Ratiometric fluorescent 3D DNA walker and catalyzed hairpin assembly for determination of microRNA.

Abstract

Using6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detectionwas constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. Thedual emission ratio fluorescent signal with a single excitation wavelength was usedas the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experimentis reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488nm and the maximum fluorescence emission wavelength of 520nm and 580nm, respectively. It has a good linear response at a microRNA concentration range of 156.0pM ~ 7.00nM and a detection limit of 42.94pM. This strategy has been successfully appliedto detect microRNAs in spiked serum samples. Graphical abstract Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis.