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.

In the growing field of capacitive deionization (CDI), a number of performance metrics have emerged to describe the desalination process. Unfortunately, the separation conditions under which these metrics are measured are often not specified, resulting in optimal performance at minimal removal. Here we outline a system of performance metrics and reporting conditions that resolves this issue. Our proposed system is based on volumetric energy consumption (Wh/m 3 ) and throughput productivity (L/h/m 2 ) reported for a specific average concentration reduction, water recovery, and feed salinity. To facilitate and rationalize comparisons between devices, materials, and operation modes, we propose a nominal standard testing condition of removing 5 mM from a 20 mM NaCl feed solution at 50% water recovery for CDI research. Using this separation, we compare the desalination performance of a flow-through electrode (fte-CDI) cell and a flow between membrane (fb-MCDI) device, showing how significantly different systems can be compared in terms of generally desirable desalination characteristics. In general, we find that performance analysis must be considered carefully so to not allow for ambiguous separation conditions or the maximization of one metric at the expense of another. Additionally, for context we discuss a number of important underlying performance indicators and cell characteristics that are not performance measures in and of themselves but can be examined to better understand differences in performance.

CRISPR-diagnostic assays have gained significant interest in the last few years. This interest has grown rapidly during the current COVID-19 pandemic, where CRISPR-diagnostics have been frontline contenders for rapid testing solutions. This surge in CRISPR-diagnostic research prompts the following question: what exactly are the achievable limits of detection and associated assay times enabled by the kinetics of enzymes such as Cas12 and Cas13? To explore this question, we here present a model based on Michaelis–Menten enzyme kinetics theory applied to CRISPR enzymes. We use the model to develop analytical solutions for reaction kinetics and develop back-of-the-envelope criteria to validate and check for consistency in reported enzyme kinetic parameters. We applied our analyses to all studies known to us, which report Michaelis–Menten-type kinetic data for CRISPR-associated enzymes. These studies include all subtypes of Cas12 and Cas13 and orthologs. We found all but one study clearly violate at least two of our three rules and therefore present data that violate basic physical limits. We performed an experimental study of reaction kinetics of LbCas12a with both ssDNA and dsDNA activators and use these data to validate our model and its predicted scaling. The validated model is used to explore CRISPR reaction time scales and the degree of reaction completion for practically relevant target concentrations applicable to CRISPR-diagnostic assays. The results have broad implications for achievable limits of detection and assay times of emerging, amplification-free CRISPR-detection methods.

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.