am405550j_si_001.doc (300 kB)
Target-Induced Conjunction of Split Aptamer Fragments and Assembly with a Water-Soluble Conjugated Polymer for Improved Protein Detection
journal contribution
posted on 2014-03-12, 00:00 authored by Xingfen Liu, Lin Shi, Xiaoxiao Hua, Yanqin Huang, Shao Su, Quli Fan, Lianhui Wang, Wei HuangRapid
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.