RESUMO
As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.
Assuntos
COVID-19 , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Pandemias , RNA Viral , SARS-CoV-2/isolamento & purificaçãoRESUMO
IMPACT STATEMENT: Humanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.
Assuntos
Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/isolamento & purificação , COVID-19 , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , SARS-CoV-2 , Saliva/virologia , Sensibilidade e EspecificidadeRESUMO
Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.
Assuntos
Astrócitos/patologia , Sistema Nervoso Central/patologia , Inflamação/patologia , Fator de Transcrição MafG/genética , Proteínas Repressoras/genética , Animais , Antioxidantes/metabolismo , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Metilação de DNA , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inflamação/genética , Masculino , Metionina Adenosiltransferase/genética , Camundongos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Fator 2 Relacionado a NF-E2/genética , Análise de Sequência de RNA , Transdução de Sinais , Transcrição GênicaRESUMO
SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy in which many workplaces and activities are completely closed, leading to substantial economy costs. Here, we present a protocol we recently developed that allows rapid and simple detection of SARS-CoV-2 for the large population, eliminating costs and involvement of professional teams and laboratories. This protocol is based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). We tested this protocol directly on patient samples, both nasal and throat clinical swabs as well as saliva. Notably, this protocol is simple, cheap and can be easily applied to other pathogens as well.
RESUMO
Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.
Assuntos
Afinidade de Anticorpos , Evolução Biológica , Aptidão Genética , Modelos Genéticos , Norovirus/genética , Animais , Anticorpos Neutralizantes , Proteínas do Capsídeo/fisiologia , Epistasia Genética , Camundongos , Dobramento de Proteína , Estabilidade Proteica , Seleção GenéticaRESUMO
A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system. In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.
Assuntos
Potenciais de Ação/fisiologia , Estimulação Elétrica/métodos , Campos Magnéticos , Neurônios/fisiologia , Técnicas de Cultura de Células , Campos Eletromagnéticos , Humanos , Potenciais da MembranaRESUMO
Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.
Assuntos
Imunoprecipitação da Cromatina/métodos , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/citologia , Análise de Célula Única/métodos , Animais , Cromatina/genética , Biologia Computacional , Código de Barras de DNA Taxonômico , Humanos , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Sequência de DNARESUMO
Metagenomic studies suggest that only a small fraction of the viruses that exist in nature have been identified and studied. Characterization of unknown viral genomes is hindered by the many genomes populating any virus sample. A new method is reported that integrates drop-based microfluidics and computational analysis to enable the purification of any single viral species from a complex mixed virus sample and the retrieval of complete genome sequences. By using this platform, the genome sequence of a 5243 bp dsDNA virus that was spiked into wastewater was retrieved with greater than 96% sequence coverage and more than 99.8% sequence identity. This method holds great potential for virus discovery since it allows enrichment and sequencing of previously undescribed viruses as well as known viruses.
Assuntos
Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodos , Sequência de Bases , DNA Viral/análise , DNA Viral/genéticaRESUMO
Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.
Assuntos
Microfluídica/métodos , Recombinação Genética/genética , Análise de Sequência/métodos , Vírus/genética , Animais , Artefatos , Células Cultivadas , Corantes Fluorescentes , Ensaios de Triagem em Larga Escala , Camundongos , Tamanho da Partícula , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/genéticaRESUMO
A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.
Assuntos
Microfluídica/métodos , Norovirus/imunologia , Ensaio de Placa Viral , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Camundongos , Microfluídica/instrumentação , Norovirus/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD) curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields.
Assuntos
Potenciais de Ação/fisiologia , Axônios/metabolismo , Cálcio/metabolismo , Cronaxia/fisiologia , Dendritos/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , 4-Aminopiridina/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Bicuculina/análogos & derivados , Bicuculina/farmacologia , Cronaxia/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Bloqueadores dos Canais de Potássio/farmacologia , Cultura Primária de Células , Ratos , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
UNLABELLED: Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at â¼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity. IMPORTANCE: RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.
Assuntos
Infecções por Caliciviridae/virologia , Norovirus/genética , Norovirus/isolamento & purificação , Recombinação Genética , Animais , Variação Genética , Genótipo , Humanos , Camundongos , Microfluídica , Dados de Sequência Molecular , Norovirus/classificação , FilogeniaRESUMO
The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.
Assuntos
Microfluídica/métodos , DNA Complementar/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA/métodosRESUMO
High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 µm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Virologia/métodos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Macrófagos/virologia , Camundongos , Norovirus/fisiologia , Carga Viral , Cultura de Vírus/métodos , Replicação ViralRESUMO
In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR(+)], a protein-based epigenetic element, allows yeast to circumvent this "glucose repression" and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR(+)]. [GAR(+)] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR(+)] presents a unique example of Lamarckian inheritance.
Assuntos
Epigênese Genética , Príons/metabolismo , Saccharomyces cerevisiae/metabolismo , Staphylococcus hominis/metabolismo , Fermentação , Glucose/metabolismo , Saccharomyces cerevisiae/genética , Staphylococcus hominis/genética , Vinho/microbiologia , Leveduras/genética , Leveduras/metabolismoRESUMO
Transcranial Magnetic Stimulation (TMS) is a promising technology for both neurology and psychiatry. Positive treatment outcome has been reported, for instance in double blind, multi-center studies on depression. Nonetheless, the application of TMS towards studying and treating brain disorders is still limited by inter-subject variability and lack of model systems accessible to TMS. The latter are required to obtain a deeper understanding of the biophysical foundations of TMS so that the stimulus protocol can be optimized for maximal brain response, while inter-subject variability hinders precise and reliable delivery of stimuli across subjects. Recent studies showed that both of these limitations are in part due to the angular sensitivity of TMS. Thus, a technique that would eradicate the need for precise angular orientation of the coil would improve both the inter-subject reliability of TMS and its effectiveness in model systems. We show here how rotation of the stimulating field relieves the angular sensitivity of TMS and provides improvements in both issues. Field rotation is attained by superposing the fields of two coils positioned orthogonal to each other and operated with a relative phase shift in time. Rotating field TMS (rfTMS) efficiently stimulates both cultured hippocampal networks and rat motor cortex, two neuronal systems that are notoriously difficult to excite magnetically. This opens the possibility of pharmacological and invasive TMS experiments in these model systems. Application of rfTMS to human subjects overcomes the orientation dependence of standard TMS. Thus, rfTMS yields optimal targeting of brain regions where correct orientation cannot be determined (e.g., via motor feedback) and will enable stimulation in brain regions where a preferred axonal orientation does not exist.
Assuntos
Hipocampo/fisiologia , Campos Magnéticos , Modelos Biológicos , Estimulação Magnética Transcraniana , Animais , Humanos , Masculino , Ratos , Estimulação Magnética Transcraniana/instrumentação , Estimulação Magnética Transcraniana/métodosRESUMO
Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Microfluidic devices can be used to produce single or multiple emulsions with remarkably precise control of both the contents and size of the drops. Since each level of a multiple emulsion is formed by a distinct fluid stream, very efficient encapsulation of materials can be achieved. To obtain high throughput, these devices can be fabricated lithographically, allowing many devices to operate in parallel. However, to form multiple emulsions using a planar microfluidic device, the wettability of its surface must switch from hydrophobic to hydrophilic on the scale of micrometers where the drops are formed; this makes the fabrication of the devices very difficult. To overcome this constraint, we introduce non-planar microfluidic devices with graduated thicknesses; these can make drops even when their wetting properties do not favor drop formation. Nevertheless, the dependence of drop formation on the device geometry, the flow rates and the properties of the fluids, particularly in the case of unfavorable wetting, is very complex, making the successful design of these devices more difficult. Here we show that there exists a critical value of flow of the continuous phase above which drop formation occurs; this value decreases by two orders of magnitude as the wetting to the device wall of the continuous phase improves. We demonstrate how this new understanding can be used to optimize device design for efficient production of double or multiple emulsions.
Assuntos
Técnicas Analíticas Microfluídicas , Emulsões/química , Desenho de Equipamento , Interações Hidrofóbicas e Hidrofílicas , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 10(8) samples to be screened in one day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays.
Assuntos
Ensaios de Triagem em Larga Escala , Microfluídica , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Bactérias/efeitos dos fármacos , DNA/análise , Enzimas/química , Enzimas/metabolismo , Humanos , Reação em Cadeia da Polimerase , Ensaio de Placa ViralRESUMO
Double emulsions are useful templates for microcapsules and complex particles, but no method yet exists for making double emulsions with both high uniformity and high throughput. We present a parallel numbering-up design for microfluidic double emulsion devices, which combines the excellent control of microfluidics with throughput suitable for mass production. We demonstrate the design with devices incorporating up to 15 dropmaker units in a two-dimensional or three-dimensional array, producing single-core double emulsion drops at rates over 1 kg day(-1) and with diameter variation less than 6%. This design provides a route to integrating hundreds of dropmakers or more in a single chip, facilitating industrial-scale production rates of many tons per year.