ABSTRACT
Current quantification methods of Escherichia coli (E. coli) contamination in water samples involve long incubation, laboratory equipment and facilities, or complex processes that require specialized training for accurate operation and interpretation. To address these limitations, we have developed a microfluidic device and portable instrument prototypes capable of performing a rapid and highly sensitive bacteriophage-based assay to detect E. coli cells with detection limit comparable to traditional methods in a fraction of the time. The microfluidic device combines membrane filtration and selective enrichment using T7-NanoLuc-CBM, a genetically engineered bacteriophage, to identify 4.1 E. coli CFU in 100 mL of drinking water within 5.5 hours. The microfluidic device was designed and tested to process up to 100 mL of real-world drinking water samples with turbidities below 10 NTU. Prototypes of custom instrumentation, compatible with our valveless microfluidic device and capable of performing all of the assay's units of operation with minimal user intervention, demonstrated similar assay performance to that obtained on the benchtop assay. This research is the first step towards a faster, portable, and semi-automated, phage-based microfluidic platform for improved in-field water quality monitoring in low-resource settings.
Subject(s)
Bacteriophages , Drinking Water , Escherichia coli , Lab-On-A-Chip Devices , LuciferasesABSTRACT
Inadequate drinking water quality is among the major causes of preventable mortality, predominantly in young children. Identifying contaminated water sources remains a significant challenge, especially where resources are limited. The current methods for measuring Escherichia coli (E. coli), the WHO preferred indicator for measuring fecal contamination of water, involve overnight incubation and require specialized training. In 2016, UNICEF released a Target Product Profile (TPP) to incentivize product innovations to detect low levels of viable E. coli in water samples in the field in less than 6 h. Driven by this challenge, we developed a phage-based assay to detect and semi-quantify E. coli. We formulated a phage cocktail containing a total of 8 phages selected against an extensive bacterial strain library and recombined with the sensitive NanoLuc luciferase reporter. The assay was optimized to be processed in a microfluidic chip designed in-house and was tested against locally sourced sewage samples and on drinking water sources in Nairobi, Kenya. With this assay, combined with the microfluidic chip platform, we propose a complete automated solution to detect and semi-quantify E. coli at less than 10 MPN/100 mL in 5.5 h by minimally trained personnel.
Subject(s)
Bacteriophages , Drinking Water , Bacteria , Escherichia coli , Kenya , LuciferasesABSTRACT
The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.
Subject(s)
COVID-19/diagnosis , Immunoassay/instrumentation , Malaria/diagnosis , Point-of-Care Testing , Tuberculosis/diagnosis , COVID-19 Serological Testing/economics , COVID-19 Serological Testing/instrumentation , Equipment Design , Humans , Immunoassay/economics , Mycobacterium tuberculosis/isolation & purification , Plasmodium/isolation & purification , Point-of-Care Testing/economics , Reproducibility of Results , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Time FactorsABSTRACT
Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.
Subject(s)
Antigens, Viral/immunology , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , COVID-19/virology , Humans , Limit of Detection , Point-of-Care Systems , RNA, Viral/immunology , Sensitivity and SpecificityABSTRACT
The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.
ABSTRACT
Rapid tests for SARS-COV-2 infection are important tools for pandemic control, but current rapid tests are based on proprietary designs and reagents. We report clinical validation results of an open-access lateral flow assay (OA-LFA) design using commercially available materials and reagents, along with RT-qPCR and commercially available comparators (BinaxNOW® and Sofia®). Adult patients with suspected COVID-19 based on clinical signs and symptoms, and with symptoms ≤7 days duration, underwent anterior nares (AN) sampling for the OA-LFA, Sofia®, BinaxNOW ™, and RT-qPCR, along with nasopharyngeal (NP) RT-qPCR. Results indicate a positive predictive agreement with NP sampling as 69% (60% -78%) OA-LFA, 74% (64% - 82%) Sofia®, and 82% (73% - 88%) BinaxNOW™. The implication for these results is that we provide an open-access LFA design that meets the minimum WHO target product profile for a rapid test, that virtually any diagnostic manufacturer could produce.
Subject(s)
Antigens, Viral/analysis , COVID-19/diagnosis , Immunoassay , SARS-CoV-2/metabolism , Area Under Curve , COVID-19/virology , Humans , Nasopharynx/virology , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , ROC Curve , Real-Time Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Sensitivity and SpecificityABSTRACT
Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.
ABSTRACT
Here, we report the complete genome sequences of 38 novel bacteriophages infecting Escherichia coli, isolated from a raw sewage source in Washington. Of these phages, 26 are under 100 kb, 11 are near 170 kb, and 1 352-kb jumbo phage was discovered.
ABSTRACT
The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.
Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/diagnosis , Immunoassay/methods , Nucleocapsid/immunology , Pneumonia, Viral/diagnosis , Point-of-Care Systems , Antibodies, Viral/blood , Antigens/immunology , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/virology , Humans , Limit of Detection , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2ABSTRACT
Convective transport can significantly distort spatial concentration gradients. Interstitial flow is ubiquitous throughout living tissue, but our understanding of how interstitial flow affects concentration gradients in biological processes is limited. Interstitial flow is of particular interest for angiogenesis because pathological and physiological angiogenesis is associated with altered interstitial flow, and both interstitial flow and morphogen gradients (e.g., vascular endothelial growth factor, VEGF) can potentially stimulate and guide new blood vessel growth. We designed an in vitro microfluidic platform to simulate 3D angiogenesis in a tissue microenvironment that precisely controls interstitial flow and spatial morphogen gradients. The microvascular tissue was developed from endothelial colony forming cell-derived endothelial cells extracted from cord blood and stromal fibroblasts in a fibrin extracellular matrix. Pressure in the microfluidic lines was manipulated to control the interstitial flow. A mathematical model of mass and momentum transport, and experimental studies with fluorescently labeled dextran were performed to validate the platform. Our data demonstrate that at physiological interstitial flow (0.1-10 µm/s), morphogen gradients were eliminated within hours, and angiogenesis demonstrated a striking bias in the opposite direction of interstitial flow. The interstitial flow-directed angiogenesis was dependent on the presence of VEGF, and the effect was mediated by αvß3 integrin. We conclude that under physiological conditions, growth factors such as VEGF and fluid forces work together to initiate and spatially guide angiogenesis.
Subject(s)
Extracellular Fluid/physiology , Neovascularization, Physiologic , Diffusion , Humans , Integrin alphaVbeta3/metabolism , Microfluidics , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Tissue engineering can potentially recreate in vivo cellular microenvironments in vitro for an array of applications such as biological inquiry and drug discovery. However, the majority of current in vitro systems still neglect many biological, chemical, and mechanical cues that are known to impact cellular functions such as proliferation, migration, and differentiation. To address this gap, we have developed a novel microfluidic device that precisely controls the spatial and temporal interactions between adjacent three-dimensional cellular environments. The device consists of four interconnected microtissue compartments (~0.1 mm(3)) arranged in a square. The top and bottom pairs of compartments can be sequentially loaded with discrete cellularized hydrogels creating the opportunity to investigate homotypic (left to right or x-direction) and heterotypic (top to bottom or y-direction) cell-cell communication. A controlled hydrostatic pressure difference across the tissue compartments in both x and y direction induces interstitial flow and modulates communication via soluble factors. To validate the biological significance of this novel platform, we examined the role of stromal cells in the process of vasculogenesis. Our device confirms previous observations that soluble mediators derived from normal human lung fibroblasts (NHLFs) are necessary to form a vascular network derived from endothelial colony forming cell-derived endothelial cells (ECFC-ECs). We conclude that this platform could be used to study important physiological and pathological processes that rely on homotypic and heterotypic cell-cell communication.
Subject(s)
Cell Communication/physiology , Cell Culture Techniques/instrumentation , Cellular Microenvironment/physiology , Extracellular Fluid/physiology , Microfluidic Analytical Techniques/instrumentation , Models, Biological , Tissue Engineering/instrumentation , Cell Culture Techniques/methods , Cells, Cultured , Equipment Design , Fibroblasts/cytology , HumansABSTRACT
Models that aim to recapitulate the dynamic in vivo features of the microcirculation are crucial for studying vascularization. Cells in vivo respond not only to biochemical cues (e.g., growth factor gradients) but also sense mechanical cues (e.g., interstitial flow, vessel perfusion). Integrating the response of cells, the stroma, and the circulation in a dynamic 3D setting will create an environment suitable for the exploration of many fundamental vascularization processes. Here in this chapter, we describe an in vivo-inspired microenvironment that is conducive to the development of perfused human capillaries.