Dataset Information

Focusing analytes from 50??L into 500 pL: On-chip focusing from large sample volumes using isotachophoresis.

ABSTRACT: The use of on-chip isotachophoresis assays for diagnostic applications is often limited by the small volumes of standard microfluidic channels. Overcoming this limitation is particularly important for detection of 'discrete' biological targets (such as bacteria) at low concentrations, where the volume of processed liquid in a standard microchannel might not contain any targets. We present a novel microfluidic chip that enables ITP focusing of target analytes from initial sample volumes of 50??L into a concentrated zone with a volume of 500 pL, corresponding to a 100,000-fold increase in mean concentration, and a 300,000-fold increase in peak concentration. We present design considerations for limiting sample dispersion in such large-volume focusing (LVF) chips and discuss the trade-off between assay time and Joule heating, which ultimately governs the scalability of LVF designs. Finally, we demonstrate a 100-fold improvement of ITP focusing performance in the LVF chip as compared to conventional microchannels, and apply this enhancement to achieve highly sensitive detection of both molecular targets (DNA, down to 10 fM) and whole bacteria (down to 100 cfu/mL).

SUBMITTER: van Kooten XF

PROVIDER: S-EPMC5585209 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Focusing analytes from 50 μL into 500 pL: On-chip focusing from large sample volumes using isotachophoresis.

van Kooten Xander F XF Truman-Rosentsvit Marianna M Kaigala Govind V GV Bercovici Moran M

Scientific reports 20170905 1

The use of on-chip isotachophoresis assays for diagnostic applications is often limited by the small volumes of standard microfluidic channels. Overcoming this limitation is particularly important for detection of 'discrete' biological targets (such as bacteria) at low concentrations, where the volume of processed liquid in a standard microchannel might not contain any targets. We present a novel microfluidic chip that enables ITP focusing of target analytes from initial sample volumes of 50 μL ...[more]

PMID: 28874694

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Project description:Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Dataset Information

Focusing analytes from 50??L into 500 pL: On-chip focusing from large sample volumes using isotachophoresis.

Publications

Focusing analytes from 50 μL into 500 pL: On-chip focusing from large sample volumes using isotachophoresis.

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