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.

Fig. 1

(Left) Photograph of a chip containing an array of three multifunctional amplification reactors. (Right) A cross section of a multifunctional amplification reactor featuring the flow-through membrane for the capture and immobilization of nucleic acids

Fig. 2

Microcluidic NAT chip CAD drawings (SolidWorks™) showing assembled, bonded structure and component layers. (a) Assembled chip. (b) Top view showing the inlets and outlets and the PMMA tape covering the reaction chambers. (c) Underside view showing the PCR tape covering the membrane isolation chamber and conduits. (d) Exploded view. (e) Top view of the main slab showing the uncovered amplification chambers. (f) Underside view of the main slab (without the PCR sealing tape) showing the isolation chambers with embedded membranes and the conduits

Fig. 3

Experimental Setup for operating microfluidic cassette showing cassette mounted on base supporting heater and blue LED. Detector or camera is positioned over cassette. Optical filter blocks blue excitation light to allow detection of green fluorescence from LAMP reaction(s) on cassette. A power/control box provides power for heater and LED, and auxilliary functions such as data logging and communication with smartphone. Inset shows photo of real-time LAMP reaction on cassette

Fig. 4

Plasma separation device: (a) whole blood inserted into the separator chamber, (b) sedimentation of blood components, (c) extraction of plasma. (d) A photograph of a standalone version of the plasma separation device

Fig. 5

Processing steps for plasma extraction and microfluidic NAT

Fig. 6

An example of real time detection. Fluorescence emission intensity as a function of time when the sample contains 0 (negative control), 10², 10³, and 10⁴ HIV copies/ml. (Inset) The threshold time as a function of HIV concentration