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

Tanya Narahari; Joshua Dahmer; Alexandros Sklavounos; Taehyeong Kim; Monika Satkauskas; Ioana Clotea; Man Ho; Julian Lamanna; Christopher Dixon; Darius G. Rackus; Severino Jefferson Ribeiro da Silva; Lindomar Pena; Keith Pardee; Aaron R. Wheeler

doi:10.1039/D1LC01068A

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

Tanya Narahari,‡^ab Joshua Dahmer,‡^a Alexandros Sklavounos,

‡^ab Taehyeong Kim,

‡^ab Monika Satkauskas,‡^a Ioana Clotea,^a Man Ho,^ab Julian Lamanna,^ab Christopher Dixon,

^a Darius G. Rackus,

§^ac Severino Jefferson Ribeiro da Silva,

¶^d Lindomar Pena,^d Keith Pardee^ce and Aaron R. Wheeler

*^abf

Author affiliations

* Corresponding authors

^a Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada
E-mail: aaron.wheeler@utoronto.ca

^b Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, Canada

^c Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada

^d Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Institute (FIOCRUZ Pernambuco), Av. Professor Moraes Rego, s/n – Cidade Universitária, Recife, PE, Brazil

^e Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, M5S 3G8 Canada

^f Institute for Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario, Canada

Abstract

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 μL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.

This article is part of the themed collection: Miniaturised Sensors & Diagnostics

Lab on a Chip

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

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