Portable sample processing for molecular assays: application to Zika virus diagnostics

This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL(-1) level from a 5 mu L sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.

Automated summary

This paper introduces a digital microfluidic platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges and programmable control instrumentation, and it can be coupled with paper-based analysis for amplified Zika RNA. Blinded laboratory evaluation and field evaluation in Brazil showed high sensitivity and specificity of the platform.