Sensitivity Enhancement of MicroRNA Detection Using a Power-free Microfluidic Chip

摘要

We present a microRNA (miRNA) detection method that achieves enhanced sensitivity by means of a power-free microfluidic chip without the requirement of an external power source. The miRNA detection is completed by sandwich hybridization between probe DNAs and target miRNA with small sample volume (0.5 μL) within 20 min. Fluorescence signals after hybridization were amplified by laminar flow-assisted dendritic amplification (LFDA) using fluorescein isothiocyanate (FITC)-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) as amplification reagents. To enhance the sensitivity of on-chip miRNA detection, the hybridization buffer solution was newly optimized with three main components—sodium dodecyl sulfate (SDS), formamide and dextran sulfate—that are known to strongly influence hybridization. An on-chip miRNA detection test in the newly optimized hybridization buffer (0.2% SDS, 5% formamide and 1% dextran sulfate) revealed dramatic increases in both the LFDA signal in the sample channel and the signal-to-background ratio (S/B ratio). Moreover, the LFDA signals in a blank reference channel remained low due to the suppression of non-specific bindings and hybridizations. By changing the hybridization buffer, we obtained an improved limit of detection (LOD) that was 0.045 pM (miRNA-196a) and 0.45 pM (miRNA-331), which are around 30- and 10-fold better than that of when control hybridization buffer was used. The improved performance of our miRNA detection system with short running time and high sensitivity could contribute to future research, including point-of-care diagnostic systems.