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.
We report a new Föster resonance energy transfer (FRET) system that uses a special dye, thioflavin T (ThT), as an energy acceptor and a water-soluble conjugated polymer (CP) with high fluorescence as an energy donor. A simple, label-free, and sensitive strategy for the detection of thrombin in buffer and in diluted serum was designed based on this new system using ThT as an efficient inducer of the G-quadruplex. The difference between the blank and the positive samples was amplified due to distinctive FRET signals because thrombin has little effect on the intercalation of ThT into the G-quadruplex. In the absence of the target, ThT induces the aptamer to form a G-quadruplex and intercalates into it with strong fluorescence. The electrostatic attractions between the negatively charged G-quadruplex and positively charged CP allow a short donor-acceptor distance, resulting in a high FRET signal. However, in the presence of the target, the aptamer forms a G-quadruplex-thrombin complex first, followed by the intercalation of ThT into the G-quadruplex. A long distance exists between the donor and acceptor due to the strong steric hindrance from the large-sized thrombin, which leads to a low FRET signal. Compared with previously reported strategies based on the FRET between the CP and dye, our strategy is label-free, and the sensitivity was improved by an order of magnitude. Our strategy also shows the advantages of being simple, rapid (about 50 min), sensitive, label-free, and low-cost in comparison to strategies based on the FRET between quantum dots and dyes.
An improved turn‐on aptasensor for thrombin detection using split aptamer fragments and graphene oxide (GO) was reported. The thrombin‐binding aptamer (Apt15) was split into two parts for target recognition, an 8‐base sequence labeled with fluorescein (FAM‐Apt‐A) and a 7‐base oligonucleotide sequence (Apt‐B). In the absence of target protein, the fluorescence of FAM‐Apt‐A/Apt‐B was quenched by GO through Π‐Π stacking between GO and single‐stranded DNA. However, when thrombin was introduced into the system, a target‐induced G‐quadruplex forms with two split aptamer fragments and thrombin. The fluorescence recovered due to weak interaction between G‐quadruplex and GO. Compared to the strategy using intact aptamer, probe concentration was lowered, and an improved sensitivity was obtained. Moreover, heating process to avoid unfavorable secondary structure was avoided due to the use of shorter split aptamer fragments.
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