AN INTEGRATED RT-LAMP AND CRISPR ASSAY FOR NUCLEIC ACID DETECTION IN A SINGLE MICROFLUIDIC CHIP

We will present a microfluidic assay to detect SARS-CoV-2 RNA from nasopharyngeal swab samples. Our method leverages isotachophoresis (ITP) to integrate sample preparation, RT-LAMP, and CRISPR-based nucleic acid detection in an automatable chip. For the first time, we use ITP to purify, pre-concentrate and isothermally amplify target nucleic acids into a ~1 µL reaction volume on-chip. The device then transitions LAMP amplicons into an on-chip zone containing Cas12-gRNA complexes and reporter molecules to measure target-activated CRISPR activity. We will use our method to automatically detect COVID-19 from nasopharyngeal swab samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

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

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.