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.
Interest in CRISPR-Cas12 and CRISPR-Cas13 detection continues to increase as these detection schemes enable the specific recognition of nucleic acids. The fundamental sensitivity limits of these schemes (and their applicability in amplification-free assays) are governed by kinetic rates. However, these kinetic rates remain poorly understood, and their reporting has been inconsistent. We quantify kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a, and LbuCas13a) and their corresponding limits of detection (LoD). Collectively, we present quantification of enzyme kinetics for 14 guide RNAs (gRNAs) and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. We validate the self-consistency of our measurements by comparing trends and limiting behaviors with a Michaelis−Menten trans-cleavage reaction kinetics model. For our assay conditions, activated Cas12 and Cas13 enzymes exhibit trans-cleavage catalytic efficiencies between order 10 5 and 10 6 M −1 s −1 . For assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, the kinetic rates at the current assay conditions result in an amplification-free LoD in the picomolar range. The results suggest that successful detection of target requires cleavage (by an activated CRISPR enzyme) of the order of at least 0.1% of the fluorescent reporter molecules. This fraction of reporters cleaved is required to differentiate the signal from the background, and we hypothesize that this required fraction is largely independent of the detection method (e.g., endpoint vs reaction velocity) and detector sensitivity. Our results demonstrate the fundamental nature by which kinetic rates and background signal limit LoDs and thus highlight areas of improvement for the emerging field of CRISPR diagnostics.
24The rapid spread of COVID-19 across the world has revealed major gaps in our ability to 25 respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular 26 diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based 27 diagnostic approaches are proving to be useful as field-deployable solutions. In a basic 28 form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA 29 (gRNA). This complex is activated when it highly specifically binds to target DNA, and 30 the activated complex non-specifically cleaves single-stranded DNA reporter probes 31 labeled with a fluorophore-quencher pair. We recently discovered that electric field 32 gradients can be used to control and accelerate this CRISPR assay by co-focusing 33Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient 34 using a selective ionic focusing technique known as isotachophoresis (ITP) implemented 35 on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for 36 automated purification of target RNA from raw nasopharyngeal swab sample. We here 37 combine this ITP purification with loop-mediated isothermal amplification, and the ITP-38 enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to 39 result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This 40 electric field control enables a new modality for a suite of microfluidic CRISPR-based 41 diagnostic assays. 42 43 3 Significance statement 44Rapid, early-stage screening is especially crucial during pandemics for early identification 45 of infected patients and control of disease spread. CRISPR biology offers new methods 46 for rapid and accurate pathogen detection. Despite their versatility and specificity, existing 47 CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid 48 extraction, large reagent volumes, and several manual steps-factors which prolong the 49 process and impede use in low resource settings. We here combine on-chip electric-field 50 control in combination with CRIPSR biology to directly address these limitations of current 51 CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-52 2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a 53 significant improvement over existing diagnostic methods for COVID-19. 54 55 56 4 Infectious diseases such as COVID-19 are a persistent global threat. Early-stage 57 screening and rapid identification of infected patients are important during pandemics to 58 treat the infected and to control disease spread. The frontline diagnostic tool for COVID-59 19 has been RT-qPCR (reverse transcription -quantitative polymerase chain reaction), 60 and protocols for this have been developed and published by the World Health 61 Organization (WHO)(1) and the Centers for Disease Control and Prevention (CDC)(2). 62 While these tests are highly specific and sensitive, they are laborious, time-consuming, 63 and are des...
We report on the design and testing of glass nozzles used to produce liquid sheets. The sheet nozzles use a single converging channel chemically etched into glass wafers by standard...
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