Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in ...resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in ...point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers.
Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common ...detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.
Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before ...whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.
The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced ...Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.
Cytokines and acute-phase proteins are promising biomarkers for inflammatory disease. Despite its potential, early diagnosis based on these biomarkers remains challenging without technology enabling ...highly sensitive protein detection immediately after sample collection, because of the low abundance and short half-life of these proteins in bodily fluids. Enzyme-linked immunosorbent assay (ELISA) is a gold-standard method for such protein analysis, but it often requires labor-intensive and time-consuming sample handling and as well as a bulky benchtop platereader, limiting its utility in the clinical site. We developed a portable microfluidic immunoassay device capable of sensitive, quantitative, and high-throughput protein detection at point-of-need. The portable microfluidic system performs eight magnetic bead-based sandwich immunoassays from raw samples in 40 min. An innovative bead actuation strategy was incorporated into the system to automate multiple sample handling steps with minimal user intervention. The device enables quantitative protein analysis with picomolar sensitivity, as demonstrated using human samples spiked with interleukin-6 and C-reactive protein. The affinity-based assays are highly specific to the target without cross-reactivity. Therefore, we envision the reported device offering ultrasensitive and field-deployable immunoassay tests for timely and accurate clinical diagnosis.
A portable microfluidic platform uses a novel programmable magnetic actuation mechanism to process samples for sensitive detection of inflammatory biomarkers.
The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools ...capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection = 1.35–5.50 ng/mL for immunoassays and 1–30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.
•Portable, microfluidic platform for detection of proteins and nucleic acids.•Simultaneous detection of toxins and pathogenic bacteria from stool.•Highly sensitive and specific assays require no sample preparation.•Disposable, plastic, microfluidic devices integrate sample handling.•Suitable for deployment in low-resource settings.
The respiratory virus responsible for coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected nearly every aspect of life worldwide, claiming ...the lives of over 3.9 million people globally, at the time of this publication. Neutralizing humanized nanobody (V
H)-based antibodies (V
H-huFc) represent a promising therapeutic intervention strategy to address the current SARS-CoV-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity V
H bacteriophage library, several potent neutralizing V
H-huFc antibodies were identified and evaluated for their capacity to tightly bind to the SARS-CoV-2 receptor-binding domain, to prevent binding of SARS-CoV-2 spike (S) to the cellular receptor angiotensin-converting enzyme 2, and to neutralize viral infection. Preliminary preclinical evaluation of multiple V
H-huFc antibody candidates demonstrate that they are prophylactically and therapeutically effective
against
SARS-CoV-2. The identified and characterized V
H-huFc antibodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic.
High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are ...typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.
is a widespread white-rot basidiomycete fungus with significance in diverse biotechnological applications due to its ability to degrade aromatic compounds, xenobiotics, and lignin using an assortment ...of oxidative enzymes including laccase. In this work, a chemical screen with 480 conditions was conducted to identify chemical inducers of laccase expression in
. Among the chemicals tested, phenothiazines were observed to induce laccase activity in
with promethazine being the strongest laccase inducer of the phenothiazine-derived compounds examined. Secretomes produced by promethazine-treated
exhibited increased laccase protein abundance, increased enzymatic activity, and an enhanced ability to degrade phenolic model lignin compounds. Transcriptomics analyses revealed that promethazine rapidly induced the expression of genes encoding lignin-degrading enzymes, including laccase and various oxidoreductases, showing that the increased laccase activity was due to increased laccase gene expression. Finally, the generality of promethazine as an inducer of laccases in fungi was demonstrated by showing that promethazine treatment also increased laccase activity in other relevant fungal species with known lignin conversion capabilities including
and
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