Rapid Multiplex Detection of Viral Pathogens in Whole Blood Using a Microfluidic Sample Processor and Smartphone-linked Handheld Instrument

An integrated two-part system for providing rapid (<30 minute) sample-to-answer simultaneous detection and quantitation of four viral pathogens (Zika, Chikungunuya, and two serotypes of Dengue) in a droplet of whole blood is described. The first part of the system is comprised of a plastic microfluidic sample processor that internally stores all required reagents to perform blood lysis, mixing of lysed material with master mix for the loop-mediated isothermal amplification (LAMP) protocol, and partitioning of the resulting volume into six fluid compartments (four virus nucleic acid tests, plus two experimental control compartments) with dried target-specific LAMP primers. We used 3D printing to optimize microfluidic design parameters to provide precisely metered volumes and thorough mixing while minimizing internal fluid retention. The optimized design was used to produce an injection molded cartridge to reduce manufacturing costs. The second part of the system is a handheld smartphone-linked, battery-powered detection instrument. The detection compartments of the microfluidic processor are inserted into the instrument, which provides uniform 65°C heating from below and LED illumination from above, while a smartphone gathers images of LAMP-generated fluorescent products in all six compartments, where image processing analysis of the spatial pattern of fluorescent product generation is used to differentiate positive from negative compartments without the need to wait for the entire compartment to become uniformly bright. The system achieves detection limits for live virus spiked into whole blood in the range of 2x105 – 6x106 PFU/ml, lack of cross reactivity between assays, and pipette-free operation, while requiring no additional equipment. The low cost of the system, rapid detection, and sensitivity provide a path toward point of care identification of the underlying cause of febrile illnesses driven by mosquito-borne pathogens.