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Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection

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Mobile Health Technologies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1256))

Abstract

Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specificity. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfluidic cassette (“chip”) has been developed for nucleic acid-based testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplification such as LAMP (Loop-mediated AMPlification), NASBA (Nucleic Acid Sequence Based Amplification), and RPA (Recombinase Polymerase Amplification); and real-time optical detection of DNA or RNA analytes. The microfluidic cassette incorporates an embedded nucleic acid binding membrane in the amplification reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplified at a constant incubation temperature. The amplification product, labeled with a fluorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion filtration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfluidic chip has also been developed. For HIV virus detection in blood, the microfluidic NAT chip achieves a sensitivity and specificity that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers.

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Acknowledgments

The work reported here was supported, in part, by NIH Grants U01DE017855 (Bau, Mauk) and K25AI099160 (Liu), and a grant from the Commonwealth of Pennsylvania’s Ben Franklin Technology Development Authority through the Ben Franklin Technology Partners of Southeastern Pennsylvania (Bau, Sadik).

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Authors and Affiliations

  1. Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, 220 South 33rd Street, 107 towne building, Philadelphia, PA, 19104-6315, USA

    Michael G. Mauk, Changchun Liu, Mohamed Sadik & Haim H. Bau

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  1. Michael G. Mauk
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  2. Changchun Liu
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  3. Mohamed Sadik
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  4. Haim H. Bau
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Correspondence to Haim H. Bau .

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Editors and Affiliations

  1. National Cancer Institute, Rockville, Maryland, USA

    Avraham Rasooly

  2. Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA

    Keith E. Herold

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Mauk, M.G., Liu, C., Sadik, M., Bau, H.H. (2015). Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection. In: Rasooly, A., Herold, K. (eds) Mobile Health Technologies. Methods in Molecular Biology, vol 1256. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2172-0_2

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  • DOI: https://doi.org/10.1007/978-1-4939-2172-0_2

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