Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis

Benjamin P Sullivan; Andrew T Bender; Duy N Ngyuen; Jane Yuqian Zhang; Jonathan D Posner

doi:10.1016/j.jchromb.2020.122494

Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis

J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 15:1163:122494. doi: 10.1016/j.jchromb.2020.122494. Epub 2020 Dec 13.

Authors

Benjamin P Sullivan¹, Andrew T Bender¹, Duy N Ngyuen², Jane Yuqian Zhang¹, Jonathan D Posner³

Affiliations

¹ Department of Mechanical Engineering, University of Washington, Seattle, WA, USA.
² Department of Chemical Engineering, University of Washington, Seattle, WA, USA.
³ Department of Mechanical Engineering, University of Washington, Seattle, WA, USA; Department of Chemical Engineering, University of Washington, Seattle, WA, USA; Department of Family Medicine, University of Washington, Seattle, WA, USA. Electronic address: jposner@uw.edu.

Abstract

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.

Keywords: Isotachophoresis; Nucleic acids; Paper-microfluidics; Protein digestion; Sample preparation.

MeSH terms

Electrophoresis, Polyacrylamide Gel
Equipment Design
Humans
Isotachophoresis / instrumentation*
Isotachophoresis / methods
Lab-On-A-Chip Devices*
Microfluidic Analytical Techniques / instrumentation*
Nucleic Acid Amplification Techniques
Nucleic Acids* / blood
Nucleic Acids* / chemistry
Nucleic Acids* / isolation & purification
Paper

Substances

Nucleic Acids

Grants and funding

R01 EB022630/EB/NIBIB NIH HHS/United States