DROPLET SPACER FOR DROPLET-BASED ASSAYS
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`Cross-References
`
`This application incorporates by reference in their entireties for all purposes the
`
`following materials: U.S. Patent No. 7,041,481, issued May 9, 2006; and U.S. Patent
`
`Application Serial No. 12/586,626, filed September 23, 2009.
`
`Introduction
`
`Many biomedical applications
`
`rely on high-throughput assays of samples
`
`combined with reagents. For example,
`
`in research and clinical applications, high-
`
`10
`
`throughput genetic tests using target-specific reagents can provide high-quality
`
`information about samples for drug discovery, biomarker discovery, and clinical
`
`diagnostics, among others. As another example,
`
`infectious disease detection often
`
`requires screening a sample for multiple genetic targets to generate high-confidence
`
`results.
`
`15
`
`The trend is toward reduced volumes and detection of more targets. However,
`
`creating and mixing smaller volumes can require more complex instrumentation, which
`
`increases cost. Accordingly,
`
`improved technology is needed to permit testing greater
`
`numbers of samples and combinations of samples and reagents, at a higher speed, a
`
`lower cost, and/or with reduced instrument complexity.
`
`20
`
`Emulsions hold substantial promise for revolutionizing high-throughput assays.
`
`Emulsification techniques can create billions of aqueous droplets that function as
`
`independent reaction chambers for biochemical reactions. For example, an aqueous
`
`sample (e.g., 200 microliters) can be partitioned into droplets (e.g., four million droplets
`
`of 50 picoliters each) to allow individual sub-components (e.g., cells, nucleic acids,
`
`
`
`Cross-References
`
`This application incorporates by reference in their entireties for all purposes the
`
`following materials: U.S. Patent No. 7,041,481, issued May 9, 2006; and U.S. Patent
`
`Application Serial No. 12/586,626, filed September 23, 2009.
`
`Introduction
`
`Many biomedical applications
`
`rely on high-throughput assays of samples
`
`combined with reagents. For example,
`
`in research and clinical applications, high-
`
`10
`
`throughput genetic tests using target-specific reagents can provide high-quality
`
`information about samples for drug discovery, biomarker discovery, and clinical
`
`diagnostics, among others. As another example,
`
`infectious disease detection often
`
`requires screening a sample for multiple genetic targets to generate high-confidence
`
`results.
`
`15
`
`The trend is toward reduced volumes and detection of more targets. However,
`
`creating and mixing smaller volumes can require more complex instrumentation, which
`
`increases cost. Accordingly,
`
`improved technology is needed to permit testing greater
`
`numbers of samples and combinations of samples and reagents, at a higher speed, a
`
`lower cost, and/or with reduced instrument complexity.
`
`20
`
`Emulsions hold substantial promise for revolutionizing high-throughput assays.
`
`Emulsification techniques can create billions of aqueous droplets that function as
`
`independent reaction chambers for biochemical reactions. For example, an aqueous
`
`sample (e.g., 200 microliters) can be partitioned into droplets (e.g., four million droplets
`
`of 50 picoliters each) to allow individual sub-components (e.g., cells, nucleic acids,
`
`
`
`proteins) to be manipulated, processed, and studied discretely in a massively high-
`
`throughput manner.
`
`Splitting a sample into droplets offers numerous advantages. Small
`
`reaction
`
`volumes (picoliters to nanoliters) can be utilized, allowing earlier detection by increasing
`
`reaction rates and forming more concentrated products. Also, a much greater number of
`
`independent measurements (thousands to millions) can be made on the sample, when
`
`compared to conventional bulk volume reactions performed on a micoliter scale. Thus,
`
`the sample can be analyzed more accurately (i.e., more repetitions of the sametest)
`
`and in greater depth (i.e., a greater number ofdifferent tests). In addition, small reaction
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`10
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`volumes use less reagent,
`
`thereby lowering the cost per
`
`test of consumables.
`
`Furthermore, microfluidic technology can provide control over processes used for the
`
`generation, mixing,
`
`incubation, splitting, sorting, and detection of droplets,
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`to attain
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`repeatable droplet-based measurements.
`
`Aqueous droplets can be suspended in oil
`
`to create a water-in-oil emulsion
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`15
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`(W/O). The emulsion can be stabilized with a surfactant
`
`to reduce or prevent
`
`coalescenceof droplets during heating, cooling, and transport, thereby enabling thermal
`
`cycling to be performed. Accordingly, emulsions have been used to perform single-copy
`
`amplification of nuclei acid target molecules in droplets using the polymerase chain
`
`reaction (PCR).
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`20
`
`Compartmentalization of single molecules of a nucleic acid target in droplets of
`
`an emulsion alleviates problems encountered in amplification of larger sample volumes.
`
`In particular, droplets can promote more efficient and uniform amplification of targets
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`from samples containing complex heterogeneous nucleic acid populations, because
`
`
`
`sample complexity in each droplet is reduced. The impact of factors that lead to biasing
`
`in bulk amplification, such as amplification efficiency, G+C content, and amplicon
`
`annealing, can be minimized by droplet compartmentalization. Unbiased amplification
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`can be critical
`
`in detection of rare species, such as pathogens or cancer cells, the
`
`presence of which could be masked by a high concentration of background speciesin
`
`complex clinical samples.
`
`Despite their allure, emulsion-based assays present technical challenges for
`
`high-throughput
`
`testing. As an example,
`
`the arrangement and packing density of
`
`droplets may need to be changed during an assay, such as after the droplets have been
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`10
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`reacted and before detection.
`
`In particular,
`
`it
`
`is frequently advantageous to thermally
`
`cycle droplets at a high packing density, in either a flow-based mode or a static (batch)
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`mode. However, detection of signals from closely packed droplets may be problematic
`
`because the signals cannot always be correctly assigned to individual droplets. Thus,
`
`there is a need for systems that space droplets after reaction and before detection for
`
`15
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`improved detection accuracy.
`
`Brief Description of the Drawings
`
`Figure 1
`
`is a flowchart
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`listing exemplary steps that may be performed in a
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`method of sample analysis using droplets and droplet-based assays, in accordance with
`
`aspects of the present disclosure.
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`20
`
`Figures 2 through 9 are views of exemplary droplet spacers (separators) that
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`may be disposed in any suitable fluidic portion of a droplet-based assay system, such
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`as in a fluid flow path from a thermal cycler to a detector of the system and/or at a
`
`
`
`position upstream of a detector in a fluid flow path to the detector, in accordance with
`
`aspects of the present disclosure.
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`Detailed Description
`
`The present disclosure provides
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`systems,
`
`including methods, apparatus,
`
`compositions, and kits,
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`for
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`the generation, mixing,
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`incubation,
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`splitting,
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`sorting,
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`separation, and/or detection of small volumes or droplets of fluid in emulsions. The
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`systemsparticularly involve a droplet separator or spacer that spaces droplets from one
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`another, and that optionally arranges droplets in single file in a flow stream that is
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`upstream of a detection site in a flow path,
`
`to permit serial detection of individual,
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`10
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`spaced droplets at the detection site. These systems may involve, among others, (A)
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`preparing a sample, such as a clinical or environmental sample, for analysis,
`
`(B)
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`separating components of the samples by partitioning them into droplets or other
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`partitions, each optionally containing only about one component (such as a single copy
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`of a nucleic acid target (DNA or RNA) or other analyte of interest (e.g., a protein
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`15
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`molecule or complex)), (C) performing an amplification and/or other reaction within the
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`droplets to generate a product(s), where successful occurrence of the amplification or
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`other reaction in each droplet is dependent on the presence of the component in such
`
`droplet, (D) detecting the product(s), or a characteristic(s) thereof, and/or (E) analyzing
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`the resulting data.
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`In this way, complex samples may be converted into a plurality of
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`20
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`simpler, more easily analyzed samples, with concomitant reductions in background and
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`assaytimes.
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`
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`Figure 1 shows an exemplary system 500 for performing such a droplet-, or
`
`partition-, based assay.
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`In brief,
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`the system may include sample preparation 502,
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`droplet generation 504,
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`reaction 506 (e.g., amplification), detection 508, and data
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`analysis 510. The system may beutilized to perform a digital PCR (polymerase chain
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`reaction) analysis. More specifically, sample preparation 502 may involve collecting a
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`sample, such asa clinical or environmental sample, treating the sample to release an
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`analyte (e.g., a nucleic acid or protein, among others)), and forming a reaction mixture
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`involving the analyte (e.g., for amplification of a target nucleic acid that corresponds to
`
`the analyte or that is generated in a reaction (e.g., a ligation reaction) dependent on the
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`10
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`analyte). Droplet generation 504 may involve encapsulating the analyte and/or target
`
`nucleic acid in droplets, for example, with about one copy of each analyte and/or target
`
`nucleic acid per droplet, where the droplets are suspendedin an immiscible carrierfluid,
`
`such as oil, to form an emulsion. Reaction 506 may involve subjecting the droplets to a
`
`suitable reaction, such as thermal cycling to induce PCR amplification, so that target
`
`15
`
`nucleic acids,
`
`if any, within the droplets are amplified to form additional copies.
`
`Detection 508 may involve detecting some signal(s) from the droplets indicative of
`
`whether or not
`
`there was amplification. Finally, data analysis 510 may involve
`
`estimating a concentration of the analyte and/or target nucleic acid in the sample based
`
`on the percentage of droplets in which amplification occurred.
`
`20
`
`These and other aspects of the system are described in further detail below,
`
`particularly with respect to exemplary droplet spacers, and in the patent documents
`
`cited above under Cross-References and incorporated herein by reference.
`
`
`
`I.
`
`Droplet Spacer Configurations
`
`This Section describes exemplary droplet spacers, also termed separators, that
`
`may be positioned in a flow path of a droplet-based assay system. A spacer may be
`
`disposed at any suitable position, such as in fluid communication with and upstream of
`
`a detection site, in fluid communication with and downstream of an incubation/reaction
`
`site (e.g., a thermal cycling region), or both, among others. The spacer may reduce the
`
`packing density of droplets in a flow stream, may reduce the droplets from a multiple file
`
`to a single file arrangement, and/or may focus droplets within the flow stream.
`
`The droplet spacer may include at least two inlets, an outlet, and a confluence
`
`10
`
`region or chamber that connects the inlets to the outlet. The at least two inlets may
`
`include a droplet inlet that receives droplets in an emulsion, and at least one carrier or
`
`dilution
`
`inlet
`
`that
`
`receives
`
`a
`
`carrier
`
`fluid,
`
`such as
`
`an oil,
`
`for diluting
`
`the
`
`droplets/emulsion. The carrier fluid received at the carrier inlet may be the sameas, ora
`
`different carrier fluid from, that in which the droplets are disposed at the dropletinlet.
`
`15
`
`The spacer may have any suitable configuration. For example, the inlets, the
`
`chamber, and the outlet collectively may form a T-shape, a cross, orthe like.
`
`The droplet
`
`inlet may have a uniform diameter or may taper toward the
`
`confluence region.
`
`If tapered, the droplet inlet may have a maximum diameter that is
`
`greater than that of the droplets (e.g., at least about 50%, 100%, 150%, or 200%
`
`20
`
`greater in diameter). The droplet inlet may taper to a minimum diameter (e.g., adjacent
`
`the confluence region) that is about the same size as the diameter of the droplets. The
`
`use of a minimum diameter that is about the same size as the droplets may permit only
`
`
`
`one droplet to enter the confluence region at a time, thereby facilitating production of a
`
`single file stream of droplets for a downstream detection site.
`
`The carrier inlet may have a diameter that is less than, about the same as, or
`
`greater than the maximum or minimum diameter of the droplet inlet.
`
`The confluence region may have any suitable structure. The confluence region
`
`may have a diameter that is greater than the minimum diameter of the inlet and greater
`
`than the diameter of the droplets. As a result, any droplets newly-formed at the droplet
`
`spacer (such as by fragmentation of a coalesced set of droplets) should be larger than
`
`the original droplets of interest. Accordingly, any droplets detected to be larger than a
`
`10
`
`threshold size by a downstream detector (and thus likely to be formed after thermal
`
`cycling) may be excluded from the analysis. The confluence region may taper toward
`
`the outlet, which may act to accelerate each individual droplet out of the confluence
`
`region. Furthermore, the droplet inlet and the outlet may be near one another, such as
`
`separated by no more than about twice, one, or one-half the droplet diameter,
`
`to
`
`15
`
`promote exit of droplets from the confluence region, thereby allowing only one droplet to
`
`be present in the confluence region at a time.
`
`ll.
`
`Examples
`
`This Section describes exemplary aspects and features of droplet spacers and
`
`advantages/disadvantagesthereof.
`
`20
`
`Figure 2 shows a butted-end droplet separator, which,
`
`in this configuration,
`
`generates low shear forces and thus is unable to separate droplets.
`
`In this figure,
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`droplets are introduced to the droplet separator from the tube on the right, exiting to the
`
`tube on the left. The separating oil comes from both the top and the bottom.
`
`In this
`
`
`
`configuration, the separating flow comes from all directions surrounding the lumens of
`
`the introduction and exit tubes. The fluid forces generally are not large enough to
`
`separate droplets until the droplet is already in the exit tube. The force can be increased
`
`by making the gap between the introduction and exit tubes smaller, thereby increasing
`
`the velocity of the separating oil.
`
`Figure 3 shows a butted-end droplet separator with a smaller gap than that
`
`shownin Figure 2. The fluid forces are now high enough to separate adjacent droplets,
`
`but also high enough that they cause significant deformation of the droplets.
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`If this
`
`deformation is too high, the existing droplets will be split
`
`into two or more droplets,
`
`10
`
`which is generally undesirable for the analysis. Additionally, to generate enough force to
`
`separate droplets, the gap must be small enough to be an effective droplet generator.
`
`So,
`
`if a large bolus of aqueous fluid (such as coalesced droplets) comes through, this
`
`droplet separator will generate new droplets of the approximate size of the original
`
`droplets, biasing the Poisson analysis. Even smaller gaps would create smaller droplets
`
`15
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`from the desired droplet size, potentially even fragmenting original droplets into newly-
`
`formed smaller droplets
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`Figure 4 showsa drilled cross used as a droplet separator, which creates similar
`
`issues to those of Figure 3, albeit to a lesser degree.
`
`In this photo, the droplets are
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`introduced from the bottom and exit the top. Separating oil comes from both the right
`
`20
`
`and left branches. The droplet separation forces are higher because the separating oil
`
`comesfrom only two directions, unlike the butted-end separator.
`
`
`
`Figure 5 shows a droplet separator formed in a tee configuration.
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`In this photo,
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`droplets are introduced from the bottom and exit to the left. Separating oil comesin from
`
`the right. The top port is plugged so that no fluid can flow. This configuration provides
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`even morefluid force for separating droplets. However, the stress on the droplets is also
`
`significant, potentially creating smaller droplets from the original droplets. Additionally, a
`
`large bolus would still create new droplets, potentially indistinguishable in size from the
`
`original droplet size.
`
`Figure 6 showsa droplet separator in a tee configuration and illustrating effective
`
`droplet separation.
`
`10
`
`Figure
`
`7
`
`shows
`
`another
`
`exemplary
`
`droplet
`
`separator
`
`that
`
`combines
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`advantageous features of different configurations, while avoiding some or all of the
`
`limitations. Droplets are introduced from the bottom port through a constriction thatwill
`
`only allow one droplet at a time to enter the droplet separator. The droplet separator
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`itself may be a chamberthat is at least 25% larger in diameter than the desired droplet
`
`15
`
`size. This feature determines that any bolus of aqueous fluid entering the chamber will
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`only generate droplets that are significantly larger than the target droplet size. The tee
`
`separator configuration maintains significant force for separating droplets at up to two
`
`times the target droplet diameter. The exit constriction is kept close to the introduction
`
`constriction so that any droplet that enters the droplet separator chamber will accelerate
`
`20
`
`downthe exit tube before the next droplet can enter the chamber, effectively separating
`
`the droplets.
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`Figure 8 showsvarious views of a CAD drawing of a droplet separator prototype.
`
`
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`Figure 9 shows a hand-made prototype of a droplet separator, modified from a
`
`standard cross. Droplets are introduced from the bottom, and exit to the left. Separating
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`oil enters from the right. The top channel
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`is plugged and there is no flow. Close
`
`inspection of the photo shows the leading edge of one droplet moving toward the
`
`detector after exiting the flow separator (the trailing edge is hidden by the tubing
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`junction). The trailing edge of the next droplet can be seen in the introduction port (the
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`leading edge is hidden by the edge of the expansion). In this prototype, the introduction
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`constriction and the exit tube are 100 um in diameter and the expanded chamber is
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`about 250 um.
`
`10
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`The disclosure set forth herein may encompass one or moredistinct inventions,
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`with independent utility. Each of these inventions has been disclosed in its preferred
`
`form(s). These preferred forms, including the specific embodiments thereof as disclosed
`
`and illustrated herein, are not intended to be considered in a limiting sense, because
`
`numerous variations are possible. The subject matter of the inventions includesall novel
`
`15
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`and nonobvious combinations and subcombinations of the various elements, features,
`
`functions, and/or properties disclosed herein.
`
`10
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`
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