Electric emulsification on microfluidic platform

Abstract

Devices and methods for conducting electric emulsification are described. Emulsions are created using an electric field to break up a large droplet or continuous fluid stream into a plurality of highly uniform smaller droplets. The electric field can be tuned in order to control the characteristics of the resulting droplets. The electric emulsification can be performed on a digital microfluidic platform thereby allowing integration of emulsification with other sample preparation and processing steps for lab-on-a-chip and point-of-care applications.

Description

ELECTRIC EMULSIFICATION ON MICROFLUIDIC PLATFORMRELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 597,784 entitled “ELECTRIC EMULSIFICATION ON MICROFLUIDIC PLATFORM,” filed November 10, 2023, the entire disclosure of which is incorporated herein by reference.BACKGROUND OF THE INVENTIONField of the Invention

[0002] Certain embodiments of the present invention are directed toward devices and methods for electric emulsification. Emulsions comprising small droplets are created using an electric field to break up the liquid surface of a large droplet or continuous fluid stream. The volume of the small droplets generated is tunable by varying the frequency and / or voltage of the electric field. Emulsions produced can be used in various applications, including food, cosmetics, pharmaceutics, vaccines, chemical reactions, oil production, diagnostics, digital assay, etc.Description of the Prior Art

[0003] Digital assays are an important tool for performing biological testing in a number of areas including quantification of nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. In particular, digital polymerase chain reaction (dPCR) is a third generation of PCR that enables quantification through partitioning the reaction. dPCR has proven to be highly sensitive and accurate in molecular detection making the technology well suited for applications such as trace DNA detection, mutation rate detection, and copy number variation.

[0004] Digital assays, such as dPCR, are performed using machined microwell plates. The reaction mixtures are loaded into the plate’s microwells and then subjected to conditions favorable for the target reaction. However, fabrication of the microwell plates and the necessity of specialized equipment for filling the microwells with thereaction mixture add expense to the dPCR technique. In some applications, liquid partitioning is performed using shear- force-based micro fluidic devices. Use of these devices requires specialized mechanical pumps to provide the strong shear force needed to emulsify the reaction mixture.

[0005] Accordingly, there is a need in the art for an alternate technique of small-volume emulsification that does not require use of specialized plates or expensive processing equipment.SUMMARY OF THE INVENTION

[0006] According to one embodiment of the present invention there is provided a microfluidic emulsification device. The microfluidic emulsification device comprises a top electrode assembly, a bottom electrode assembly, and a fluid-receiving passage located between the top and bottom electrode assemblies. The top and bottom electrode assemblies are configured to apply an electric field to a fluid located within the passage and operable to generate a plurality of droplets from the fluid located within the passage.

[0007] According to another embodiment of the present invention there is provided a method of electric emulsification. The method comprises applying an electric field to a liquid to be emulsified and generating a plurality of smaller emulsion droplets (EDs) from the liquid.

[0008] According to still a further embodiment of the present invention there is provided a method of continuous electric emulsification. A continuous stream comprising at least one liquid is provided. The continuous stream comprises a downstream liquid boundary transverse to the direction of flow of the continuous stream. An electric field is applied to the continuous stream at the liquid boundary. A plurality of droplets of the at least one liquid is generated at the liquid boundary of the continuous stream.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A is a schematic depiction of electric emulsification illustrating a principle of the present invention;

[0010] FIG. IB is a schematic depiction of an exemplary digital microfluidic chip;

[0011] FIG. 2A is a schematic depiction of electric emulsification via one-step emulsification;

[0012] FIG. 2B is a schematic depiction of an example of one-step emulsification with a scale bar of 40 pm;

[0013] FIG. 2C is a graph comparing ED (emulsion droplet) diameter as a function of MD (mother droplet) volume;

[0014] FIG. 2D is a schematic depiction of electric emulsification via two-step emulsification;

[0015] FIG. 2E is a schematic depiction of an example of two-step emulsification involving emulsification and reorganization; and

[0016] FIG. 2F is a graph comparing ED diameter as a function of signal voltage;

[0017] FIG. 3 A is an image of single particle compartmentation with a MD of polystyrene particle suspension;

[0018] FIG. 3B is an image of single particle compartmentation with emulsified 1.6k EDs;

[0019] FIG. 3C is an image of single particle compartmentation with single particles compartmentation;

[0020] FIG. 3D is a graph depicting particle count statistics.

[0021] FIG. 3E is an image of cell compartmentation via a bright filed; and

[0022] FIG. 3F is an image of cell compartmentation via fluorescence images of emulsified EDs with Alexa Fluor 488-E. coir,

[0023] FIG. 4 A is a digital bright field PCR image;

[0024] FIG. 4B is a digital PCR FAM fluorescence image;

[0025] FIG. 5 is a schematic depiction of an electrode configuration;

[0026] FIG. 6A is a schematic depiction of a cross-section view of a microfluidic chip according to an embodiment of the present invention;

[0027] FIG. 6B is a schematic depiction of a top view of a microfluidic chip according to an embodiment of the present invention;

[0028] FIG. 7A is a schematic top view depiction of a device configured for continuous production of emulsion droplets from a continuous fluid stream;

[0029] FIG. 7B is a schematic cross-sectional depiction of a device configured for continuous production of emulsion droplets from a continuous fluid stream;

[0030] FIG. 8 A is an image of a water-in-oil emulsion with mineral oil;

[0031] FIG. 8B is a graph depicting the droplet size distribution from electric emulsification of the resulting water-in-oil emulsion with mineral oil;

[0032] FIG. 8C is an image of a water-in-oil emulsion with diethylhexyl carbonate (DEC);

[0033] FIG. 8D is a graph depicting the droplet size distribution from electric emulsification of the resulting water-in-oil emulsion with diethylhexyl carbonate (DEC);

[0034] FIG. 8E is an image of a water-in-oil emulsion with isopropyl palmitate (IPP);

[0035] FIG. 8F is a graph depicting the droplet size distribution from electric emulsification of the resulting water-in-oil emulsion with isopropyl palmitate (IPP);

[0036] FIG. 8G is an image of a water-in-oil emulsion with HFE-7500;

[0037] FIG. 8H is a graph depicting the droplet size distribution from electric emulsification of the resulting water-in-oil emulsion with HFE-7500;

[0038] FIG. 81 is an image of a water-in-oil emulsion with silicone oil;

[0039] FIG. 8 J is a graph depicting the droplet size distribution from electric emulsification of the resulting water-in-oil emulsion with silicone oil;

[0040] FIG. 9A is a graph showing the ED diameter as a function of signal voltage, frequency, and MD volume of 1 uL;

[0041] FIG. 9B is a graph showing the ED diameter as a function of signal voltage, frequency, and MD volume of 150 nL;

[0042] FIG. 9C is a graph showing the ED diameter as a function of signal voltage, frequency, and MD volume of 22.5 nL;

[0043] FIG. 9D is a graph showing the ED diameter as a function of signal voltage, frequency, and MD volume of 1.76 nL;

[0044] FIGS 10A-10F depict digital PCR quantifications of single-cell Shiga Toxinproducing E. coli (STEC) images at various concentrations;

[0045] FIG. 10G is a graph comparing the measured concentration to the expected concentration;

[0046] FIGS 11A-11F depict images of digital PCR quantifications of EGFR T790M mutant DNA at various concentrations; and

[0047] FIG. 11G is a graph comparing the electric emulsification concentration to the QX200 concentration.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0048] In one aspect, a device and method for performing controllable electric emulsification are provided. The device and method operate to create emulsions with an electric field. The device and method are operable to generate a plurality of emulsion droplets (EDs) from a liquid to be emulsified. The EDs are generated at a liquid boundary of the liquid through the action of the electric field. In one embodiment, the liquid can be provided in the form of a larger droplet generated from a liquid reservoir, also referred to as a mother droplet (MD). As shown in FIG. 1 A, a plurality of emulsion droplets (ED) 2 may be derived from a mother droplet (MD) 4. In an alternate embodiment, the liquid to be emulsified can be provided as a continuous stream having a leading edge of fluid boundary where the EDs are generated.

[0049] As can be seen in FIG. IB, a micro fluidic emulsification device 10 is illustrated. The device 10 comprises a top electrode assembly 12, a bottom electrode assembly 14, and a fluid-receiving passage 16 located therebetween.

[0050] The top electrode assembly 12 comprises a top plate upon which a top emulsification electrode, or “common” electrode, is located. As used herein, the term “plate” refers to a thin material that can serve as a support for additional materials, including electrode materials. The top electrode assembly can further comprise a top hydrophobic layer located over at least a portion of the top emulsification electrode. The top hydrophobic layer faces the fluid-receiving passage 16.

[0051] The bottom electrode assembly 14 comprises a bottom plate upon which a bottom emulsification electrode is located. In certain embodiments, the bottom electrode assembly further comprises a dielectric layer located over at least a portion of the bottom emulsification electrode and a bottom hydrophobic layer located over at least a portion of the dielectric layer. The bottom hydrophobic layer faces the fluid-receiving passage.

[0052] A variety of materials are available for use in fabricating the top and bottom electrode assemblies. The top and bottom plates are preferably formed from aninsulating material unless a passivation layer is coated between the emulsification electrodes and the plate when the plate holds a plurality of electrodes. The electrodes are formed from a conductive material. The dielectric material of the bottom electrode assembly also comprises an insulating material. It is noted that the dielectric layer and hydrophobic layers can help facilitate emulsification in certain applications but need not always be present. Only the electrodes themselves are necessary for generation of the electric field needed for emulsification to occur. In some particular embodiments, optical observation of the droplets is necessary, therefore, some materials used to construct the electrode assemblies can be transparent.

[0053] Exemplary emulsification devices can be constructed based upon glass plate materials or PCB plate materials. For glass-based devices, the top and bottom plates are made of glass. Indium tin oxide (ITO) can be used as the electrode material due to its conductivity and transparency. SU-8 photoresist can be used as a dielectric layer. However, numerous dielectric materials and fabrication techniques, such as physical vapor deposition (PVD), chemical vapor deposition (CVD), spin coating, dip coating, spray coating, screen printing, and 3D printing, can be used. Teflon AF and polydimethylsiloxane (PDMS) can be used to form the hydrophobic layer. As mentioned above, depending upon the application and liquid properties, the dielectric and hydrophobics layers are not always required.

[0054] For PCB-based devices, the bottom plate can be made from a printed circuit board (PCB) material and the top plate can be made from polycarbonate (PC), polymethyl methacrylate (PMMA) or other polymer materials. Copper or another conductive metal, such as silver, can be used as the bottom electrode material, while transparent, conductive poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) can be used as the top electrode. Polyimide (PI) can be used as the dielectric layer, and Teflon AF and PDMS can be used as hydrophobic materials. As with the glassbased devices, the dielectric and hydrophobics layers are not always required.

[0055] Generally, the composition of the liquid to be emulsified does not affect the selection of materials from which the top and bottom electrode assemblies are constructed. If the liquid is not sensitive to an electric current (i.e., no electrolysis will occur), the dielectric layer may not be needed. If the liquid is alreadyhydrophobic to the electrode or dielectric layer, the hydrophobic layer need not be present. However, using dielectric and / or hydrophobic layers may impart some degree of universality to the device enabling its use in a wide range of applications.

[0056] Table 1, below, lists exemplary materials from which the various embodiments of the micro fluidic device can be constructed.

[0057] The micro fluidic emulsification device can comprise additional structure that is operable to dispense, hold, and / or transport the fluid to the top and bottom electrode assemblies for emulsification. In one embodiment, a reservoir of the fluid is provided. The fluid is held in place within the fluid-receiving passage by one or more reservoir electrodes. A top reservoir electrode assembly, which may be a continuation of the common electrode of the top emulsification electrode assembly, is provided opposite a bottom reservoir electrode. The bottom reservoir electrode assembly may be constructed similarly to the bottom emulsification electrode described above.

[0058] One or more driving electrodes can be located between the reservoir electrodes and the emulsification electrodes and configured to generate the MD and transport the MD, therebetween. The one or more driving electrodes can be constructed similarly to the reservoir and emulsification electrodes. The driving electrodes can be configured to generate an electric field, the cycling of which can cause the MD to move toward the emulsification electrode assemblies and particularly the portion of the fluid-receiving passage located therebetween.

[0059] In one or more embodiments, the emulsification electrodes are configured to completely emulsify the MD. In particular embodiments, the side length of the emulsification electrodes is at least twofold larger than that of the driving electrode, which determines the volume of the MD. The gap height between the opposedemulsification electrodes and / or driving electrodes can vary from a few pm to hundreds of pm. In one or more embodiments, the gap height can be from 3 to 500 pm, from 5 to 400 pm, from 10 to 250 pm, or from 25 to 150 pm. In certain embodiments, the side length of the driving electrodes is typically 5 -fold to 20-fold larger than the gap heigh to enable efficient droplet manipulations.

[0060] FIG. 5 depicts a schematic depiction of an electrode configuration containing a reservoir electrode 20, an emulsification electrode 22, and a plurality of driving electrodes 24. As noted above, the driving electrodes 24 may be configured to generate the MD 4 and transport the MD 4 from the reservoir electrode 20 to the emulsification electrode 22 to thereby emulsify the MD 4 and form the EDs 2. As shown in FIG. 5, the MD 4 may be dispensed from the reservoir electrode 20 and driven via the driving electrodes 24 to the emulsification electrode 22, which then breaks up and emulsifies the MD 4 into a plurality of EDs 2.

[0061] In one or more embodiments, and as depicted in FIGS. 6A and 6B, the device 10 can comprise one or more spacers 26 that are operable to maintain separation between the top and bottom electrode assemblies thereby defining a height of the fluid-receiving passage. FIG. 6A also demonstrates the presence of the top plate 28, a common emulsification electrode 30, a top hydrophobic layer 32, a bottom hydrophobic layer 34, a dielectric layer 36, and a bottom plate 38.

[0062] In certain embodiments, the spacing between the top and bottom electrode assemblies is such that the liquid, for example in the form of an MD, is sandwiched therebetween, contacting both the top and bottom electrode assemblies. Thus, the spacing between the top and bottom electrodes may be selected based upon likely MD size. When formed, the EDs, being of much smaller dimension than the MD from which they are generated, generally contact only one electrode assembly, such as the bottom electrode assembly. However, this need not always be the case.

[0063] Devices according to one or more embodiments of the present invention are parallel-plate devices that are easily fabricated without requiring machining of microwells in a testing plate. Similarly, the devices are capable of performing liquid emulsification without using shear forces. Thus, the need for mechanical pumps to provide the strong shear forces needed for mechanical emulsification is eliminated.Also, by being of a parallel-plate construction, the emulsification devices are easily integrated and implemented on a digital microfluidic platform capable of upstream and downstream assay procedures with versatile liquid manipulations, including extraction, washing, mixing, splitting, thermal cycling, detection, etc. This construction enables development of one-stop digital assay or single-cell analysis instruments.

[0064] In certain embodiments, the devices can be configured for single use or for multiple uses. In applications in which the composition of the liquid is unchanged, the liquid to be emulsified (e.g., MDs) and EDs can be purged, and the device can be reused for subsequent emulsification. However, if the liquids to be emulsified are different, the carryover contamination may need to be evaluated and eliminated in some applications (e.g., digital PCR for diagnosis), thereby rendering the devices more appropriate for single use.

[0065] Methods of electric emulsification generally involve emulsification of a liquid into a plurality of EDs. The liquid can be provided in the form of an MD or the process can comprise the continuous emulsification of a liquid stream into a plurality of fine EDs. To perform electric emulsification, a liquid can be placed on the bottom electrode and surrounded by air or an immiscible liquid such as oil. When an electric signal is applied, the liquid, at its liquid boundary continuously ejects a plurality of EDs from its periphery into the surrounding environment (air or oil), breaking up the liquid into an emulsion.

[0066] In one or more embodiments, an MD, preferably having a volume in the nanoliter range (e.g., from about 1 to about 1000 nl, from about 5 to about 750 nl, from about 15 to about 500 nl, or from about 25 to about 250 nl) can be broken into a plurality of EDs with electric-tunable volumes in the pico- and femtoliter range (e.g., from 10 fl to 500 pl, from 50 fl to 250 pl, from 100 fl to 100 pl, from 250 fl to 50 pl, or from 500 fl to 10 Pl).

[0067] In one or more embodiments, the MD is generated, such as through the action of the one or more driving electrodes described above. However, it is within the scope of the present invention for other means of forming MDs to be used, such as through the use of piezoelectric devices. Once formed, the MD is positioned in between a pair of substantially parallel emulsification electrodes, such as those described above. TheMD can be surrounded by a continuous phase comprising air or an immiscible liquid, such as an oil. An electric signal is applied to one electrode, using a signal generator, for example, for the purpose of generating an electric field. The electric field acts upon the MD to generate a plurality of EDs having a diameter (and thus a volume) that is less than the MD from which they were ejected. The EDs then form the disperse phase of the emulsion.

[0068] It is believed that the mechanism for electric emulsification is analogous to droplet breakup from a stream of water by Plateau-Rayleigh instability. The formation of EDs originates when an electric field perturbates the fluid boundary of a liquid provided either as an MD or as a continuous stream, and generates periodic waves along its surface. The peak of the surface wave further proceeds to expand until it ejects a satellite droplet and resets the surface of the liquid for the next ejection. In certain embodiments, a high throughput of ED generation is attributed to the repeating process and multiple peaks / ejects along the liquid boundary, for example the MD circumference.

[0069] The perturbation of the surface can be described with mathematical models, while the growth and the settled volume of the satellite droplet can be analyzed by the Laplace pressure and the electric pressure. Equation (1) describes the balance between Laplace pressure, EP apiace, and the electric pressure, APE, in a droplet ejected (i.e., the DD) during the electric emulsification process.

[0070] On the left side of Equation (1), the Young-Laplace equation indicates the Laplace pressure as resistance against the creation of a ED, where yWater-oU is the water-oil interfacial tension, and Ri and R are the two principal radii of curvature of the ED. On the right side of Equation (1), the required creation pressure to overcome the resistant Laplace pressure is provided by the electric pressure, as shown in FIG. IB. EMD and 8env are the permittivity of the MD and the environment (oil or air), and E is the appliedelectric field strength between electrodes. Accordingly, the composition of the MD and the property of the oil / air affect the selection of frequency and voltage for the electric field, as both the electric pressure and Laplace pressure are compositionsensitive.

[0071] In an alternate embodiment, it is not necessary to generate EDs from an MD. As shown in FIGS. 7A and 7B, emulsion droplets (EDs) 102 can be formed during a continuous electric emulsification process from a continuous stream 104 comprising at least one liquid. The continuous stream may be provided from a fluid reservoir and flowed toward the emulsification electrodes 106, which operate as discussed above. Driving electrodes, such as those described above, may be employed to facilitate the movement of the continuous stream, or alternate means may be employed such as microfluidic pumps and the like. The continuous stream comprises a downstream liquid boundary that is transverse to its direction of flow. The liquid boundary is preferably located between the emulsification electrodes 106 and within the fluidreceiving passage 108 of a microfluidic emulsification device 100, such as that described above. FIG. 7B also depicts the upper plate 110, the upper dielectric layer 112, the lower dielectric layer 114, and the bottom plate 116.

[0072] An electric field is then applied to the continuous stream at the liquid boundary, much in the same way that an electric field is applied to a MD in the previously- described embodiment. The electric field causes a plurality of droplets to be ejected from the liquid boundary of the continuous stream according to the same principles described above. In this way, emulsion droplets can continuously be formed thereby increasing droplet total throughput through the device.

[0073] Optionally, the EDs formed can undergo additional processing in order to reorganize the EDs, if desired. During ED formation, an emulsification signal, having a first voltage and a first frequency, is used to generate the EDs. In the reorganization step, a reorganization signal is applied to the emulsification electrodes to generate an electric field that causes accumulation of two or more EDs into a larger diameter droplet. The reorganization signal can comprise usage of a second voltage and / or a second frequency that are different from the voltage and / or frequency of the emulsification signal. In one or more embodiments, the second frequency is greaterthan the first frequency and / or the second voltage is less than the first voltage. This reorganization step permits a greater control over the final ED dimensions as required for a particular application. Initial ED formation and the optional reorganization of those EDs into larger diameter EDs is depicted in FIGS. 2A, and 2D.

[0074] The emulsions generated by embodiments of the present invention have extensive applications. In one aspect, the emulsions can be used in various assays, such as single cell, ELISA, PCR, etc., and in extraction applications, such as crude oil, etc. Taking digital PCR as an example, MDs can be mainly made of a PCR master mix containing buffer, ion, enzyme, etc. For the immiscible liquid environment, a surfactant-supplemented oil can be used. The choice of oil-surfactant is also vast. Table 2 shows some examples in electric emulsification for the application of digital PCR.

[0075] Apart from dPCR, electric emulsification can also be used for digital immunoassay or enzyme-linked immunosorbent assay (ELISA) and single-cell RNA-seq for diagnosis applications. Emulsification is also widely used in chemical extraction, food products, cosmetics, pharmaceutics, vaccines, etc.

[0076] In one or more embodiments, the MD or continuous stream can comprise a plurality of suspended particles, including DNA, RNA, or protein molecules, or cells. It has been discovered that single particle / cell compartmentation can be achieved with the electric emulsification processes described herein. In these embodiments, the EDs formed contain a single particle or cell of the plurality once contained within the MD or continuous stream.

[0077] It has been discovered also that the EDs formed via the processes described herein exhibit better size homogeneity than those obtained from mechanical vortex shearing of liquids. When these processes are implemented on a digital microfluidic platform, it allows the integration of emulsification with other essential sample preparation and processing steps, such as extraction, washing, mixing, and splitting, for the fulfillment of a lab-on-a-chip and point-of-care applications.EXAMPLES

[0078] The following examples describe methods of electric emulsification according to the present invention. It is to be understood, however, that these examples are provided by way of illustration, and nothing therein should be taken as a limitation upon the overall scope of the invention.EXAMPLE 1

[0079] Electric emulsification is performed by breaking up an MD into smaller EDs. As a part of a one-step emulsification process, a signal with a proper frequency is used to break up the mother droplet into numerous final daughter droplets (FIG. 2A). For example, by applying 1 kHz and 160 VRMS for 7 s, an original 3.44-nl MD was emulsified into -38100 EDs with an average diameter of 5.6 pm (volume 90 fl) (FIG. 2B). Other than electric signal, the diameter of EDs can be adjusted by controlling the MD volume. Given a constant electrode size, by varying the MD volume from 0.44 nl to 14.4 nl, the average diameter of EDs increased from 4.7 pm to 7.0 pm (FIG. 2C).

[0080] Optionally, EDs formed from an MD can be subjected to a secondary reorganization step to locally accumulate several EDs into a final larger ED. Thus, this process comprises a two-step emulsification in which an emulsification signal (e.g., 1 kHz) is used to break up the mother droplet into intermediate emulsion droplets, and then a reorganization signal (e.g., 30 kHz) is used to accumulate several emulsion droplets into a final larger emulsion droplet (FIG. 2D). For example, by applying 1 kHz and 160 VRMS for 3 s, a 0.44-nl MD was emulsified into 8.2k EDs (volume 54 fl) with an average diameter of 4.7 pm (FIG. 2E). Then, by applying a reorganization signal of 30 kHz and 75 VR S for 2 s, EDs were locally accumulated, finally forming 3.2k EDs (volume 138 fl) with an average diameter of 6.4 pm. The final diameter of EDs can befurther adjusted by controlling the voltage of the reorganization signal. By varying the voltage of the reorganization signal from 75 VRMS to 95 VRMS, the average diameter of final EDs increased from 6.4 gm to 7.9 gm (FIG. 2F). Emulsification examples mentioned above were performed in hexadecane with 2 wt.% Span 80.EXAMPLE 2 - Single Cell Emulsification

[0081] Single parti cl e / cell compartmentation can be achieved with electric emulsification, which simplifies the single-cell procedure. A 6.79-nl MD containing 372 polystyrene (PS) particles (55 particles / nl) was emulsified into 1.6k (volume 4.2 pl) EDs with an average diameter of 22.2 pm (see FIGS. 3A-C). 2.6% (41 / 1585) EDs contained single particles see FIG. 3D). Similarly, a 300-nl MD containing 3000 Alexa Fluor 488-E. coli (104 cells / pl) was emulsified into DDs with single cells (FIGS. 3E and 3F).EXAMPLE 3 - Digital Assay

[0082] Digital assay can be improved and performed in EDs formed by electric emulsification. A 1 -nl MD of reaction mixture was emulsified into 11.1k EDs (volume 90 fl) with an average diameter of 5.6 pm. Then, dPCR (digital PCR) was carried out on a thermal cycler (Benchmark TC9639) with the following thermal protocol: 95°C for 300 s, and 40 cycles of 95°C for 15 s and 60°C for 60 s. The endpoint digital readout of dPCR can be obtained by a fluorescence microscopic image (see FIGS. 4A and 4B).EXAMPLE 4 - Emulsions Formed with Different Oils

[0083] Various water-in-oil emulsions were created using mineral oil, diethylhexyl carbonate (DEC), isopropyl palmitate (IPP), HFE-7500, or silicone oil utilizing the process steps described herein. Images of these emulsions are provided in FIGS. 8A, 8C, 8E, 8G, and 81, while graphs depicting the droplet size distribution of the emulsions are provided in FIGS. 8B, 8D, 8F, 8H, and 8J.EXAMPLE 5 - Comparing ED Diameters from MD Size

[0084] FIGS. 9A-9D provide graphs showing the droplet diameter (microns) of the EDs derived from MDs of various volumetric sizes. The graphs also depict droplet diameter as a function of signal voltage and frequency.EXAMPLE 6 - Analyzing PCR Quantifications

[0085] FIGS. 10A-10F depict digital images of PCR quantification of single-cell Shiga Toxin-producing E. coli (STEC) at various concentrations. FIG. 10G provides a graph comparing the measured concentration with the expected concentration.EXAMPLE 7 - Analyzing PCR Quantifications

[0086] FIGS. 11A-11F depict digital images of PCR quantification of EGFR T790M mutant DNA at various concentrations quantified with a commercial digital PCR system (QX200). FIG. 11G provides a graph comparing the measured electric emulsification concentration with the QX200 concentration.

[0087] The preferred forms of the invention described above are to be used as illustration only and should not be used in a limiting sense to interpret the scope of the present invention. Modifications to the exemplary embodiments, set forth above, could be readily made by those skilled in the art without departing from the spirit of the present invention.

[0088] The inventors hereby state their intent to rely on the Doctrine of Equivalents to determine and assess the reasonably fair scope of the present invention as it pertains to any apparatus not materially departing from but outside the literal scope of the invention as set forth in the following claims.

Claims

We claim :

1. A microfluidic emulsification device comprising: a top electrode assembly; a bottom electrode assembly; a fluid-receiving passage located between the top and bottom electrode assemblies, the top and bottom electrode assemblies being configured to apply an electric field to a fluid located within the passage and operable to generate a plurality of droplets from the fluid located within the passage.

2. The microfluidic emulsification device of claim 1, wherein the top electrode assembly comprises a top plate upon which a top emulsification electrode is located.

3. The microfluidic emulsification device of claim 2, wherein the top electrode assembly comprises a top hydrophobic layer located over at least a portion of the top emulsification electrode, the top hydrophobic layer facing the fluid-receiving passage.

4. The microfluidic emulsification device of claim 1, wherein the bottom electrode assembly comprises a bottom plate upon which a bottom emulsification electrode is located.

5. The microfluidic emulsification device of claim 4, wherein the bottom electrode assembly comprises a dielectric layer located over at least a portion of the bottom emulsification electrode.

6. The microfluidic emulsification device of claim 5, wherein the bottom electrode assembly comprises a bottom hydrophobic layer located over at least a portion of the dielectric layer, the bottom hydrophobic layer facing the fluid-receiving passage.

7. The microfluidic emulsification device of claim 1, wherein the device further comprises one or more spacers operable to maintain separation between thetop and bottom electrode assemblies thereby defining a height of the fluid-receiving passage.

8. The microfluidic emulsification device of claim 1, further comprising a reservoir electrode assembly configured to dispense and / or hold a mother droplet of the fluid.

9. The microfluidic emulsification device of claim 8, further comprising one or more driving electrodes configured to transport the mother droplet from the reservoir electrode assembly to the fluid-receiving passage.

10. A method of electric emulsification comprising: providing a liquid to be emulsified having a liquid boundary; applying an electric field to the liquid; and generating a plurality of emulsion droplets from the liquid boundary of the liquid.

11. The method of claim 10, wherein the liquid to be emulsified is provided as a mother droplet.

12. The method of claim 11, wherein the mother droplet is located within an immiscible continuous phase.

13. The method of claim 10, wherein at least a portion of the plurality of emulsion droplets form a disperse phase of an emulsion.

14. The method of claim 10, wherein the liquid comprises a PCR master mix.

15. The method of claim 10, wherein the liquid is sandwiched between a top electrode and a bottom electrode of a microfluidic emulsification device.

16. The method of claim 15, wherein the top and bottom electrodes are used to apply the electric field to the liquid.

17. The method of claim 10, wherein the electric field generating the emulsion droplets has a first frequency and first voltage.

18. The method of claim 17, further comprising, subsequent to generation of the plurality of emulsion droplets, changing the electric field to a second frequency and / or a second voltage and causing two or more emulsion droplets to accumulate into a larger emulsion droplet.

19. The method of claim 18, wherein the second frequency is greater than the first frequency and / or the second voltage is less than the first voltage.

20. The method of claim 10, wherein the liquid comprises a plurality of particles or cells.

21. The method of claim 19, wherein a plurality of the emulsion droplets formed contain a single particle or cell of the plurality of particles or cells.

22. A method of continuous electric emulsification comprising: providing a continuous stream comprising at least one liquid, the continuous stream comprising a downstream liquid boundary transverse to the direction of flow of the continuous stream; applying an electric field to the continuous stream at the liquid boundary; generating a plurality of droplets of the at least one liquid at the liquid boundary of the continuous stream.

23. The method of claim 22, wherein the continuous stream is flowed within a fluid-receiving passage of a microfluidic emulsification device, the liquid boundary being located between a top electrode and a bottom electrode of the microfluidic emulsification device.

24. The method of claim 23, wherein the top and bottom electrodes are used to apply the electric field to the continuous stream at the liquid boundary.

25. The method of claim 22, wherein the electric field generating the plurality of droplets has a first frequency and first voltage.

26. The method of claim 25, further comprising, subsequent to generating the plurality of droplets, changing the electric field to a second frequency and / or a second voltage and causing two or more of the plurality of droplets to accumulate into a larger droplet.

27. The method of claim 26, wherein the second frequency is greater than the first frequency and / or the second voltage is less than the first voltage.

28. The method of claim 22, wherein at least one of the two or more immiscible liquids comprises a plurality of particles or cells.

29. The method of claim 28, wherein a quantity of the plurality of droplets formed contain a single particle or cell of the plurality of particles or cells.

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