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Automated sample-to-answer nucleic acid testing with frequency controlled reagent release from cartridge integrated stickpacks

For the first time we demonstrate an automated centrifugal Lab-on-a-Disk system for sample-to-answer point-of-care testing of multiple nucleic acid targets that features pre-storage of all required liquid reagents for nucleic acid extraction as well as primers and probes and magnetic beads. Highly wetting and thus hardly controllable liquid buffers were pre-stored in stickpacks with frequency controlled on-demand reagent release enabling automated addition of binding buffer after sample lysis. The self-contained Lab-on-a-Disk system automates all necessary assay steps for PCR-based pathogen detection: RNA extraction, aliquoting of the RNA and geometrically multiplexed real-time RT-PCR. As a proof-of-principle, we demonstrated detection of as little as 15 plaque forming units (pfu) of RNA bacteriophage MS2 in a 200 μL sample in 3.5 hours.