Smartphone Clip-On Instrument and Microfluidic Processor for Rapid Sample-to-Answer Detection of Zika Virus in Whole Blood Using Spatial RT-LAMP

We report an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release RNA from the Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered instrument heats the compartments from below, while LEDs illuminate from above. Fluorescence generation in the compartments is dynamically monitored by a smartphone camera. We characterize the assay time and detection limits for detecting Zika RNA and gamma-irradiated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent "bloom" events for positive samples. We show that dynamic image analysis reduces the time required to designate an assay as a positive test to 22 minutes, compared to similar to 30-45 minutes for conventional analysis of the average fluorescent intensity of the entire compartment. We achieve a total sample-to-answer time in the range of 17-32 minutes, while demonstrating a viral RNA detection as low as 2.70x10(2) copies/ul, and a gamma-irradiated virus of 10(3) virus particles in a single 12.5 microliter droplet blood sample.