Graphene Enhanced Fluorescence Anisotropy For Copper Ion And Potassium Ion Analysis

Fluorescence anisotropy is a polarization-based attractive phenomenon that has been applied in many fields, such as immunoassays, enzyme assays, protein-protein interactions and single nucleotide polymorphism (SNP) analysis. Only a few Fluorescence anisotropy methods for the detection of metal ions or small molecules have been reported because their molecular masses are too small to produce detectable fluorescence anisotropy changes. In this paper, we developed a novel graphene oxide (GO) amplifying fluorescence anisotropy strategy, and applied to analyse of metal ions including copper ions (Cu2+) and potassium ions (K+), and investigated the mechanism of our strategy. The main contents are as follows:(1) By introducing GO as the fluorescence anisotropy enhancer, a DNAzyme-based fluorescence anisotropy system for Cu2+detection is constructed. In the absence of copper ions, GO can assemble with the hybrid of DNAzyme (Cu-Enz) and substrate DNA strand (Cu-Sub) through the π-π stacking and the electrostatic interaction, thus the rotation of TAMRA can be coupled with the entire formation to obtain high anisotropy. In the presence of Cu2+, Cu-Sub undergoes irreversible cleavage, and the cleaved short TAMRA-labeled DNA fragment is kept away from the surface of GO because the π-π stacking interaction between short single-strand DNA (ssDNA) and GO is much weaker than that of long ssDN A. As a result, the fluorescence anisotropy of TAMRA gets reduced dramatically. It was found that a detection range of1-32nM could be achievable. The strategy for Cu2+detection is sensitive, simple and selective. (2) A label-free GO enhanced fluorescence anisotropy strategy for G-quadruplex-based assay of K+is explored. In the absence of K+, both probe ssDNA and acridine orange (AO) dye are assembled on the surface of GO, and the rotation of AO is coupled with the formed complex and high fluorescence anisotropy is obtained. In the presence of K+, however, a quadruplex of DNA gets formed, and thus AO can bind with G-quadruplex, keeping away from GO surface because of a competitive binding of AO with G-quadruplex, and a more stable AO-G-quadruplex complex than AO-GO complex is formed. As a result, fluorescence anisotropy of AO gets reduced. This is a label-free method and has a wide detection range of10μM-2mM, which is simple and pratical.In this study, a GO enhanced fluorescence anisotropy strategy has been proposed and successfully applied to the assays of metal ions.This method has overcome the limitations of conventional fluorescence anisotropy. Given that a similar system can be constructed with DNA, antibodies, peptides as well as other molecules, we expect that the novel strategy will find universal use in the areas of small molecule detection, protein analysis, pathogen detection and other target sensing.