Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 47; pp. 29518 - 29525
• Main Authors: Ramachandran, Ashwin, Huyke, Diego A., Sharma, Eesha, Sahoo, Malaya K., Huang, ChunHong, Banaei, Niaz, Pinsky, Benjamin A., Santiago, Juan G.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 24.11.2020
• Subjects: Assaying; Coronaviruses; COVID-19; COVID-19 Nucleic Acid Testing - methods; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA probes; Electric fields; Gene amplification; gRNA; Humans; Isotachophoresis; Isotachophoresis - methods; Microfluidics; Microfluidics - methods; Nasal Mucosa - virology; Physical Sciences; Purification; Ribonucleic acid; RNA; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Severe acute respiratory syndrome coronavirus 2; Single-stranded DNA; COVID-19; rapid testing; CRISPR diagnostics; microfluidics; isotachophoresis
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2010254117

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR–Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore−quencher pair.We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12–gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.