Based on this digital microfluidic platform, we recently developed a methodology for highthroughput purification of singlecell genomes and multipleallele sequencing of single cells 36. Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures. We present a dropletbased microfluidic technology that enables highthroughput screening of single mammalian cells. Highthroughput microfluidic singlecell rtqpcr article pdf available in proceedings of the national academy of sciences 10834. Microfluidic techniques for high throughput single cell. Fluorescence data acquired with the ultrahighthroughput detector was plotted using a heat map. Apr 18, 2019 the disclosed embodiments relate to method, apparatus and system for high throughput single cell dna sequencing with droplet microfluidic. Highthroughput automated microfluidic sample preparation. Most singlecell gene sequencing methods use the polymerase chain reaction pcr to increase the concentration of the target gene for detection, thus requiring a barcoding process for cell identification and creating a challenge for realtime, largescale screening of sequences in cells to rapidly profile physiological samples.
In this study, we combined mica emulsion generation with emda micaemda, achieving high throughput and overcoming the technical. Highthroughput cellbased screens can benefit considerably from the unique liquidhandling capabilities offered by microfluidic systems. We present a dropletbased microfluidic technology that enables high throughput screening of single mammalian cells. Linked optical and gene expression profiling of single. Protocol singlecell analysis and sorting using dropletbased. This protocol describes the use of twophase, dropletbased microfluidics systems for highthroughput singlecell analysis and sorting.
Single cell rnaseq is a powerful and general method for analyzing cells, but not all traits of interest are observable in gene expression data alone. Singlecell quantitative realtime pcr qrt pcr combined with highthroughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h. Highperformance single cell genetic analysis using microfluidic emulsion generator arrays. Sperling, michael schnalllevin, huidan zhang, anindita basu, bradley e. Microfluidic systems facilitate single cell analyses by leveraging the micronscale geometry for improving sensitivity, decreasing reaction time, decreasing reagents consumption, and improving parallelization and automation for high throughput. The microfabrication of microfluidic control systems and the development of increasingly sensitive molecular amplification tools have enabled the. Here, we piloted an automated microfluidic system that integrates at high throughput all the key steps in ngs sample preparation. As a subset of microfluidics, droplet microfluidics involves the production of microscale droplets typically on the order of tens to hundreds of m in diameter of one fluid within a second immiscible carrier fluid in a highthroughput manner, leading to promising applications in a variety of fields including directed evolution, tissue printing, and polymerase chain reaction pcr 24,25,26. Pdf development of droplet microfluidics enabling high. Abstract the human gut and other environmental samples contain large populations of diverse bacteria that are poorly characterized and unculturable, yet have many functions relevant to human health.
Microfluidic double merger device for singlecell genome barcoding. Pcr polymerase chain reaction technology has been evolved with the. Our method, known as indrops for indexing droplets, makes use of droplet microfluidic technology to index individual cells. The resulting emulsion is collected in fractions about 40. Sims1,3,4 abstract many important biological questions demand singlecell transcriptomics on a. Droplet microfluidics reverse transcription and pcr towards single cell and exosome analysis 2.
Highthroughput automated microfluidic sample preparation for. Highthroughput singlecell labeling hiscl in drops is a novel method that uses dropbased libraries of oligonucleotide barcodes to index individual cells in a population. Highthroughput single copy dna amplification and cell. Raindance technologies is making complex genetics simple. High throughput single cell applications set to benefit. The nanoliter volume droplets uniquely enable quantitative highyield amplification of dna targets suitable for longrange. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets 1 pl to 10 nl volumes dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very highthroughput. Linked optical and gene expression profiling of single cells. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a microfluidics chip. Single cell measurement of telomerase expression and splicing.
Droplet microfluidics is particularly well suited and extensively used for high throughput single cell analysis. Microfluidic singlecell mrna isolation and analysis. The architecture of the microfluidic chip is designed to merge two streams of aqueous fluids. Droplet microfluidics reverse transcription and pcr towards single cell and exosome analysis. Workflow for single cell isolation, culture and analysis. Here, we present a microfluidic system that can overcome these limitations, making use of a dualcolor signal normalization approach. Jan 27, 2017 here, we piloted an automated microfluidic system that integrates at high throughput all the key steps in ngs sample preparation. Protocol singlecell analysis and sorting using droplet. Highperformance single cell genetic analysis using.
Highthroughput microfluidic singlecell rtqpcr pnas. A multiplex single cell pcr method is developed to detect and quantify both wild. An ultrahighthroughput microfluidic platform for singlecell. Temperature control and fluorescent detection during pcr amplification were performed using a ccd. Highthroughput singlecell measurement technologies are therefore of intense interest and have broad application in clinical and research settings. Livedead staining of spheroids after release from emulsion at 6 h using 1h,1h,2h,2hperfluoro1octanol. Mar 10, 2017 the encapsulation and isolation of single cells in a dropbydrop fashion has opened up new opportunities for costeffective and ultrahighthroughput singlecell genetic studies in applications such as wholegenome amplification fu et al. Live cells were labeled with calcein am green, and dead. Aug 23, 2011 highthroughput singlecell measurement technologies are therefore of intense interest and have broad application in clinical and research settings.
Highperformance single cell genetic analysis using microfluidic. Individual cells are encapsulated with dna amplifiers in waterinoil droplets and then lysed. From singlemolecule detection to nextgeneration sequencing. This userfriendly, high throughput system offers simple, straightforward encapsulation. Highthroughput singlecell pcr using microfluidic emulsions. High throughput single cell labeling hiscl in drops is a novel method that uses dropbased. A powerful, cultivationfree method for identifying specific phage hosts is singlecell pcr in individual microfluidic chambers tadmor et al. This approach enables singlecell based highthroughput. Single molecule quantitation and sequencing of rare. Dropletbased microfluidics in high throughput single. Microfluidic droplet platform for ultrahighthroughput singlecell screening of biodiversity. Isolation of single infected host cells into the individual microwells of the biochip was achieved by onestep vacuum. This application note reports some results of encapsulation of cells with barcoded beads for singlecell rna sequencing using the dolomite bio cell encapsulation system.
Dropletbased microfluidics in highthroughput singlecell analysis. Dolomite bio application note high throughput single cell. Pdf the importance of singlecell level data is increasingly appreciated, and significant advances in this direction have been made in recent years find, read and cite all the research you. Inertial flowbased cell spacing and singlecell encapsulation using a a. Microfluidic emulsification is a high throughput route to single cell genomic classification. Highthroughput microfluidic singlecell analysis pipeline. Hansen, centre for high throughput biology and department of physics and astronomy, university of british columbia, 3072125 east. Dmf technologies 56 including slip chip 57, centrifugal step emulsification droplets 58. The msp method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for. Microfluidic technologies for quantitative single cell analysis. Highthroughput singlecell wholegenome amplification through.
Tremendous amounts of new data could now be uncovered to redefine our understanding of cell omics. Although these systems provide a high throughput qpcr readout, they do not address the front end sample preparation and require single cell isolation by facs or micropipette followed. High throughput singlecell cultivation on microfluidic. Dropletbased microfluidic technologies have proved themselves to be of significant utility in the performance of highthroughput chemical and biological experiments. Three droplet microfluidic technologies allow single. Development of droplet microfluidics enabling highthroughput. Droplet microfluidics, reverse transcription, droplet pcr, high throughput biology, single cell analysis, exosomes.
Dropletbased microfluidics has in this way originated major advances in the field at the interface between technological advancements and biomedical applications of singlecell sequencing. Method, systems and apparatus for highthroughput singlecell. The use of drops as containers, and a microfluidics platform to manipulate them enmasse, yields a highly scalable methodological framework. The basic principle of droplet microfluidic systems. In addition, this approach was optimized to realize singlecell rt pcr with two aqueous inlets flushed with cells and rt pcr reagentscell lysis buffer, respectively. A microfluidic platform for highthroughput singlecell. Microfluidic highthroughput encapsulation and hydrodynamic.
High throughput production of single core double emulsions. Ultrahighthroughput droplet microfluidic device for. Single cell measurement of telomerase expression and. Microfluidic technologies for quantitative single cell. The distinctive solgel properties of agarose allow for. Each array unit consists of i a reagent inlet, ii a 0. Here we describe a highperformance single cell genetic analysis scga technique that combines highthroughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction pcr. Pdf singlemolecule emulsion pcr in microfluidic droplets. Chip contains independent compartments and 3574 elastomeric valves. Here we present a fully integrated microfluidic device capable of performing highprecision rtqpcr measurements of gene expression from hundreds of single cells per run. Highthroughput singlecell cultivation on microfluidic. Passive, high throughput microfluidic systems enable single cell subpopulation classification.
In this study, a rapid, pcr free, singlecell mirna assay is developed from a continuousflow microfluidic process employing a dna hybridization chain reaction to amplify the target mirna signal. The biochip is simply structured with a twodimensional array of 900 microwells, one inlet, and one outlet microchannels. Method open access scalable microfluidics for singlecell rna printing and sequencing sayantan bose1, zhenmao wan2, ambrose carr2, abbas h. Pcr tubes, the oil under the buoyant droplets is removed with a pipette and. Multidrug resistance analysis represents a great challenge in cancer chemotherapy, drug development and pathological study. A highthroughput microfluidic biochip to quantify bacterial. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets 1 pl to 10 nl volumes dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high throughput. A highthroughput platform for linked optical phenotype and gene expression of single cells. Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion pcr at the singlecell level.
Fluorescence signal from droplets is interrogated and used to selectively dispense a cell and bead to indexed locations on a nanowell array. Highthroughput singlecell dna sequencing of acute myeloid leukemia tumors with droplet microfluidics. Singlecell quantitative realtime pcr qrt pcr combined with highthroughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample. Encapsulation uses a celltriggered rayleighplateau instability in a flowfocusing geometry, and selfsorting puts to work two extra hydrodynamic mechanisms. Singlemolecule emulsion pcr in microfluidic droplets. A droplet of pcr mix is first generated and merged with a barcode droplet in the region shown in yellow using. High throughput single cell pairing of antibody or tcr. Nov 26, 2019 in this section, droplet microfluidic technologies for single.
Ultrahighthroughput mammalian singlecell reversetranscriptase polymerase chain reaction in microfluidic drops. Here, we demonstrate an approach that allows gene expression to be linked to optical data in a high throughput format scalable to thousands of single cells. In contrast, dropletbased microfluidic systems enable the handling of millions of droplets and cells in a single experiment, including sorting at high speed45 and recovery of viable cells from the droplets22. Highthroughput droplet digital pcr system for absolute quantitation of. Ultrahighthroughput droplet microfluidic device for single. Singlecell droplet microfluidic screening for antibodies. Leveraging this approach, singlecell pcr was conducted to identify a single pathogen e. Mar 04, 2008 we present a purely hydrodynamic method for the highthroughput encapsulation of single cells into picoliter droplets, and spontaneous selfsorting of these droplets. High single cell isolation efficiency and longterm culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. Pdf droplet microfluidics has revolutionized the study of single cells.
In previous work we have developed the use of microfluidic droplet generators to produce highly monodisperse agarose droplets for emulsion pcr epcr 6. Highthroughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Scalable microfluidics for singlecell rna printing and. A twostep merger operation pairs barcode droplets with tagmented genomes at a highthroughput. In this approach, taqman pcr interrogates individual microbes for phage sequences such that, if the sequences are present, a fluorescent signal is produced. A droplet of pcr mix is first generated and merged with a barcode droplet in the region shown in yellow using saltwater electrodes. Pcractivated cell sorting as a general, cultivationfree. Currently, the throughput of our singlemoleculecell sequencing procedure is limited by the use of secondround pcr to amplify the beadbound dna for the. Artificial compartments of double emulsions were produced with high. The modern biological lab is thus full of highthroughput tools, from robotic. Pdf singlecell analysis using droplet microfluidics researchgate. Microfluidic droplet platform for ultrahighthroughput singlecell. We demonstrate the benefit of this approach to screen antibodies for the specific binding of cancer target cells in a high throughput single cell format, which should have immense clinical potential.
Emulsions, consisting of water, oil, and a surfactant, are. Droplet microfluidic technology for singlecell high. Microfluidics as a strategic player to decipher singlecell. Microfluidics has emerged as a major technological player in this new era and is gradually increasing in use among biology laboratories, mainly due to the single cell high throughput handling solutions it offers. Droplet microfluidicsenabled highthroughput screening. Your story matters citation rotem, assaf, oren ram, noam shoresh, ralph a. Highthroughput singlecell cultivation on microfluidic streak plates article in applied and environmental microbiology 827. We present a parallel numberingup design for microfluidic double emulsion devices, which. High throughput single cell labeling hiscl for rnaseq using dropbased microfluidics the harvard community has made this article openly available.
H7 in the high background of 100,000 excess normal k12 cells. In this paper, multifunctional gradientscustomizing microfluidic devices were developed for high throughput single cell multidrug resistance mdr analysis. In an exemplary embodiment, a microfluidic apparatus is used. Droplet microfluidics reverse transcription and pcr. Highthroughput singlecell labeling hiscl for rnaseq. To overcome cell lysatemediated inhibition of rt pcr in microdroplets, a new microfluidic workflow was. Dropletbased singlecell sequencing the microfluidic circle.
The importance of single cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. A highthroughput single copy genetic amplification scga process is developed that utilizes a microfabricated droplet generator dg to rapidly encapsulate individual dna molecules or cells together with primer functionalized microbeads in uniform pcr mix droplets. The small microwell array is designed for precisely isolating single cells, and the large microwell array is used for single cell clonal culture in the microfluidic chip. A microfluidic device for highthroughput singlecell rtqpcr. An ultrahighthroughput microfluidic platform for single. The integration of multiplex pcr with highthroughput emulsion generation is valuable for the analysis. Highthroughput single cell multidrug resistance analysis. This paper describes the microfluidic streak plate msp, a facile method for highthroughput microbial cell separation and cultivation in nanoliter sessile droplets. A highthroughput microfluidic polydimethylsiloxane biochip was developed to quantify bacterial adhesion to single host cells by realtime pcr assay. Droplets microfluidics for single cell encapsulation in. Microfluidic singlecell realtime pcr for comparative.
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. Highthroughput single copy dna amplification and cell analysis in engineered nanoliter droplets. Dropletbased microfluidics in high throughput singlecell. Existing methods for measuring transcript levels in single cells include rtqpcr, single molecule counting using digital pcr or hybridization probes 3, 4, and next generation sequencing. For singlecell genomics studies, droplet microfluidics can bring a much needed throughput necessary to profile large 10,000 numbers of cells. This paper describes the microfluidic streak plate msp, a facile method for high throughput microbial cell separation and cultivation in nanoliter sessile droplets. Our device executes all steps of singlecell processing, including cell capture, cell lysis, reverse transcription, and quantitative pcr. Compared to the continuous microfluidic devices, the main advantage of digitaldroplet microfluidic for single cell rt pcr is the possibility of highthroughput analysis. Silicone microfluidic array chip for highthroughput single cell assays. The companys ultrasensitive genomic tools enable research of novel noninvasive liquid biopsy applications that should result in more accurate, reliable, costeffective, and early detection of cancer and other inherited and infectious diseases. Particletemplated emulsification for microfluidicsfree.