title: Microfluidic devices for nucleic acid (NA) isolation, isothermal NA amplification, and real-time detection.
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.
authors.affiliation: 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.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Mauk
authors.forename: Changchun
authors.initials: C
authors.lastname: Liu
authors.forename: Mohamed
authors.initials: M
authors.lastname: Sadik
authors.forename: Haim H
authors.initials: HH
authors.lastname: Bau
mesh_terms: D000942:Antigens, Bacterial / Q000097:blood / Q000302:isolation & purification; D000956:Antigens, Viral / Q000097:blood / Q000302:isolation & purification; D040421:Cell Phone; D004247:DNA / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D008567:Membranes, Artificial; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation* / Q000379:methods; D019095:Point-of-Care Systems; D012313:RNA / Q000097:blood* / Q000302:isolation & purification; D052616:Solid Phase Extraction; D013997:Time Factors
chemical_list: D000942:Antigens, Bacterial; D000956:Antigens, Viral; D008567:Membranes, Artificial; D012313:RNA; D004247:DNA