Self-actuated microfluidic chiplet for two-stage multiplex nucleic acid amplification assay

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Point-of-Care Testing

Background:

• Accurate diagnosis of diseases with overlapping symptoms requires assays that can detect multiple pathogens simultaneously.
• Existing methods often lack the speed and specificity needed for timely clinical decisions.
• The Penn-RAMP assay was developed for multiplexed, isothermal amplification to address these diagnostic challenges.

Purpose of the Study:

• To develop a self-actuated chiplet for the Penn-RAMP assay, enabling point-of-need pathogen detection.
• To integrate automated sample handling and multiplexed amplification onto a single, user-friendly device.
• To demonstrate the chiplet's capability for co-detection and differentiation of multiple targets.

Main Methods:

• Development of a self-actuated chiplet incorporating temperature-controlled phase change and capillary valves.
• Integration of a two-stage isothermal amplification process: recombinase polymerase amplification (RPA) followed by multiplexed loop-mediated isothermal amplification (LAMP).
• Automated self-aliquotting of RPA amplicons into five distinct LAMP reaction chambers for target-specific amplification.

Main Results:

• The chiplet successfully demonstrated co-detection of plant pathogens, validating its functionality.
• Analytical performance of the chiplet was comparable to the benchtop Penn-RAMP assay.
• The chiplet outperformed standalone LAMP assays in terms of sensitivity and specificity.

Conclusions:

• The self-actuated Penn-RAMP chiplet provides a robust platform for point-of-need, multiplexed nucleic acid amplification and detection.
• This technology facilitates precision medicine by enabling rapid differentiation of co-endemic pathogens.
• The chiplet can be used independently or integrated into broader sample preparation workflows for diverse diagnostic applications.