Target-Induced Conjunction of Split Aptamer Fragments and Assembly with a Water-Soluble Conjugated Polymer for Improved Protein Detection

Rapid and sensitive detection of proteins is crucial to biomedical research as well as clinical diagnosis. However, so far, most detection methods rely on antibody-based assays and are usually laborious and time-consuming, with poor sensitivity. Herein, we developed a simple and sensitive fluorescence-based strategy for protein detection by using split aptamer fragments and a water-soluble polycationic polymer (poly­{[9,9-bis­(6′-(N,N,N-diethylmethylammonium)­hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis­(3′-(N,N,N-diethylmethylammonium)-1′-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP)). The thrombin-binding DNA aptamer was split into two fragments for target recognition. The PFEP with high fluorescence emission was used as energy donor to amplify the signal of dye-labeled DNA probe. In the absence of target, three DNA/PFEP complexes were formed via strong electrostatic interactions, resulting in efficient Föster resonance energy transfer (FRET) between two fluorophores. While the presence of target induces a conjunction of two split aptamer fragments to form G-quadruplex, and subsequent assemble with PFEP leading to the formation of G-quadruplex/thrombin/PFEP complex. The distance between the PFEP and dye increased due to protein’s large size, leading to a remarkable decrease of the FRET signal. Compared with the intact aptamer, the use of shorter split aptamer fragments increases the possibility of forming G-quadruplex upon target. Thus, the rate of change of FRET signal before and after the addition of target improved significantly and a higher sensitivity (limit of detection (LOD) = 2 nM) was obtained. This strategy is superior in that it is rapid, has low cost and homogeneous detection, and does not need heating to avoid an unfavorable secondary structure of DNA probe. With further efforts, this method could be extended to a universal way for simple and sensitive detection of a variety of biomolecules.