The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.

The acceptability and feasibility of large-scale testing with lateral flow tests (LFTs) for clinical and public health purposes has been demonstrated during the COVID-19 pandemic. LFTs can detect analytes in a variety of samples, providing a rapid read-out, which allows selftesting and decentralized diagnosis. In this Review, we examine the changing LFT landscape with a focus on lessons learned from COVID-19. We discuss the implications of LFTs for decentralized testing of infectious diseases, including diseases of epidemic potential, the 'silent pandemic' of antimicrobial resistance, and other acute and chronic infections. Bioengineering approaches will play a key part in increasing the sensitivity and specificity of LFTs, improving sample preparation, incorporating nucleic acid amplification and detection, and enabling multiplexing, digital connection and green manufacturing, with the aim of creating the next generation of high-accuracy, easy-to-use, affordable and digitally connected LFTs. We conclude with recommendations, including the building of a global network of LFT research and development hubs to facilitate and strengthen future diagnostic resilience. Sections• Bioengineering approaches, such as the use of nano-and quantum materials, nucleic-acid-based LFTs, CRISPR and machine learning, will improve the sensitivity, specificity, multiplexing and connectivity features of LFTs.• We recommend investing in an international LFT research and development hub network to spearhead the development of a pipeline of innovative bioengineering approaches to design next-generation LFTs.

Double-stranded DNA translocates through sufficiently large nanopores either in a linear, single-file fashion or in a folded hairpin conformation when captured somewhere along its length. We show that the folding state of DNA can be controlled by changing the electrolyte concentration, pH and PEG content of the measurement buffer. At 1 m LiCl or 0.35 m KCl in neutral pH, single-file translocations make up more than 90% of the total. We attribute the effect to the onset of electroosmotic flow from the pore at low ionic strength. Our hypothesis on the critical role of flows is supported by the preferred orientation of entry of a strand that has been folded into a multi-helix structure at one end. Control over DNA folding is critical for nanopore sensing approaches that use modifications along a DNA strand and the associated secondary current drops to encode information.

Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific analysis of a DNA sample. Solid-state nanopore sensors are especially promising for this type of single-molecule sensing because of the ability to analyze patterns in the ionic current signatures of DNA molecules. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify 1 two different DNA targets in a background mixture of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.

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