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

Area of Science:

• Biotechnology
• Molecular Biology
• Diagnostics

Background:

• Disease pathogenesis is linked to complex gene regulation by microRNAs (miRNAs).
• Current miRNA detection methods are often limited to single-sample analysis, hindering accurate diagnosis of complex diseases.
• Simultaneous detection of multiple miRNAs is crucial for understanding disease mechanisms and improving diagnostic accuracy.

Purpose of the Study:

• To develop a simple, reliable, and simultaneous detection method for multiple microRNAs (miRNAs).
• To enable room-temperature, equipment-free miRNA detection without enzymatic reactions.
• To establish a sensitive and sequence-specific assay for clinical diagnostics.

Main Methods:

• A liquid flow-based microfluidic optical assay utilizing catalytic hairpin assembly for signal amplification.
• Simultaneous amplification of two distinct dimeric DNA probes, each specific to a target miRNA.
• Formation of i-motif-driven DNA hydrogels in microfluidic channels for visual detection using gold nanoparticles.

Main Results:

• The assay successfully detected two different target miRNAs simultaneously at room temperature.
• Visual confirmation of miRNA presence via naked-eye observation of flow blockage caused by gold nanoparticles.
• Demonstrated sensitive and sequence-specific detection, distinguishing single-base mismatch mutants within 1.5 hours.

Conclusions:

• The developed microfluidic assay provides a powerful platform for simple and simultaneous miRNA detection.
• This method eliminates the need for analytical equipment and enzymatic reactions, facilitating point-of-care diagnostics.
• The assay holds significant potential for advancing clinical diagnostics and understanding diseases associated with miRNA dysregulation.