Abstract
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.
Highlights
Captured on the membrane, which eliminates the need for a separate elution step and simplifies flow control; (3) Isothermal amplification such as loop mediated isothermal amplification (LAMP), which simplifies the instrumentation compared to PCR and is less energy demanding than thermal cycling
POC devices and operation are significantly simplified by (1) a multifunctional reaction chamber that includes an embedded membrane for the capture of DNA and RNA, storage of reagents, and the in situ amplification of nucleic acids (NAs) on the membrane which circumvents the need for a separate elution step and decouples sample size from amplification reaction volume; and (2) isothermal amplification such as LAMP (Loop mediated amplification) with real-time detection, using for example, a smartphone CCD camera as detector
POC molecular diagnostics devices were first reported in the mid-1990s [45], single-chamber PCR
Summary
In the last decade there has been considerable interest and research in addressing the needs and opportunities for on-site Nucleic Acid-based Tests (NATs) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. The chip combines the following unit operations in a single reactor: (1) A porous silica or cellulose NA binding “membrane” is used to load sample lysate in a flow-through mode, allowing NA to be isolated from large sample volumes, such that the amplification volume is decoupled from the sample size, which is crucial for detecting low abundance targets This feature is absent in many previously-reported POC NAAT devices; (2) in situ amplification of NA captured on the membrane, which eliminates the need for a separate elution step and simplifies flow control; (3) Isothermal amplification such as loop mediated isothermal amplification (LAMP), which simplifies the instrumentation compared to PCR and is less energy demanding than thermal cycling.
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