A novel multiplex signal amplification strategy based on microchip electrophoresis platform for the improved separation and detection of microRNAs

Abstract

A multiplex fluorescence signal amplification method based on microchip electrophoresis (MCE) platform was developed for the improvements in the separation and detection of microRNAs. The method used two kinds of fluorescein amidite labeled DNA signal probes to hybridize with its target microRNAs, utilizing T7 exonuclease assisting target circling realized the fluorescence signal amplification. Then, two kinds of fluorescein amidite labeled DNA segments with different size were separated and detected on the MCE-laser induced fluorescence detection platform. The microRNAs-126 and microRNAs-141 were used as model analytes in the proof-of-concept experiments. Two calibration curves between the fluorescence intensity and microRNAs concentration all showed good linearity in the range of 0.025–20 nM. The correlation coefficients obtained were 0.9975 and 0.9925, respectively. The limits of detection for two kinds of microRNAs were estimated to all be 15 pM. By spiking T24 cell lysate samples with varying amounts of miRNA-126 and miRNA-141, the recovery of analytes ranged from 96.0% to 115%, and the relative standard deviations are lower than 5.5%. The present method showed high sensitivity and selectivity, which has a promising application in biomedical research.