A Low-Cost Handheld Centrifugal Microfluidic System for Multiplexed Visual Detection Based on Isothermal Amplification

A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA.