WO2018227204A1

WO2018227204A1 - Controlled encapsulation in droplets by liquid-liquid interfacial shearing

Info

Publication number: WO2018227204A1
Application number: PCT/US2018/036952
Authority: WO
Inventors: Gopakumar KAMALAKSHAKURUP; Abraham P. Lee
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2017-06-09
Filing date: 2018-06-11
Publication date: 2018-12-13
Anticipated expiration: 2019-12-09

Abstract

A passive, hydrodynamic technique to perform co-encapsulation of one cell-one bead in droplets is described herein. The hydrodynamic technique utilizes laminar flows and high shear liquid-liquid interfaces at a microfluidic junction to achieve a one-one-one encapsulation efficiency of about 30%. This technique may he implemented using a microfluidic device to significantly improve the efficiency of droplet sequencing and other bead based single cell assays.

Description

CONTROLLED ENCAPSULATION IN DROPLETS BY LIQUID-LIQUID INTERRACIAL SHEARS

CROSS REFERENCE

{99811 This application claims priority to U.S. Provisional Ap lication No. 82/517,775 filed June 09, 2017. the speeiScation{s) of which is are Incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

(99921 The present invention relates to microfluldic devices, namely, to encapsulation of samples using droplet-based microfiuidic devices.

GOVE NMENT SUPPORT

[9093 j The inventions were made with government support under Grant Mo, 1362185 awarded - by the National Science Foundation, The government may have certain rights in the inventions.

BACKGROUND OF THE INVENTION HWj $ingte-cet{ analysis is a field that studies the genomics, transcriptomics, proteomics and metaooiomics at the single cell level. Conventional techniques to perform single ceil analysis are flow cytometry and automated microscopy. To address the wide range of applications for single eel analysis, these conventional methods are often coupled with microfluldic devices. Microfiuidic devices and systems are configured to process (e.g., move, mix. separate) small volumes of fluid, typically In the range of picoilters to microliters, in addition, the microfiuidic devices can spatially collect single eels in micro wells, patterned surfaces, and various traps based on mechanical, magnetic, hydrodynamlc, optical, dieieetrophoretic, and acoustic principles. These microfiuidic devices can be used for various applications including printing, bio-chemical assays, drug discovery, etc, A class of microfiuidic devices and systems includes microfiuidic droplet generating and manipulating devices configured to manipulate discrete droplets. Droplet-based microfiuidic devices can be configured to perform a variety of operations., such as. for example, transportation of droplets, storage of droplets, mixing of droplets, analysis of droplets, etc. For example, these devices can be used as mieroreaetors to achieve controlled and rapid mixing of fluids and/or to synthesize droplets and encapsulate various biological entitles for biomedicine and biotechnology applications.

19995} Droplet-based single ceil assays are based on the ability to encapsulate and confine single cells in individual droplets and enaibie genome wide expression profiling. One-one-one encapsulation in droplets is a critical unit operation in single ceil high-throughput screening and droplet sequencing, Most single cell: encapsulations in droplets are performed randomly and are dictated by Poisson statistics. One of the current challenges in performing droplet sequencing (drop-seq) operation In droplets is achieving high efficiency one cell-one-bead encapsulation, it has been recently reported thai for genome wide expression profiling, the encapsulation efficiency is as low as 0.1%, or 1 In 1000 droplets will have a cell therein. Hence, there is a need for improved microfiuidic device and method for encapsulation in single droplets.

(09961 The present invention features a passive, hydrodynamlc technique to perform encapsulation in droplets utilizing laminar flows and high shear liquid-liquid interface at a microfiuidic function, wit an encapsulation efficiency of 30%, which could significantly improve the efficiency of drop-seq and other bead based single cell assays. um y OF THE INVENTION

hWilTj It is an objective of the present invention to provide fo microfiuidic devices and methods for encapsulating bsomolecuSes in droplets. Embodiments of the invention are given in the dependent dates. Embodiments of t e present invention can be freely combined wsth each other 8 they are not mutually exclusive^'.

\WW) in some aspects, the present invention provides an snterfaclai hydrodynamic technique that combines the effects of laminar flow and liquid-liquid inferfacial shearing, resulting in one-one-one encapsulation in droplets. Beads, cells and aqueous phase introduced through upper, lower and middle inlets respectively, create distinct laminar flow streams at a Junction. The flow rates at three inlets are kept equal to prevent the bead/cell migration across the streamlines due to Magnus forces. The beads and ceils self-assemble in a single f fe along the channel wall while moving toward the droplet generation junction. Upon reaching the droplet generation junction, the beads and cells get pulled from either side of the channel wall toward the symmetrical high shear interfaces into the droplet. The droplet diameter has to be large enough to accommodate one ceil and one bead in it. This can foe achieved by precisely tuning the dispersed to continuous phase pressure ratio (Φ). MWj Various embodiments discussed herein comprise microfiuidic devices that are configured to encapsulate single particle or cells with high through-put. Various embodiments of the microfiuidic devices can be configured to provid encapsulation efficiencies o 30% or higher. For example, various microfiuidic devices discussed herein can be configured to encapsulate particles or cells with an encapsulation efficiency of about 30% or greater. In some embodiments, the encapsulation efficiency is maximized in the squeezing regime to near dripping regime, where Ca < 10^"\ and ψ is about 0,5-1.0.

I8010J One of the unique inventive features of the present invention is the formation of high shear interfaces between the continuous phase and dispersed phase fluid streams can be formed, which increases the encapsulation efficiency. Without wishing to limit the present invention to a particular mechanism or theory, laminar flow guides the pariieies eeiis along the channel wail toward the high shear interface, and the high shear interface draws the cells or particles toward it at a higher velocity resulting in self-spacing of the cells, thereby reducing and even eliminating the possibility of doublets. An important advantage of this technique is that it can be modified based on the desired application including single cell or bead encapsulation (1 -1 ), and 1 ceil~1 bead-1 droplet encapsulation or 1 ceiM cell-1 droplet encapsulation (1 -1-1 ) for different cell types and ceil sizes.

(88! ij in some aspects, the method of encapsulating one or more solid samples {e.g., biological material comprising cellular material, one or more cells, one or more particles, one or more beads, etc.) In a droplet of a fluid (e.g., water) may comprise flowing a dispersed phase fluid stream comprising a solid sample (e.g., a biological sampl comprising cellular material or one or more cells) dispersed in a fluid (e.g., water) through a combining channel; controlling t e flow rates of the flow stream to establish laminar flow through the combining channel; flowing a continuous phase fluid through a continuous phase channel that intersects the combining channel the continuous phase fluid being immiscible with the dispersed phase fluid; controlling the flow rate of the continuous phase fluid to shear the laminar flow of the dispersed phase fluid stream and generating droplets encapsulating the solid sample in an output microfluidic channel jWS2j Another innovative aspect of the subject matter of this application is embodied In a microfluidic device, comprising: a combining channel configured to transport a dispersed phase fluid stream comprising a solid sample (e.g., a biological sample comprising cellular material or one or more cells) dispersed In a fluid (e.g., water); and continuous phase channels intersecting the combining channel to form an intersection region. Each continuous phase channel is configured to transport a continuous phase fluid stream. The intersection region is configured to open into an output microfluidic channel through an orifice. The microfluidic device further comprises a fluid controller to control the flow rates of the fluid streams to generate droplets encapsulating the solid sample. The flow rate of the dispersed phase flow stream can be controlled to establish laminar flow, and the flow rate of the continuous phase fluid stream can be controlled to shear the laminar flow of the dispersed phase fluid stream in the intersection region, thus generating droplets that enter the output microfluidic channel through the orifice. fWOj Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims,

BRIEF DESCRIPTION OF THE DRAWINGS

{00141 The features and advantages of the present invention will become apparent from a considerafion of the following detailed description presented in connection with the accompanying drawings in which:

{OtHSj FIG:. 1A shows an exemplary mechanism of one-ceii-one-bead■encapsulation in droplets using Ihterfacial shearing technique, according to an embodiment of the present invention. Beads and cells introduced from uppe and lower inlets self-assemble along the channel wail while moving toward high shear interfaces. At the droplet generation junction, both the heads and cells get pulled toward the high shear interface symmetrically f rom both the channel boundaries resulting in one-οηβ-οηβ encapsulation, {98161 FIG. 18 is a schematic of the encapsulation process using interfacia! shearing.

|WI7| FIG. 2 shows a non-limiting computational fluid dynamic model {GFD) of interfaelai shearing.

{0818| FIG. 3 shows a non-limiting embodiment of one-one-one (1-1-1) encapsulation of 10 pm beads and K-562 cells. The 10 pm beads were introduced from an upper inlet while the k-562 cells are flowed in through a lower inlet. Both the beads and ceils align close to the channel wall on the way toward a flow focusing junction. At the Junction, the heads and the ceils are pulled toward a symmetrical shear interface and encapsulated into droplets, FIGs, 4A-4D show steps of 1-1-1 encapsulation of 10 pm beads from the top and He!a sells from the b ttom:. The encircled droplet indicates the 1 -1-1 droplet

fiMOisj FIG, 5A shows the 10 m beads seif-assembie along the top channel wall while Hela ce!!s align along the bottom wall,

19921) FIG, 5B shows the one cell-one bead encapsulation in droplets. The encircled droplets Indicate the 1-1-1 droplets.

j0622j FIGs. 6A-6E sho steps of one-one-one encapsulation of 10 pro beads, in FIG. 6A, beads self- align along the channel wall. In FIG. 8B, beads gets pulled by the high shear flow at the boundary, in FIGs, 6C-6D, beads enter the droplets from two sides and are encapsulated in FIG, 6E, The encircled droplet shows the 1-1-1 droplets.

[88231 FiG, 7,A is a graph of encapsulation efficiency vs. droplet diameter. The encapsulation: efficiency Increases with the dropiel diameter, reaches a maximum, and decreases thereafter due io multiple encapsulations sn one droplet.

f«Mi24| FIG. 7B is a graph of 1-1-1 encapsulation efficiency vs. pressure ratio of dispersed phase to continuous phase (φ . The encapsulation efficiency increases with the dispersed io continuous pressure ratio ( $}, ead es a maximum, and decreases thereafter due to multiple encapsulations in one droplet. |8825| FiG. 7C Is a graph of concentration optimization for encapsulation efficiency vs. ceil concentration. The encapsulation efficiency increases with cell concentration, reaches a maximum, and decrease ^'thereafter due to multiple encapsulations i one droplet.

002*1 FiG, 8A is a schematic illustration of single cell encapsulation in droplets (1-1) using interacial shearing. The ceils were introduced from a single nlet,

8827j FiG, SB shows one cell encapsulation in droplets of cells introduced from a single inlet, The circles Indicate the single cells.

|8828 j FiG. 9A is an alternative schematic of single cell encapsulation in droplets (1-1) using interfacial shearing. The cells were introduced from two inlets.

[8829 j FiG. 9B shows one cell encapsulation in droplets of ceils introduced from two in!eis. The circles Indicate the single ceils,

{m i DESCRIPTION OF PREFERRED EMBODIMEN S

Following lists elements corresponding to a particular element referred to herein:

18832 j 100 microfluidic device

[8833| 102 solid sample

|8834j 104 droplet

[8835j 108 dispersed phase fluid

|88.¾j 10? flow stream

£6037 j 108 continuous phase fluid

f083«j 109 high shear interface

[β039| 110 combining channel

fO840j 112 channel side ail

(8841 j 114 first dispersed phase channel mm) 118 second dispersed phase channel

iH 3j 117 aqueous phase channel

] 118 aqueous phase fluid

us\ 119 laminar interface stream

0046} 120 first coniinuous phase channel

130 second continuous phase channel

m \ 140 intersection region

mw\ 145 droplet shearing junction

ww\ 147 orifice

§ \ 150 output channel

«052 j 160 fluid flow controller f¾ 3j As used herein, the .^:.micro&iidtc devices employ fluid volumes on the scale of microliters: to picoSters (1Q^"'!¾ that are contained within sur>miiiimeter scale channels. The structural or functional features may be dimensioned on the order of mro-sea!e or less, preferably in the micron scale or less. For example, a diameter or width of a channel or a dimension of an intersection or Junction may range from <0,1 pm to greater than 1000 pm. Alternatively or in addition, a length of channel may range from 0.1 μηι to greater than cm-scale. The rnscroflusdic device may employ active or passive techniques for fluid transport and droplet production. Compared to the active approach, which fluid manipulation involves the use of micropumps and microvalves, the passive approach takes advantage of the characteristic flow fleid in micfoRuidics to control the interface and capillary instability, and consequently to produce droplets. f¾§S j As used herein, the term "high shear interface" refers to a high velocity iiquid-iiquid interface formed between two immiscibte liquids. Generally, the continuous phase flow rate is greater than the flow rate of the dispersed phase. For instance, the continuous phase flow rate may be about 2-5 times greater. At the aqueous-oil interface, the high continuous phase Sow rate imparts the same velocity to the dispersed phase at the interface. Hence, the dispersed phase at the interface is at a higher velocity {shear} than the bulk. As used herein, the term "laminar Sow" refers to flow of a fluid in layers that do not mix. One of ordinary skill in that art would understand that at lower Reynold's numbers ( 10), a laminar flow is always established In the microfiuidic channel. The fluid flows in parallel layers with no lateral mixing but with some minor diffusion, WSS) As known to one of ordinary skill in the art, in a geometry-mediated regime, or squeezing regime, the droplet generation depends only on the size of the orifice and the flow rate ratio of the dispersed phase to the continuous phase flow rate, whereas Intetfaeial tension and viscosity has no significant influence. The transition between the geometry-mediated regime to a dripping regime Is dictated by the Capillary number (Ca), Ca ~ ~~ , where μ is the viscosity, v is the velocity of the continuous phase, and σ

Is the Snferfaciai tension between the wo fluid phases. Generally, in the geometry-mediated regime, Ca is <1G~\ In the dripping regime. Ca may be≥ 10^"' and interfaciai tension and viscosity can predict the formation of droplets. \mm) SAMPLES FOR ENCAPSULATION

p*857| in various embodiments of the present invention, the samples for encapsulation may be microparticfes. The microparticies may be beads. Examples of beads include, but are not limited to, polymer beads, bar-coded beads, functsonaiized beads, and magnetic beads. In some embodiments, the beads may have a size or dimension, such as a diameter or width, ranging from about 0.01 μχη to about 20 pm. hWSSj in other various .^'embodiments of the present invention, the samples tor encapsulation may be cells. Any particular ceil type from any o ganism may be used in the methods and systems of the present invention:. The ceiis may have a size or dimension, such as a diameter or width, ranging from about 0.1 pm to about 20 m - So some embodiments, the cells mayb wild type ceils or genetically modified ceiis. In other embodiments, the cells may be ceils harboring one or more mutations, healthy cells, diseased cells or unhealthy ceiis, etc. For example, in some embodiments, the cells may be prokaryotse cells (e.g., bacteria, arehaebaeteria, etc.). in other embodiments, the cells ma be eukaryotic ceils such as single- celled eukaryotes, fungal cells (e,g, yeast, moid, etc), animal cells, mammalian cells (e.g. cells from a human, non-human primate, rodent, rabbit, sheep, dog, cat, ete), and non-mammalian ceiis (e.g. cells from insects, reptiles, amphibians, birds, etc.). in some embodiments, the cells used in the present invention may be other eukaryotic cells such as plant ceiis or algal ceiis, on-iimiting and non-exhaustive examples of plant cells include cells from corn, soybean, wheal, cotton, grass, flowering plants, fruit-bearing plants, trees, tuberous plants, potatoes, root plants, carrots, peanut, nuts, beans, legumes, and squashes, it is to be understood that the term "plant ceil' encompasses ail types and stages of plant ceiis and is not limited to the aforementioned examples. Non-limiting and non-exhaustive examples of algal cells include ceiis from Chlorelia sp., Mannochloropsis sp, and Boiryococcus sp. It is to be understood that the term "algal ceil" encompasses all types of algal ceiis and is not limited to the aforementioned examples. One of the distinguishing characteristics that plant and aigal ceils have over animal ceiis is a cei! wall that surrounds a cell membrane to provide rigidity, strength, and structure to the cell. The ceil wall may be comprised of polysaccharides including celiuiose, hemicelluiose, and pectin. Similar to plant and algal cells, the fungal cells also have a ceii wail, which ma be comprised of polysaccharides including glucaos, mannans, and chitin. jlM½0l in other embodiments, the ceiis used in the present invention may be protoplasts, which are intact plant, bacterial or fungal ceiis that had its cell wall completely or partially removed using either mechanical or enzymatic means. f886lj in yet other embodiments, the ceiis used in the present invention ma be a tetrad. The term "tetrad" is used to herein to refer to a single structure comprised of four individual physically attached components. A "microspore" is an individual hapioid structure produced from diploid sporogenous cells {e.g., microsporoyte, pollen mother ceii, or meiocyte) following meiosls, A microspore tetrad refers to four individual physically attached microspores. A "pollen grain" Is a mature gametophvte containing vegetative {non-reproductive} eels and a generative (reproductive) ceil. A pollen tetrad refers to four individual physically attached pollen grains.

Wi<>2] ENCAPSULATION

h¾M3| Microfiuidic devices including droplet generatio portions can be used to create droplets of a fluid {e.g., oil or water).. Microfiuidlc -devices that include droplet generation portions can be used: to study chemical reactions, in drug delivery, in drug discovery, ©to. One method of generating droplets in microiiuidle devices comprises flowing a .first liquid {e.g., water) through a first- channel and a second liquid (e.g., oil) that is immiscible with the first liquid through channels Intersecting the first channel. The first liquid flowing through the first channel {e.g., water) is broken up to form discrete droplets as a result of shear forces from the second liquid. The s-f¾e of the generated first liquid droplets generated can depend on a variety of factors including velocity of the second liquid. For example, as the velocity of the second liquid is increased, the size of the first liquid droplets is reduced. )6 j Referring now to FIG, 1A-1B, in one embodiment, the present invention features a method for encapsulating a solid sample (102) in a droplet ( 04). The method may comprise flowing a first fluid {106) through a first microituidic channel (1 0) at a first flow rate (v^) such that flow of the first fluid is laminar, and co-flowing a second fluid (108) through each of a second microfiuidic channel (120) and a third microfiuidlc channel (130) at a second flow rate (v_c). In one embodiment, the first fluid (108} may comprise at least two flow streams (107). One o both of said flow streams (107} may comprise dispersed solid samples ( 02} that seif-assernhie near a sidewali (112) of the first microfiuidlc channel while flowing towards an intersection region (140), The second and third rnlcrofiuidic channels (120, 130} can intersect the first microfiuidlc channel (1 0) at the intersection region {140} such that the second fluid streams (108) intersect the first fluid (106) and merge to form a droplet shearing junction (145) within the intersection region (140), in some embodiments, the method further comprises adjusting v_a, v_e. or both such that each of the second fluid streams {108} forms a high shear interface (109) with the first fluid (106), and the solid samples (102) are drawn to the high shear interface (109), and gene-rating droplets (104) at the droplet shearing junction (145) such that each droplet (104} is substantially sized to encapsulate one solid sample or co-encapsulate two different soiid samples.

{®Μ5\ According to some embodiments, the method for encapsulating a solid sample (102) in a droplet (104) may include providing a microfiuidic device {100}. In some embodiments, the microfiuidlc device (100) may comprise a combining channel (110), a first continuous phase channel (120) having a portion thereof disposed on one side of the combining channel, a second continuous phase channel ( 30) having a portion thereof disposed on an opposite side of the combining channel, and an output channel {150}, The portions of the first and second continuous phase channels can intersect at a terminal end of the combining channel to form an intersection region {140} to which the output channel {150} Is fluidly coupled thereto, in one embodiment, the portions of the first and second continuous phase channels can intersect the combining channel ( 10) orthogonally such that the continuous phase channels and combining channel form a T-junction. Alternatively, the continuous phase channels can intersect the combining channel {110} at an acute angle such that the continuous phase channels and output channel form a V- junction, )66j in some embodiments, the mierofiuidie device (100) ma further comprise a first dispersed phase channel {114} comprising one of the flow streams (107) forming the dispersed phas fluid (106). and a second dispersed phase channel (11S) comprising the othe Sow stream (107), The first ami second dispersed phase channels (114, 116} can me ge to form the combining channel {110}. in other embodiments, the fnierofiuidfe device (TOD) may further compose an aqueous phase channel {117} intersecting with the first and second dispersed phase channels (114, 116). The aqueous phase channel (11 ?) may comprise aqueous phase fluid (118), which flows to the combining channel {1 0} such thai the aqueous phase fluid (118} forms a laminar interface stream (119) between the two flow steams {10?}.

$86 ) in other embodiments, the device {100} ma further comprise a fluid flow controlle (160} configured to perform operations. These operation can include adjusting of the dispersed phase fluid to establish laminar fiow in the combining channel (110} such that the solid samples (102) assembl near a sidewaii {112} of the combining channel while Sowing towards the Intersection region {140}, adjusting v_¾, v_c, or both such that each continuous phase fluid stream {106} forms a high shear interface {108} with the dispersed phase fluid (106) at the intersection region {140} and the solid samples {102) are drawn to the high shear interface (109} while flowing through the intersection region {140}, and adjusting ¾, vv, or both to generate droplets (104) at the droplet shearing junction (145) such that each droplet {104} is substantially sized to encapsulate a solid sample (102). in one embodiment the fiow in the microfiuidic device (100) and adjustment of the flow rates ma be pressure-driven. Preferably, the microfiuidic device {100} utilizes passive techniques to control fluid flow.

(0*68) in various embodiments, the width of the various microfiuidic channels (e.g., the first and second dispersed phase and aqueous phase channels (1 4, 116, 117); tie combining channel (1 0); and the continuous phase channels (120, 130» can range from about 25 pm to about 75 pm. For examples, the width of the various microfiuidic channels can be in a range between about 30 pm to about 80 pm. M9j in other embodiments, a width and/or length of the Intersection region can be about 3-6 times the width of the various microfiuidic channels (e.g., the combining channel, the first continuous phase channel, or the second continuous phase channel). For example, the width of the intersection region may be about 150 pm_« which is about three times the width of a 50 pm incoming microfiuidic channel. In another embodiment, the length of the intersection region may be about 200 pm , which is about four times the width of a 50 pm incoming microfiuidic channels. fW?¾j in some ernhodiments, the width of the orifice may b about 5-40 pm.. For example, In one embodiments, the width of the orifice may be about 5-15 pm, about 10-20 pm, about 20-30 pm, or about 30-40 pm . in other embodiments, the width of the output channel may widen from the width of the orifice to a maximum width. The maximum width of the output channel can be about 2-18 times th width of the orifice. For examples, for a 30 pro orifice, the output channel widens from a minimum width of 30 pm to a maximum width of about 120 pm. In further embodiments, the width of the output channel may be reduced after reaching its maximum. p)?.l s Consistent with the embodiments described above, an exemplary implementation of the method may comprise flowing a dispersed phase fluid (106) t rough the combining channel (110) at a first flow rate (v_d)< and adjusting ¾ of the dispersed phase fluid (108) to establish Seminar flow in the combining channel (110) such that the soiid samples (102) assemble near a sidewaii {112} of the combining channel while flowing towards the intersection region (140). In one embodiment, the dispersed phase fluid (106) ma comprise ..at least two flow streams (107), with one or both of the flow streams (107) having dispersed solid samples (102). Continuous phase fluid streams (106) co-flow through each of the first and second continuous phase channels (120_; 130} at a second flow rate (v₀)« The continuous phase fluid streams (108) can intersect the dispersed phase fluid (106) at the intersection region (140) such^' that a droplet shearing Junction (145} is formed within the intersection region {140) as the continuous phase fluid streams (108) merge with the dispersed phase fluid (108). The droplet shearing junction (145) can comprise an orifice (1 7) that fiuidiy couples the output channel (150} to the intersection region (140). The method continues with adjusting ¾, v_¾, or both such that each continuous phase fluid stream (108) forms a high shear interface (109) with the dispersed phase fluid (106} at the intersection region (140}. The solid samples (102} are drawn to the high shear interface (109) while flowing through the intersection region (140}. ¾, v_c, or both are further adjusted to generate droplets {104} encapsulating one soiid sample (102) at the droplet sheeting junction (145). Preferably, each droplet (104) can be substantially sized to encapsulate said solid sample (102). t?2j in one embodiment, as shown in FIG. 3~8E_< the method and mlorofiuldie device can b adapted to co-encapsulate two different samples in one droplet. For example, the dispersed soiid samples {102} may comprise a plurality of ceils flowing in one of the flow streams {107}, and a plurality of particles flowing in the other flow stream (107), When flowing through the combining channel (110), laminar flow of the dispersed phase fluid causes the ceils to assemble near the sidewaii (112a} and the particles to assemble near an opposing sidewaii (112b). At the intersection region {140}, the ceils are drawn to one high shear interface (109a} and the particles are drawn io the other high shear interface (109b), thereby enabling one ceil and one particle to be co-encapsuiated in one droplet {104} as said droplet (104) is formed at the droplet shearing Junction {145). The droplet (104) co-encapsulating the one eel and one particle can then be released from the orifice (147) info the output channel {150}. fiMi73| in another embodiment, as shown in FIGs. 8A-9B, the method and microfluidic device can be adapted to encapsulate a single sample in one droplet. The dispersed solid samples (102) may comprise either ceils or particles. The ceils or particles enter the combining channel (110) from one or both of the first and second dispersed phase channels, and one solid ceil or particle {102} is encapsulated as the droplet (104) is formed at the droplet shearing junction (145). The droplet {104} encapsulating the one solid sample {102} is released from the orifice (147) into the output channel (150).

|{H ?4j Mierofiuidie droplet generators utilizing the droplet generation method described above can b used to compartmentalize or encapsulate a single cell or a bead comprising single ceil, cellular material or some other biological material in a single water droplet Droplets encapsulating a single eel! or bead can be useful for single cell assays of ceils (e.g., cancer cells or immune cells) that exhibit biological heterogeneity for which assays that provide a population average may be insufficient. Encapsulation of a single eel (one cell) and/or a single bead (one-bead) in a single droplet can be useful for high-throughput screening of single ceii. As previously described of prior technoofogies, the efficiency of encapsulating a single ceil {one ceil) and/or a single bead (one-bead) in a single droplet can be as low as 0.1 %, i.e. 1 in 1000 droplets may have a single ceii (one cell) and/or a single bead (one-bead) while the remaining droplets may have no ceils and/or beads or have more than one ceii and/or one bead. Without wishing to limit the present invention, this application provides a passive, hydrodynamie technique which can achieve a 'one-one-one' {one cell and/or one bead in one droplet) encapsulation efficiency of 30% or higher, which could significantly improve the bfomoleeuiar captur efficiency of various bead-based single cell assays.

|ββ75 The device can be configured to encapsulate one ceil and/or one bead in a single droplet of a fiuid (e.g., water) by the combined effect of laminar flow and the high shear liquid-liquid interfaclai boundary. In the illustrated device of FIG. 1A, a first fluid stream comprising a first solid sample (e.g., cells or cellular material) dispersed in a first fluid (e.g., water) is introduced through a first incoming microfluidic channel and a second fluid stream comprising a second so!id sample (e.g., beads or particles) dispersed in the firs fluid {e.g., water) is introduced through the second incoming microfluidic channel. A third fluid stream comprising the first fluid (e.g., water) is introduced through the third incoming microfluidic channel. The first, second and third flow streams, collectively referred to as a dispersed phase fiuid stream, flow into the combining channel.

\W76) in some embodiments, the velocities of the first, second and third flow streams can be adjusted such that laminar flow is established in the combining channel. For example, the flow rates of the first, second and third: flow streams can be equal to each other such that laminar flow is established in the combining channel. By maintaining equal flow rates in the three Incoming microfluidic channels, bead cell migration across the streamlines due to Magnus force can be prevented. The first and the second fluid streams in the combining channel can be separated by a laminar Interface as a result of the laminar flow. The constituents of the first solid sample {e.g., cells or cellular material) self-assemble on one side of the laminar interface and the constituents of the second solid sample {e.g., particles or beads) self-assemble on another side of the laminar interface, for example, beads or particles self-assemble in a single row along a channel wall of the combining channel adjacent to the incoming microfluidic channel of the bead or particles, and cells self-assemble in a single row along the opposite channel wall of the combining channel adjacent to the incoming microftuidie channel of the cells.

The laminar flow of the dispersed phase fiuid stream enters the intersection region. In the intersection region, the flow rate of the continuous phase fluid streams can be adjusted to create a high shear interface between the laminar flow of the dispersed phase fluid stream. Cells in the first flow stream and the beads or particles in the second flow stream are pulled towards the high shear interface as shown In FIG. 1B. The flow rates of the dispersed phase fluid stream and the continuous phase fiuid streams can be adjusted to generate droplets having a droplet size large enough to encapsulate a single cell from the first flow stream and a single bead/particle from the secohd fluid stream. The stee of the droplet can depend an the capillary number Ca » μν/σ, where μ Is the viscosity of the continuous phase comprising the second fluid, V is the superficial velocity (flow rate) of the continuous phase comprising the second fluid, and a is the equilibrium surface tension between the continuous phase and the dispersed phas fluid streams. To generate droplets having an appropriate size to encapsulate a single cell andfor single head, the capillary number can foe in the range of about 0.01 and about 1 (e.g.. about 0,1 ). In various embodiments, the velocity of the continuous phase fluid streams can be about 2-10 times greater that tie velocity of the dispersed phase fluid stream.

The droplet size can also be controlled by controlling the pressure ratio between the dispersed phase fluid stream and the continuous phase fluid stream. In various embodiments, a droplet encapsulating a single ceil and a single bead/particle can be achieved by controlling the pressure ratio (φ) and/or the flo rate ratio between the dispersed phase and the continuous phase. In some embodiments, the pressure ratio and/or the fiow rate ratio between the dispersed phase and the continuous phase may be about 0.1 to about 0.5 (e.g., about 0,3) in order to maximize encapsulation efficiency. Depending on the pressure ratio and/or the fiow rate ratio, the generated droplets can be configured to have a diameter of about 20 pm to about 00 prn to match the size and/or concentration of the incoming ceils and/or beads.

|W?9j in various embodiments, the height of the various icrofiutdie channels Is less than twice the diameter of the solid samples (e.g., cells, beads, particles, etc.) that are configured to be dispersed in dispersed phase fluid. Restricting the height of the various microfiuidic channels to be less than twice the diameter of the solid samples can advantageously reduce the chance that the soiid samples roil over each other and/or stack over each other. iwm\ FiGs. 4A-4D and 5A-5B illustrate experimental results showing encapsulation of single beads and cells in a single droplet using a microti uidic device of the present: invention, in the experiment, the first flow stream comprises Hela cells having a size of about 10 microns dispersed in water and the second fiow stream comprises particles/beads having a size of about 10 microns dispersed in wafer. The concentration of the ceils or beads in water can be in the range between about 10^s - 10⁸ cell or beads in 1 ml of water. Fie. 5A illustrates the seff-assemb!y of 10 pm beads along *he channel wail and the self- assembly of the Hela ceiis along the channel wall. FIG. SB Illustrates the droplets encapsulating a single 10 micron particle/bead and a single Heia cell.

|θβ8ϊ I FIG. 8A Illustrates another embodiment of a microfiuidic device that is configured to encapsulate a single eel! in a single droplet. In contrast to the device depicted in FIG. 1A, the device of FIG. 8A comprises only two incoming microfiuidic. channels instead of three. Ceils dispersed in a first fluid {e,g,, wafer} is introduced through a first microfiuidic channel and the first fluid is introduced through the through a second microfiuidic channel. Laminar fiow is established in the combining channel such that the cells self-assemble along the channel wall. By controiiing the pressure ratio between the dispersed phase and the continuous phase, the concentration of cells in the dispersed phase and the droplet size, encapsulation of a single ceii in a single droplet can be achieved as shown In the droplets of FIG, 88. )K2j !t is noted that ceiis can be introduced through both the microfluldic channels. For example, as shown in FIG. 8A, cells can be Introduced through both the first and the second Incoming channels, in such embodiments, the pressure ratio between the dispersed phase and the continuous phase can be adjusted to change the size of the generated droplets to facilitate encapsulation of a single cell In a single droplet, as shown in the droplets of FIG. SB. in various embodiments, the size of the generated droplets can be tuned by adjusting the droplet generatio regimes, Tfte encapsulation of a..single cell in a single droplet and/or a single bead and a single ceil In single droplet can be achieved !ii both .geometry- mediated and dripping regimes. For example, when the droplets am generated In the geOTtetry-mediated regim , the size of the droplet can be greater t an or equal to the size of ohfiee diameter^'. In the dripping regime, where the droplet break-off occurs due to interfacial instability, the droplet size can be less than the size of the orifice, in both regimes, the heads and ceiis that assemble in single row along the channel wali are pulled into the droplets by the symmetrical high shear zone resulting in encapsulation. The droplet size can be tailored to the size of the incoming ceils anoVor concentrations by controlling the pressure and/or flow rate ratio between the dispersed phase and the continuous phase and the capillary number. Without wishing to limit the present invention, the encapsulation efficiency achieved using the methods described herein can be 10% or higher. More preferably, the encapsulation efficiency achieved using the methods described herein can be 30% or higher. f»«83j EXAMPLES

9884 { The following are non-limiting examples of encapsulation using the interfacial shearing technique of the present invention, it is to be understood that the examples are for illustrative purposes only and are not intended to limit the present invention in any way. Equivalents or substitutes are withi the scope of the invention,

(0085) Ma erials and Methods

86| The mferoffuldfc; devices were fabricated in poiydimethylsiioxane (POMS) using soft lithography. The POMS molded imprints and another piain POMS !ayer were plasma treated and brought together to form a permanent seal. The device was left in art oven at 120 "C overnight to regain its natural hydrophobic^. Ethyl oSeate and 2% ABiL EM 80 formed the continuous phase, and mixture of water, !ipids (DSPC and DSPE-PEG 2000), glycerol and surfactant (Plutonic F-68) form the dispersed phase. Hela cells and 10 pm beads were suspended in freshly prepared dispersed phase,

19087} Both the continuous phase and the dispersed phase were Introduced into the microffufdfc chip using a constant pressure source via high speed solenoid valves controlled by a custom-built lab view program. One-one-one encapsulation was monitored using a Nikon 10D-S inverted microscope and recorded using a Phantom camera V-310. Image J software was used to analyze the videos frame by frame, and yield the encapsulation data.

£6088! Results (098$) A one cell-one bead encapsulation process is shown in FIGs. A-4D and F!Gs. 5A-5B. To demonstrate the one-ceii-one-bead encapsulation in droplets, 10 pm beads were introduced through an upper inlet while He!a ceils entered through a lower inlet. Referring to FIG. SA, the heads and cells then assemble single file atong the channel wail white moving towards a symmetrical high shear zone. Referring to FIGs, 7A-7B, the droplet diameter can be tuned by adjusting (Dp/Cp) to achieve maximum encapsulation efficiency. The encapsulation efficiency increases with the droplet diamete or (Dp/Cp); however, it starts decreasing beyond a threshold because of the multiple encapsulations. By arranging the calls/ beads single file along the channel wail using laminar flow, along wilh the high shear Interface, the randomness involved in the encapsulation process is overcome to a considerable extent,

§86 8j Referring now to FIGs. 6A-8E; "10 urn beads were Introduced from both the upper and lower inlets and were encapsulated into the droplets from either side of the channel Referring As shown in FiGs, 8A- 8S, the ceils were introduced from only one inlet and were encapsulated into single-ceil droplets. As shown in FIGs. 9A-9B, the ceils were introduced from two inlets and were encapsulated into singie-celi droplets.

|909i I Based on the examples described herein, the present Invention has been demonstrated to perform one-one or one-one-one encapsulation in droplets utilizing the combined effect of laminar flow and high shear liquid-liquid interface at the mierofiuidie Junction. These results suggest that this technique can be applied to droplet-based high-throughput genomic workflows. M 2) As used herein, the term "about" refers to plus or minus 10% of the referenced number,

I9093J Various modifications of the invention,- in addition to those described herein,, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to faii within the scope of the appended claims. Each reference cited In the present application is Incorporated herein by reference in its entirety.

(8894J Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the below claims are soiely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. The figures are understood to be representative only and the claims are not limited by the dimensions of the figures, in some embodiments, descriptions of the Inventions described herein using the phrase *com prising* includes embodiments that could he described as "consisting of, and as such the written description requirement for claiming one or more embodiments of the present Invention using the phrase "consisting of is met.

Claims

WHAT IS CLAIMED IS;

1. A method for encapsulating a solid sample {102} in a droplet {1^'94}_> comprising:

a. providing a microfiuidic device {100} comprising:

I. a combining channel (110);

II.. a first continuous phase channel (120) having a portion thereof disposed on one side of the combining channel;

ill. a second continuous phase channel ( 30) having a portion thereof disposed on an opposite side of the combining channel, wherein said portions of the first and second continuous phase channels intersect at a terminal end of the combining channel to form an intersection region (140); and

iv. an output channel (150) fluidly coupled to the intersection region{ 0); b. fiowing a dispersed phase fluid (106) through the combining channel (110) at a first flow rate (Y_d), wherein th dispersed phase fluid {108} comprises at least two flow streams (107), wherein one or both of said flow streams ^'{107} comprises dispersed solid samples (102):

c. adjusting ¾ of the dispersed phase fluid {108} to establish laminar flow in the combining channel {110} such that the solid samples {102} assemble near a sidewa!! {112} of the combining channel while fiowing towards the intersection region {1 0);

d. co-flowing a continuous phase fluid stream (108) through each of the first and second continuous phase channels (120, 130} at a second flow rate (v«)_> wherein the continuous Phase fluid streams (108) intersect the dispersed phase fluid {108} at the intersection region (140), wherein a droplet shearing junction (145) is formed within the intersection region (140) as the continuous phase fluid streams (108) merge with the dispersed phase fluid^■{106}, wherein the droplet shearing junction (145) comprises, an orifice {147} fluidiy coupling the output channel {160} to the intersection region (140);

e. adjusting v_c, or both such that each continuous phase fluid stream (108) forms a high shear interface {109} With the dispersed phase fluid {^' 06) at the intersection regio {140}, wherein the solid samptes {102} are drawn to the high shear interface (109) while flowing through the intersection region (140); and

f. adjusting ¾, y_c, or Both to generate droplets (104) encapsulating one solid sample { 02} at the droplet shearing junction (145), wherein each droplet {104} is substantially sized to encapsulate said solid -sample {102}.

2. The method of claim 1, wherein the microfiuidic device {100} comprises a first dispersed phase channel {114} comprising one of the flow streams {107} forming the dispersed phase fluid {106}, and a second dispersed phase channel (116) comprising the other flow stream (107), wherein the first and second dispersed phase channels (114, 1 6) merge to form the combining channel (110).

3. The method of claim 2, wherein the microfiuidic device {100} further comprises an aqueous phase channel {117} Intersecting with the first and second dispersed phase channels {114, 116), wherein the aqueous phase channel (117) comprises aqueous phase fluid {118), wherein the aqueous phase fluid (1 8) flews in the combining channel (110) such thai the aqueous phase fluid ( 18} forms a laminar interface stream (119) between the two flow streams (107).

4. The method of claim 2, wherein the dispersed solid samples (102) comprises a plurality of ceils flowing in one of the flow streams {107), and a plurality of particles Sowing in the other flow stream (107), wherein when flowing through the combining chahnei {110}, laminar flow of the dispersed phase fluid causes the cells to assemble nea the sidewaii (1 a) and the particles to assemble near an opposing sldewail (112b), wherein at the intersection region (140), the cells are drawn to one high shear interface {109a} and the particles are drawn to the other high shear interface (109h) wherein one cell and one particle are ca-enoapsuiated In one droplet (104) as said droplet {104} is formed at the droplet shearing junction (145), wherein the droplet {104} co- encapsulating the one cell and one particle is released from the orifice {147} into the output channel {150}.

5, The method of claim 2, wherein th dispersed solid samples (102) are either ceils or particles. wherein the dispersed solid samples {102} en er the combining channel (110) from one or both of the first and second dispersed phase channels, wherein one solid sample (102) is encapsulated as the droplet {104} is formed at the droplet shearing function {145}, wherein the droplet {104} encapsulating the one solid sample (102) is released from the orifice (147) into the output channel {1S0}.

8. The method of claim 4, wherein the ceils are eukaryoile ceils, prokafyotic ceils, or a combination thereof.

7. The method of claim 6, w erein the eukaryotlc ceils are animal cells, plant ceils, algae ceils, fungal ceils, or a combination thereof,

8. The method of claim 8, wherein the prokaryotic ceils are bacterial cells,

9. The method of claim 6, wherein the ceils are protoplasts, pollen grains, microspores, or tetrads, 0. The method: of claim 4, wherein the particles are beads.

11. The method of claim 1 , wherein the portions of the first and second continuous phase channels intersect the combining channel { 10} orthogonally.

12. The me od^ of claim 1, wherein flow in the microfiufcfic device (100) is pressure-driven.

13. The method of claim 1 , wherein a width of the combining channel, the first continuous phase channel, or the second continuous phase channel ranges from about 30 pm to about 80 pm.

14. The method of claim 13, wherein a length and a width of the Intersection region are each about 3 to 5 times the width of the combining channel, the first continuous phase channel or the second continuous phase channel

15. A method for encapsulating a solid sample (102) in a droplet (104), comprising:

a. flowing a first fluid (1 OS) through a first microfiuidic channel {110} at a first flow rate |v_d) such that flow of the first fluid Is laminar, wherein the first fluid ( 06) comprises^' a least two flow streams (107), wherein one or both of said flow streams (107) comprises dispersed solid sarapfes (102)., wherein the solid samples (102) assemble near a sidewali (112) of the first micfofiuidic channel while flowing towards an intersection mgksn (140)^*

0, εο-flowlng a second fluid (108) through each of a second mscrofluidic channel (120) and a third microfluidic channel (130) at a second flow rate {%), wherein the second and third microfluidie channels (120, 130) intersect the first microfluidie channel (110) at the intersection region (140) such thai the second fluid streams (108) intersect the first fluid (106) and merge to form a droplet shearing iunction (145) within the intersection region (140);

c. adjusting v_¾, v_e, or both such that each of the second fluid streams (108) forms a high shear interface ( 09) with the first fluid (108), wherein the solid samples (102) are drawn to the high shear interface (109); and

d. generating droplets (104) at the droplet shearing Junction (145) such that each droplet

16. A microfluidic device (100) for encapsulating a solid sample (102) in a droplet (104), said microfiuidic device (100) comprising:

a. a combining channel ( 1 ) having a dispersed phase fluid (106) flowing therein at a first flow rate (v_d), wherein the dispersed phase fluid (106) comprises at least two flow streams (107). wherein one or both of said flow streams (1 7) comprises dispersed solid samples (1 2);

b. a first continuous phase channel (120) having a continuous phase fluid stream (108) flowing therein at a second Sow rate (v_c)_; wherein a portion thereof is disposed on one side of the combining channel;

c. a second continuous phase channel (130) having a continuous phase fluid stream (108) flowing therein at the second flow rate. {v_c}_s wherein a portion thereof is disposed on an opposite side of the combining channel;

d. an intersection region (1 0) formed by said portions of the first and second continuous phase channels intersecting at a terminal end of the combining channel, wherein the continuous phase fluid streams (108) intersect the dispersed phase fluid (106) at the intersection region (140), wherein a droplet shearing junction (145) is formed within the intersection region (140) as the continuous phase fluid streams (108) merge with the dispersed phase fluid (106), wherein the droplet shearing junction (145) comprises an orifice (147); e. an output channel {150} fluidiy coupled to the intersection region {140} via the orifice (147); and

f. a fluid flow controller {160} configured to perform operations comprising:

i. adjusting ¾ of the dispersed phase fluid to establish laminar flo In the combining channel (110) such that the solid samples (102) assemble near a sidewall {112} of the combining channel w ile flowing towards the intersection region ( 0); ii. adjusting v_d; v_t, or both such that each continuous phase fluid stream {108} forms a high shear interface (109) with the dispersed phase fluid {106} at the intersection region (140), wherein the solid samples (102) are drawn to the high shear interface (109) white Slowing through the intersection region {140}; and ill, adjusting y_a, , or both to generate droptets {104} at the droplet shearing junction (145) such that each droplet {104} is substantially sized to encapsulate a solid^' sample (162).

17, The device of ciaim 16 further comprising a first dispersed phase channel (114} comprising one of the flow streams {107} forming the dispersed phase fluid (106), and a second dispersed phase channel (116) comprising the other flow stream (107), wherein the first and second dispersed phase channels (114, 116) merge to form the combining channel (110).

18. The device of claim 17 further comprising an aqueous phase channel (117) Intersecting with the first and second dispersed phase channels (114. 116), wherein the aqueous phase channel (117) comprises aqueous phase fluid (118), wherein the aqueous phase fluid (118) flows i the combining channel {110} such that the aqueous phase fluid (118) forms a laminar Interface stream ( 9) between the two flow streams (107)

19, The device of claim 17, wherein the dispersed solid samples (102) comprises a plurality of cells flowing in one of the flow streams (10?), and a plurality of particles flowing In the other flow stream (107), wherein when flowing through the combining channel (iiO), laminar flow of the dispersed phase fluid causes the ceils to assemble near the sidewall (112a) and the particles to assemble near an opposing sidewall {112b}, wherein at the intersection region {140}, the cells are drawn to one high shear interface (109a) and the particles are drawn to the other high shear interface {109b}, wherein one ceil and one particle are co-encapsulated in one droplet (104) as said droplet (104) is formed at the droplet shearing junction (145), wherein the droplet (104) co- encapsulating the one ceil and one particle is released from the orifice (147} into the output channel (ISO),

20. The device of ciaim 17, wherein the dispersed solid samples (102) are either cells or particles, wherein the dispersed solid samples enter the combining channel (110) from one or both of the first and second dispersed phase channels, wherein one solid sample is encapsulated as the droplet {104} is formed at the droplet shearing junction (145), wherein the droplet encapsulating the one soiid sample is released from the orifice (14?) into the output channel (150),