Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification
Microfluidic 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 represe...
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doaj-4cb6932833e5474a959da5cfc7f2ecef2020-11-25T00:30:44ZengMDPI AGMicroarrays2076-39052015-10-014447448910.3390/microarrays4040474microarrays4040474Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon QuantificationMichael G. Mauk0Changchun Liu1Jinzhao Song2Haim H. Bau3School of Engineering and Applied Sciences, University of Pennsylvania, 220 S 33 rd Street, Philadelphia, PA 19104, USASchool of Engineering and Applied Sciences, University of Pennsylvania, 220 S 33 rd Street, Philadelphia, PA 19104, USASchool of Engineering and Applied Sciences, University of Pennsylvania, 220 S 33 rd Street, Philadelphia, PA 19104, USASchool of Engineering and Applied Sciences, University of Pennsylvania, 220 S 33 rd Street, Philadelphia, PA 19104, USAMicrofluidic 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.http://www.mdpi.com/2076-3905/4/4/474microfluidicslab on a chipisothermal nucleic acid amplification |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Michael G. Mauk Changchun Liu Jinzhao Song Haim H. Bau |
spellingShingle |
Michael G. Mauk Changchun Liu Jinzhao Song Haim H. Bau Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification Microarrays microfluidics lab on a chip isothermal nucleic acid amplification |
author_facet |
Michael G. Mauk Changchun Liu Jinzhao Song Haim H. Bau |
author_sort |
Michael G. Mauk |
title |
Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification |
title_short |
Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification |
title_full |
Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification |
title_fullStr |
Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification |
title_full_unstemmed |
Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification |
title_sort |
integrated microfluidic nucleic acid isolation, isothermal amplification, and amplicon quantification |
publisher |
MDPI AG |
series |
Microarrays |
issn |
2076-3905 |
publishDate |
2015-10-01 |
description |
Microfluidic 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. |
topic |
microfluidics lab on a chip isothermal nucleic acid amplification |
url |
http://www.mdpi.com/2076-3905/4/4/474 |
work_keys_str_mv |
AT michaelgmauk integratedmicrofluidicnucleicacidisolationisothermalamplificationandampliconquantification AT changchunliu integratedmicrofluidicnucleicacidisolationisothermalamplificationandampliconquantification AT jinzhaosong integratedmicrofluidicnucleicacidisolationisothermalamplificationandampliconquantification AT haimhbau integratedmicrofluidicnucleicacidisolationisothermalamplificationandampliconquantification |
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