Simultaneous and Ultraspecific Optical Detection of Multiple miRNAs Using a Liquid Flow-Based Microfluidic Assay

Recent studies have reported that the cause and progression of many diseases are closely related to complex and diverse gene regulation involving multiple microRNAs (miRNAs). However, most existing methods for miRNA detection typically deal with one sample at a time, which limits the achievement of high diagnostic accuracy for diseases associated with multiple gene dysregulations. Herein, we develop a liquid flow-based microfluidic optical assay for the simple and reliable detection of two different target miRNAs simultaneously at room temperature without any enzymatic reactions. This assay utilizes the catalytic hairpin assembly cycling reaction in a mixture containing four types of hairpin DNAs to amplify two different dimeric DNA probes, each of which specifically recognizes one of the two different target miRNAs. The resultant two dimeric DNA probes effectively hybridize with anchor DNA grafted into two outlet channels of a microfluidic device, thus enabling i-motif-driven compact DNA hydrogels to form in the channels under acidic conditions. With this setup, the presence of two target miRNAs can be confirmed by the naked-eye observation of red-colored gold nanoparticles encountering a flow blockage in the two outlet channels. Notably, the developed assay demonstrates sensitive and sequence-specific detection that can precisely distinguish a single base mismatch mutant miRNA within 1.5 h. Our assay thus has the potential to serve as a powerful sensing platform for the simple and simultaneous detection of multiple miRNAs in clinical diagnostics at room temperature without analytic equipment or enzymatic reactions.