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A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids.

Analytical and bioanalytical chemistry (2018-08-30)
Haowen Yang, Zhu Chen, Xiaobao Cao, Zhiyang Li, Stavros Stavrakis, Jaebum Choo, Andrew J deMello, Philip D Howes, Nongyue He
ABSTRACT

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.

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Sigma-Aldrich
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane, 97%