7 research outputs found
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Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore Sensing.
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 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
Lateral flow test engineering and lessons learned from COVID-19
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 self-testing 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
Evaluating the Binding of Selected Biomolecules to Cranberry Derived Proanthocyanidins Using the Quartz Crystal Microbalance
Despite cranberry being associated
with the prevention of bacterial
infections for over a century, our understanding of the bioavailability
and mechanisms by which cranberry prevents infection is limited. This
study investigates the interactions between cranberry proanthocyanidins
(CPAC) and human serum proteins (albumin, α-1-acid glycoprotein,
and fibrinogen) that may be encountered during CPAC metabolism following
ingestion. To better understand how CPAC might interfere with bacterial
infection, we also examined the interactions between CPAC and selected
bacterial virulence factors; namely, lipopolysaccharide (LPS) and
rhamnolipid. The binding of CPAC to the serum proteins, rhamnolipids
and LPS from Escherichia coli O111:B4
can be described by Langmuir-type isotherms, allowing the determination
of the apparent adsorption affinity constants, with CPAC interacting
most strongly with fibrinogen with a binding constant of 2.2 ×
10<sup>8</sup> M<sup>–1</sup>. These binding interactions will
limit the bioavailability of the CPAC at the site of action, an important
consideration in designing further clinical trials. Furthermore, CPAC
interacts with Pseudomonas aeruginosa 10 LPS, E. coli O111:B4 LPS, and P. aeruginosa rhamnolipids in fundamentally different
manners, supporting the theory that cranberry prevents bacterial infections
via multiple mechanisms
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Source Data for Nanopore sensing with DNA nanostructures reveals Guide-Intrinsic Mismatch Tolerance of CRISPR/dCas9
This is the source data for Nanopore sensing with DNA nanostructures reveals Guide-Intrinsic Mismatch Tolerance of CRISPR/dCas9 in Nature Biomedical Engineering. The data was generated by mixing a DNA nanostructure with dCas9 and measuring the change in current generated from translocation in solid-state nanopores. Data is originally in TDMS format (which can be given upon request) and then converted from TDMS to hdf5 using https://bitbucket.org/nikaer/nanopyre/src/master/ - from this translocationfinder is used which writes files from TDMS (labview) to hdf5. Processing and further data analysis from the hdf5 is done using https://github.com/sarahsandler/nanopro. TDMS files are from labview, however there is a nptdms software which can be used to read them into python. Please see the manuscript for more details
Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants
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