Multiplexed miRNA Quantitation Using Injectionless Microfluidic Thermal Gel Electrophoresis

MicroRNAs (miRNAs) are a class of biomolecules that have high clinical and pharmaceutical significance because of their ability to regulate protein expression. Better methods are needed to quantify target miRNAs, but their similar sequence lengths and low concentrations in biomedical samples impede analysis. This report aimed to develop a simple, rapid method to directly quantify multiple miRNAs using microfluidic thermal gel electrophoresis (TGE). Fluorescent probes were designed complementarily in sequence to four target miRNAs that also contained variable DNA overhangs to alter their electrophoretic mobilities. Samples and probes were directly added into thermal gel and loaded throughout a microchannel. Applying voltage resulted in an inline preconcentration and separation of the miRNAs that did not require a sample injection nor user intervention to switch between modes. Baseline resolution was achieved between four double-stranded miRNA-probe hybrids and four excess single-stranded probes. Analytical performance was then improved by designing an innovative microfluidic device with a tapered channel geometry. This device exhibited superior detection limits and separation resolution compared to standard channel devices without increasing the complexity of microfabrication or device operation. A proof-of-concept demonstration was then performed, showing that target miRNAs could be detected from cell extracts. These results demonstrate that TGE provides a simple, inexpensive means of conducting multiplexed miRNA measurements, with the potential for automation to facilitate future clinical and pharmaceutical analyses.