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.
The plant polymer lignin is the most abundant renewable source of aromatics on the planet and conversion of it to valuable fuels and chemicals is critical to the economic viability of a lignocellulosic biofuels industry and to meeting the DOE’s 2022 goal of $\$2.50$/gallon mean biofuel selling price. Presently, there is no efficient way of converting lignin into valuable commodities. Current biological approaches require mixtures of expensive ligninolytic enzymes and engineered microbes. This project was aimed at circumventing these problems by discovering commensal relationships among fungi and bacteria involved in biological lignin utilization and using this knowledge to engineer microbial communities capable of converting lignin into renewable fuels and chemicals. Essentially, we aimed to learn from, mimic and improve on nature. We discovered fungi that synergistically work together to degrade lignin, engineered fungal systems to increase expression of the required enzymes and engineered organisms to produce products such as biodegradable plastics precursors.
We present a field-deployable microfluidic immunoassay device in response to the need for sensitive, quantitative, and high-throughput protein detection at point-of-need. The portable microfluidic system facilitates eight magnetic bead-based sandwich immunoassays from raw samples in 45 minutes. An innovative bead actuation strategy was incorporated into the system to automate multiple sample process steps with minimal user intervention. The device is capable of quantitative and sensitive protein analysis with a 10 pg/ml detection limit from interleukin 6-spiked human serum samples. We envision the reported device offering ultrasensitive point-of-care immunoassay tests for timely and accurate clinical diagnosis.
We present a field-deployable microfluidic immunoassay device in response to the need for sensitive, quantitative, and high-throughput protein detection at point-of-need. The portable microfluidic system facilitates eight magnetic bead-based sandwich immunoassays from raw samples in 45 minutes. An innovative bead actuation strategy was incorporated into the system to automate multiple sample process steps with minimal user intervention. The device is capable of quantitative and sensitive protein analysis with a 10 pg/ml detection limit from interleukin 6-spiked human serum samples. We envision the reported device offering ultrasensitive point-of-care immunoassay tests for timely and accurate clinical diagnosis.
Targeting host factors for anti-viral development offers several potential advantages over traditional countermeasures that include broad-spectrum activity and prevention of resistance. Characterization of host factors in animal models provides strong evidence of their involvement in disease pathogenesis, but the feasibility of performing high-throughput in vivo analyses on lists of genes is problematic. To begin addressing the challenges of screening candidate host factors in vivo, we combined advances in CRISPR-Cas9 genome editing with an immunocompromised mouse model used to study highly pathogenic viruses. Transgenic mice harboring a constitutively expressed Cas9 allele (Cas9tg/tg) with or without knockout of type I interferon receptors served to optimize in vivo delivery of CRISPR single-guide RNA (sgRNA) using Invivofectamine 3.0, a simple and easy-to-use lipid nanoparticle reagent. Invivofectamine 3.0-mediated liver-specific editing to remove activity of the critical Ebola virus host factor Niemann-Pick disease type C1 in an average of 74% of liver cells protected immunocompromised Cas9tg/tg mice from lethal surrogate Ebola virus infection. We envision that immunocompromised Cas9tg/tg mice combined with straightforward sgRNA in vivo delivery will enable efficient host factor loss-of-function screening in the liver and other organs to rapidly study their effects on viral pathogenesis and help initiate development of broad-spectrum, host-directed therapies against emerging pathogens.
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.
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.
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.
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.
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.
The emerging field of metagenomics seeks to assess the genetic diversity of complex mixed populations of bacteria, such as those found at different sites within the human body. A single person's mouth typically harbors up to 100 bacterial species, while surveys of many people have found more than 700 different species, of which {approx}50% have never been cultivated. In typical metagenomics studies, the cells themselves are destroyed in the process of gathering sequence information, and thus the connection between genotype and phenotype is lost. A great deal of sequence information may be generated, but it is impossible to assign any given sequence to a specific cell. We seek non-destructive, culture-independent means of gathering sequence information from selected individual cells from mixed populations. As a first step, we have developed a microfluidic device for concentrating and specifically labeling bacteria from a mixed population. Bacteria are electrophoretically concentrated against a photopolymerized membrane element, and then incubated with a specific fluorescent label, which can include antibodies as well as specific or non-specific nucleic acid stains. Unbound stain is washed away, and the labeled bacteria are released from the membrane. The stained cells can then be observed via epifluorescence microscopy, or counted via flow cytometry. We have tested our device with three representative bacteria from the human microbiome: E. coli (gut, Gram-negative), Lactobacillus acidophilus (mouth, Gram-positive), and Streptococcus mutans (mouth, Gram-positive), with results comparable to off-chip labeling techniques.
Uncultivable microorganisms likely play significant roles in the ecology within the human body, with subtle but important implications for human health. Focusing on the oral microbiome, we are developing a processor for targeted isolation of individual microbial cells, facilitating whole-genome analysis without the need for isolation of pure cultures. The processor consists of three microfluidic modules: identification based on 16S rRNA fluorescence in situ hybridization (FISH), fluorescence-based sorting, and encapsulation of individual selected cells into small droplets for whole genome amplification. We present here a technique for performing microscale FISH and flow cytometry, as a prelude to single cell sorting.
We present concepts for self-assembly of diffractive optics with potential uses in biosensors and biocomputers. The simplest such optics, diffraction gratings, can potentially be made from chemically-stabilized microtubules migrating on nanopatterned tracks of the motor protein kinesin. We discuss the fabrication challenges involved in patterning sub-micron-scale structures with proteins that must be maintained in aqueous buffers to preserve their activity. A novel strategy is presented that employs dry contact printing onto glass-supported amino-silane monolayers of heterobifunctional crosslinkers, followed by solid-state reactions of these cross-linkers, to graft patterns of reactive groups onto the surface. Successive solution-phase addition of cysteine-mutant proteins and amine-reactive polyethylene glycol allows assembly of features onto the printed patterns. We present data from initial experiments showing successful micro- and nanopatterning of lines of single-cysteine mutants of kinesin interleaved with lines of polyethylene, indicating that this strategy can be employed to arrays of features with resolutions suitable for gratings.