Cocktail-Inspired All-in-One Magnetofluidic Cartridge Platform for Versatile Nucleic Acid Testing

Low-cost, portable, and high-performance nucleic acid detection technologies are essential for point-of-care diagnosis of infectious diseases. Here, we report an ultralow-cost (~$0.1) magnetofluidic cartridge system featuring a novel tapered cylindrical-like cartridge with stable vertical separation of multiple reagents using a combination of silicone oil and wax layers in a cocktail-like configuration. We systematically investigate the mechanisms that enable shake-proof vertical isolation of multiple immiscible reagent layers, independent of aqueous reagent density, thereby allowing the stable maintenance of arbitrary numbers and arrangements of reagent layers. This innovation facilitates fully automated magnetic bead-based nucleic acid extraction coupled with multiplex PCR or CRISPR‒Cas-assisted isothermal detection in a handheld analyser. The unique reagent isolation in the cartridge overcomes the limitations of previous magnetofluidic systems by physically separating elution and amplification reagents, allowing broader assay compatibility. SARS-CoV-2 and influenza A/B detection reached 0.1 copies/µL sensitivity after 60 minutes (PCR) and 30 minutes (CRISPR). Clinical testing of 33 samples showed 100% concordance with standard methods. This low-cost integrated cartridge system offers a robust platform for rapid, sensitive, and multiplexed nucleic acid testing in resource-limited settings.