadruplex and hemin to form a sandwich structure. Both the G1-quadruplex/hemin complex and the Pt NPs/G2-quadruplex/hemin complex associated with thrombin act as enzyme mimetics and catalyze the oxidation of hydroquinone by H2O2 to form 2-hydroxy-p-benzoquinone (HPB). The HPB produced quenches the fluorescence of the CdTe QDs via a PET and an inner filter effect, thus causing large signal amplification. The effects were exploited to design a highly sensitive and selective thrombin assay. Under optimized conditions, a linear fluorescence response is achieved in the 0.05 pmol·L−1 to 10 nmol·L−1 concentration range, with a lower detection limit of 15 fmol·L−1. This approach, in our perception, represents a promising platform for sensitive detection of biomolecules for which appropriate aptamers can be found.