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.