Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification
AbstractMicrofluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed. View Full-Text
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Mauk, M.G.; Liu, C.; Song, J.; Bau, H.H. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification. Microarrays 2015, 4, 474-489.
Mauk MG, Liu C, Song J, Bau HH. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification. Microarrays. 2015; 4(4):474-489.Chicago/Turabian Style
Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H. 2015. "Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification." Microarrays 4, no. 4: 474-489.