“Paper Machine” for Molecular Diagnostics

• Published in: Analytical chemistry (Washington) 2015-08, Vol.87 (15), p.7595-7601
• Main Authors: Connelly, John T, Rolland, Jason P, Whitesides, George M
• Format: Article
• Language: English
• Subjects: Amplification; Analytical chemistry; Cells; Deoxyribonucleic acid; Devices; DNA; E coli; Humans; Lamps; Microfluidic Analytical Techniques - economics; Microfluidic Analytical Techniques - instrumentation; Molecular Diagnostic Techniques - instrumentation; Molecular Diagnostic Techniques - methods; Molecules; Paper machines; Point-of-Care Systems; Reproduction
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.5b00411

Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a “paper machine”, that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.