Methods, systems, and apparatuses for generating and dispensing emulsion droplets

Abstract

Disclosed herein are novel methods, systems, and apparatuses for generating and dispensing emulsion droplets, which beneficially eliminates the use of microfluidics chips typically used in emulsion droplet generation, thereby reducing the cost and complexity of emulsion droplet generation; and improving the ease of performance, efficacy, and reliability of droplet-based assays. Disclosed herein are high-throughput methods of contacting liquid droplets with an oil to form emulsion droplets, and simultaneously transferring said emulsion droplets to an amplification vessel. Systems and apparatuses suitable to perform the methods of the invention are also provided. Droplets generated accordingly are suitable for a reaction e.g. an amplification reaction, and detection of one or more reaction characteristics or outputs. The method may be particularly useful for the formation of droplets required for amplification reactions such as dPCR.

Description

[0001] METHODS, SYSTEMS, AND APPARATUSES FOR GENERATING AND DISPENSING EMULSION DROPLETS

[0002] Background of the Invention

[0003] Droplet-based assays play a crucial role in many biomedical applications due to their high-throughput and highly sensitive nature, such as clinical diagnostics (e.g. genetic tests of one or more targets) and drug and biomarker discovery, offering a versatile platform for a wide variety of molecular biology applications. Droplet-based assays require the encapsulation of minute volumes (e.g. pL to nL) volumes of sample and reaction mixture in emulsion droplets that function as independent reaction chambers for biochemical reactions, facilitating the high-throughput discrete processing and analysis of multiple targets in a sample, such as multiple genetic targets, or individual sub-components of a sample (e.g., cells, nucleic acids, proteins).

[0004] Current techniques can create billions of emulsion droplets, however generation of emulsion droplets presents technical challenges, which are often resolved by complex instrumentation, and / or multiple liquid handling steps, which drives the complexity, cost, and duration of these assays. For example, current techniques of emulsion droplet generation typically utilise a microfluidics chip, combining an aqueous phase stream, such as a sample, and an organic phase stream, such as an oil, to form a plurality of emulsion droplets which are deposited in a collection well and separately transferred to an amplification vessel. The generation of emulsion droplets in this manner typically consumes a microfluidics chip, and the transfer from the collection well to an amplification vessel requires liquid handling, wherein droplets may be lost and / or deformed during the transference, impacting the efficacy of the process, and wherein a pipettor may require cleaning after transference, or wherein disposable pipette tips may be consumed. Furthermore, disposable pipette tips introduce multiple alignment issues between the tip and microfluidics chip during dispensing of the droplets into an amplification vessel. Thus, there is a need for improved technology for the generation and transference of emulsion droplets to permit increased droplet generation speed, lower cost, and / or with reduced instrument complexity.

[0005] Having regard to the prior art, therefore, it would be desirable to have a method for which eliminates the use of the microfluidics chip. Such a method may be particularly beneficial for reducing the number of processing steps in emulsion droplet generation, improving both speed and efficiency of emulsion droplet generation.

[0006] The present invention seeks to overcome one or more of the problems found in the prior art.

[0007] Summary of the Invention

[0008] In various aspects and embodiments, the disclosure relates to methods, systems, and apparatuses for generating and dispensing emulsion droplets, which beneficially eliminates the use of microfluidics chip typically used in this process, therefore eliminating a liquid handling step. Consequently, the invention provides a reduction or elimination of the number of droplets lost and / or deformed during use of a microfluidics chip-based system, in particular during the transference from a microfluidics chip to an amplification vessel; pipettor cleaning after transference; and consumption of disposable pipette tips. In this way, the methods, systems, and apparatuses for generating and dispensing emulsion droplets may be facilitated by one or more of reduced cost and complexity, ease of performance, increased efficacy and / or improved reliability.

[0009] In aspects of the invention, a system for generating emulsion droplets is provided, the system comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid entering the main channel, and a distal end portion comprising an outlet for fluid exiting the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and a pipettor configured to generate and extrude at least one liquid droplet, the pipettor comprising: a nozzle having a nozzle tip from which the at least one liquid droplet is extruded; and wherein the pipettor is arranged such that the nozzle tip is located within the side port of the manifold at a first location spaced from the main channel of the manifold.

[0010] In embodiments, the nozzle tip is spaced from the main channel of the manifold by a distance ‘E’, wherein distance E defines an air gap between the channel and nozzle tip. In embodiments, the pipettor is arranged to generate and extrude at least one liquid droplet comprising a first fluid at a first location, and wherein the at least one liquid droplet passes through the air gap between the first location and the main channel before contacting a second fluid flowing through the main channel at a second location. In embodiments, the first fluid is immiscible in the second fluid, and wherein the at least one liquid droplet is immersed in the second fluid, at least one emulsion droplet of the first fluid within the second fluid is thereby generated. In embodiments, the first fluid comprises an aqueous liquid, which may comprise water, and / or a reaction mixture, and / or a sample, wherein the sample may comprise at least one target oligonucleotide. In embodiments, the second fluid comprises an organic liquid, which may comprise an oil.

[0011] In embodiments, the main channel may have an elongate axis (ex) defined between the inlet and the outlet, and the pipettor may have a proximal end defined by the nozzle and the nozzle tip, and a distal end, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end of the pipettor. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 1 1 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0012] In embodiments, the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body. In embodiments, the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port. In embodiments, the side port and the elongate body of the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0013] In embodiments, the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex) intersect.

[0014] In embodiments, the at least one liquid droplet may be extruded from a nozzle tip of a pipettor at the first location. In embodiments, the second fluid may be provided as a continuous stream, wherein the second location may be at a surface of the continuous stream of the second fluid. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises contacting the at least one liquid droplet with the continuous stream of the second fluid.

[0015] In embodiments, the main channel may comprise a channel region of reduced diameter and / or cross- sectional area, wherein the aperture in the wall of the elongate body may directly communicate with the channel region of reduced diameter and / or cross-sectional area. In embodiments, the channel region of reduced diameter and / or cross-sectional area may have a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm.

[0016] In embodiments, the main channel outlet communicates with a third location, wherein the third location may comprise an amplification vessel, and / or comprise a monolayer, and / or comprise an imaging chip. In embodiments, the at least one emulsion droplet is transferred to a third location. In embodiments, transferring the at least one emulsion droplet to a third location may comprise transferring the at least one emulsion droplet from the second location to the outlet via a region of the main channel, and from the outlet to the third location by the continuous stream of second fluid. In embodiments, the at least one emulsion droplet comprises a partitioned sample, and / or comprises the reaction mixture. In aspects of the invention, a method for generating emulsion droplets is provided, the method comprising: (a) forming at least one liquid droplet comprising a first fluid; (b) extruding the at least one liquid droplet from a first location; (c) passing the at least one liquid droplet through an air gap; and (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; and wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally (e) transferring the at least one emulsion droplet to a third location.

[0017] In aspects of the invention, a method for generating emulsion droplets in a stagnant body of liquid is provided, the method comprising: (a) forming at least one liquid droplet comprising a first fluid; (b) extruding the at least one liquid droplet from a first location; (c) passing the at least one liquid droplet through an air gap; and (d) contacting the at least one liquid droplet with a second fluid at a second location,; wherein the first fluid is immiscible in the second fluid; wherein the second fluid is provided as a stagnant body; wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally (e) transferring the at least one emulsion droplet to a third location.

[0018] In aspects ofthe invention, a method for generating emulsion droplets in a stream of liquid is provided, the method comprising: (a) forming at least one liquid droplet comprising a first fluid; (b) extruding the at least one liquid droplet from a first location; (c) passing the at least one liquid droplet through an air gap; and (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; wherein the second fluid is provided as a stream; wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet, and (e) transferring the at least one emulsion droplet to a third location.

[0019] In embodiments, the first fluid is immiscible in the second fluid, and wherein the at least one liquid droplet is immersed in the second fluid, at least one emulsion droplet of the first fluid within the second fluid is thereby generated. In embodiments, the first fluid comprises an aqueous liquid, which may comprise water, and / or a reaction mixture, and / or a sample, wherein the sample may comprise at least one target oligonucleotide. In embodiments, the second fluid comprises an organic liquid, which may comprise an oil.

[0020] In embodiments, the method comprises extrusion of the liquid droplet from the pipettor; optionally wherein the liquid droplet is extruded from a nozzle tip ofthe pipettor. In embodiments, the first location from which the at least one liquid droplet is extruded is spaced vertically above the second location.

[0021] In embodiments, the liquid droplet has a direction of travel, and the second fluid may have a direction of travel, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect at the second location. In embodiments, the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect in the main channel of the apparatus according to an apparatus of the invention. In embodiments, the direction of travel of the liquid droplet and the direction of travel of the second fluid intersects at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the direction of travel of the liquid droplet and the direction of travel of the second fluid. In embodiments, the direction of travel of the liquid droplet and the second fluid intersect.

[0022] In embodiments, the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv), and the second fluid has a speed and direction of travel defining a second velocity vector (sv), wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv), wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect in the main channel of an apparatus according to the invention. In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0023] In embodiments, the second fluid may be provided as a continuous stream, wherein the second location is at a surface of the continuous stream of the second fluid. In embodiments, the second fluid may be provided as a stagnant body, wherein the second location is at a surface of the stagnant body of the second liquid. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises contacting the at least one liquid droplet with the continuous stream of the second fluid, or contacting the at least one liquid droplet with the stagnant body of the second liquid.

[0024] In embodiments, transferring the at least one emulsion droplet to a third location comprises transferring the at least one emulsion droplet from the second location to the outlet via a region of the main channel, and from the outlet to the third location by the continuous stream of second fluid. In embodiments, the third location comprises an amplification vessel, and / or comprises a monolayer, and / or comprises an imaging chip. In embodiments, the at least one emulsion droplet comprises a partitioned sample, and / or comprises a reaction mixture.

[0025] In aspects of the invention, an apparatus for generating emulsion droplets is provided, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a pipettor comprising a nozzle having a nozzle tip, the nozzle tip arranged at a first location spaced from the main channel of the manifold, and wherein the nozzle of the pipettor is arranged at an angle relative to the elongate body.

[0026] In aspects of the invention, an apparatus for generating emulsion droplets is provided, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and wherein the side port is arranged at an angle relative to the elongate body.

[0027] In aspects of the invention, an apparatus for generating emulsion droplets is provided, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body; and a pipettor comprising a nozzle having a nozzle tip arranged within the side port, the nozzle tip positioned at a first location spaced from the main channel of the manifold, and wherein the nozzle of the pipettor is arranged at an angle relative to the elongate body.

[0028] In embodiments, an apparatus of the invention may further comprise a pipettor comprising a nozzle having a nozzle tip, and wherein the nozzle tip is located within the side port.

[0029] In embodiments, an apparatus of the invention may further comprise a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, wherein the nozzle tip of the pipettor is located within the side port.

[0030] In embodiments, wherein the pipettor is configured to dispense a liquid droplet from the nozzle tip, wherein the nozzle tip is separated from the main channel by a distance ‘E’, wherein distance E defines an air gap between the channel and nozzle tip, wherein the distance E may be a vertical distance.

[0031] In embodiments, an apparatus of the invention may further comprise a pump to drive the flow of the second fluid, wherein the pump pushes the second fluid through the manifold or draws the second fluid through the manifold. In embodiments, the main channel may be substantially linear and has an elongate axis (ex) defined between the inlet and the outlet. In embodiments, the pipettor has a proximal end comprising the nozzle and the nozzle tip, and a distal end, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0032] In embodiments, the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body, wherein the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port. In embodiments, the side port and the elongate body of the manifold intersect at an angle, wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0033] In embodiments, the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex) intersect.

[0034] In embodiments, the pipettor is directed axially-downwardly, wherein downwardly relates to a vertical direction, and comprises a piezoelectric membrane.

[0035] In embodiments, the main channel comprises a channel region of reduced diameter and / or cross- sectional area, wherein the aperture in the wall of the elongate body may directly communicate with the channel region of reduced diameter and / or cross-sectional area. In embodiments, the channel region of reduced diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm. In embodiments, the main channel comprises a channel region of increased diameter and / or cross-sectional area, wherein the channel region of increased diameter and / or cross- sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, between about 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800 pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm. In embodiments, the main channel has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400 mm. In embodiments, the main channel comprises one or more narrowing channel regions of reducing diameter and / or cross-sectional area, wherein the cross- sectional diameter of the one or more narrowing channel regions is gradually decreased relative to an adjacent region of the main channel. In embodiments, a narrowing channel region has a tapering cross-sectional diameter which connects to a narrowed channel region, wherein the one or more narrowing channel regions are configured to facilitate mixing of the liquid droplet and the second fluid. In embodiments, the channel comprises one or more widening channel regions of increasing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more widening channel regions is gradually increased relative to an adjacent region of the channel. In embodiments, a widening channel region has an inverse-tapering cross-sectional diameter which connects to a widened channel region.

[0036] In embodiments, the main channel outlet communicates with a third location, wherein the third location may comprise an amplification vessel, and / or comprises a monolayer, and / or comprises an imaging chip.

[0037] In aspects of the invention, an apparatus for generating emulsion droplets is provided, the apparatus comprising: a manifold, the manifold comprising a fluid flow path extending between an inlet and an outlet, and a liquid droplet inlet arranged to communicate with the fluid flow path at a location between the inlet and the outlet; and a pipettor configured to generate a liquid droplet, the pipettor comprising a nozzle having a nozzle tip from which a liquid droplet can be dispensed, the pipettor configured such that the nozzle tip is arranged to dispense a liquid droplet comprising a first fluid through the liquid droplet inlet of the manifold, in use, to contact a second fluid flowing along the fluid flow path.

[0038] In embodiments, the fluid flow path is defined by a channel through the manifold, which may be substantially linear, wherein the channel is configured to accept a continuous stream of the second fluid.

[0039] In embodiments, the nozzle tip of the pipettor is located within the liquid droplet inlet, and may be separated from the fluid flow path by a distance ‘E’, which may define an air gap between the fluid flow path and the nozzle tip.

[0040] In embodiments, the fluid flow path has an elongate axis (ex) defined between the inlet and the outlet of the manifold. In embodiments, the pipettor has a proximal end comprising the nozzle and the nozzle tip and a distal end, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle. In embodiments, the liquid droplet inlet has a proximal end connected to the wall of the manifold and a distal end directed away from the manifold. In embodiments, the liquid droplet inlet has a longitudinal axis (px) extending between an aperture in the wall of the manifold at the proximal end of the liquid droplet inlet to the distal end of the liquid droplet inlet and running parallel with the liquid droplet inlet. In embodiments, the liquid droplet inlet and the manifold intersect at an angle, wherein the angle is defined between the longitudinal axis (lx) of the liquid droplet inlet and the elongate axis (ex) defined between the inlet and the outlet of manifold.

[0041] In embodiments, the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex).

[0042] In embodiments, the pipettor is directed axially-downwardly, wherein downwardly relates to a vertical direction, and may comprise a piezoelectric membrane.

[0043] In embodiments, the manifold comprises a fluid flow path region of reduced diameter and / or cross- sectional area, wherein the region of the manifold comprising the liquid droplet inlet may directly communicate with the fluid flow path region of reduced diameter and / or cross-sectional area. In embodiments, the manifold comprises a fluid flow path region of increased diameter and / or cross- sectional area. In embodiments, the fluid flow path has a length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400mm.

[0044] In embodiments, the fluid flow path comprises one or more narrowing fluid flow path regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more narrowing fluid flow path regions is gradually decreased relative to an adjacent region of the fluid flow path. In embodiments, a narrowing fluid flow path region has a tapering cross-sectional diameter which connects to a fluid flow path region of reduced diameter and / or cross-sectional area, which may be configured to facilitate mixing of the liquid droplet and the second fluid.

[0045] In embodiments, the outlet communicates with a third location, which may comprise an amplification vessel, and / or comprise a monolayer, and / or comprise an imaging chip.

[0046] It will be appreciated that any features of one aspect or embodiment of the invention may be combined with any combination of features in any other aspect or embodiment of the invention, unless otherwise stated, and such combinations are envisaged and are intended to be directly and unambiguously disclosed herein, and to fall within the scope of the present invention.

[0047] Brief Description of the Drawings

[0048] A better understanding of the nature and advantages of the embodiments of the present invention may be gained with reference to the following detailed description and the accompanying drawings. Those skilled in the art will appreciate that the invention described in the drawings is susceptible to variations and modifications other than those specifically described in the drawings. It is to be understood that the invention includes all such variations and modifications that fall within the scope of the present disclosure. The present invention is not to be limited in scope by the specific drawings described herein, which are intended for the purpose of exemplification only.

[0049] Figure 1 Overview of an embodiment of an emulsion droplet generation and dispensing apparatus according to the disclosure.

[0050] Figure 2 Cross sectional view of an embodiment of an apparatus for droplet generation and dispensation, depicting a side port connecting with an elongate body, configured for fluid communication with a main channel of the elongate body through an aperture in the wall of the elongate body.

[0051] Figure 3 Image of exemplary emulsion droplets generated by dispensing liquid droplets comprising a dark blue liquid into a stagnant body of a translucent oil.

[0052] Figure 4 A schematic of an embodiment of an emulsion droplet generation and dispensing apparatus.

[0053] Figure 5 Visualisation of second fluid flow simulations in the main channel of an embodiment of an emulsion droplet generation and dispensing apparatus.

[0054] Figure 6 Visualisation of liquid droplet trajectory into the second fluid.

[0055] Figure 7 Visualisation of liquid droplet trajectory from the pipettor tip.

[0056] Detailed Description of the Invention

[0057] All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, such as molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization.

[0058] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology, organic chemistry, and nucleic acid chemistry and hybridization, which are within the capabilities of a person of ordinary skill in the art. Standard techniques are used for nucleic acid synthesis.

[0059] The techniques and procedures are generally performed according to conventional methods in the art (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. 30 (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference).

[0060] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps and features referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only.

[0061] As used herein, the term "comprising" means any of the recited elements are necessarily included and other elements may optionally be included as well. "Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. "Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention. It should also be understood that the term "and / or" as used herein refers to and encompasses any or all possible combinations of one or more associated listed items.

[0062] Droplets, droplet formation, and partitions

[0063] In aspects of the invention, a method for generating emulsion droplets is provided, the method comprising: (a) forming at least one liquid droplet comprising a first fluid; (b) extruding the at least one liquid droplet from a first location; (c) passing the at least one liquid droplet through an airgap; and (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; and wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally (e) transferring the at least one emulsion droplet to a third location.

[0064] In aspects of the invention, a method for generating emulsion droplets in a stagnant body of liquid is provided, the method comprising: (a) forming at least one liquid droplet comprising a first fluid; (b) extruding the at least one liquid droplet from a first location; (c) passing the at least one liquid droplet through an air gap; and (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; wherein the second fluid is provided as a stagnant body; wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally (e) transferring the at least one emulsion droplet to a third location.

[0065] In aspects ofthe invention, a method for generating emulsion droplets in a stream of liquid is provided, the method comprising: (a) forming at least one liquid droplet comprising a first fluid; (b) extruding the at least one liquid droplet from a first location; (c) passing the at least one liquid droplet through an air gap; and (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; wherein the second fluid is provided as a stream; wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet, and (e) transferring the at least one emulsion droplet to a third location.

[0066] The term ‘emulsion droplet’ as used herein, refers to a droplet comprising an aqueous phase, comprising an aqueous fluid (e.g. water), and an organic phase, comprising an organic fluid (e.g. an oil), to form an emulsion. In embodiments, an emulsion droplet may be an aqueous droplet that is encapsulated by an organic fluid, such as an oil. In embodiments, a droplet may be an oil droplet that is encapsulated by an aqueous solution. In embodiments, the emulsion droplet may be formed by contacting an oil with an aqueous droplet comprising a sample and reaction components. In embodiments, the emulsion droplet may be formed by contacting an aqueous liquid with an oil droplet comprising a sample and reaction components.

[0067] The terms ‘first fluid’ and ‘second fluid’ as used herein, each refer to a fluid, defined by their immiscibility with each other. In embodiments wherein the second fluid is an organic fluid, the first fluid may be any suitable aqueous fluid which forms an aqueous phase in an organic fluid. In embodiments wherein the second fluid is an aqueous fluid, the first fluid may be an organic fluid which forms an organic phase in an aqueous fluid. In embodiments wherein the first fluid is an aqueous fluid, the second fluid may be an organic fluid which forms an aqueous phase in an aqueous fluid. In embodiments wherein the first fluid is an organic fluid, the second fluid may be an aqueous fluid which forms an aqueous phase in an organic fluid.

[0068] In embodiments, the first fluid is immiscible in the second fluid, and wherein the at least one liquid droplet is immersed in the second fluid, at least one emulsion droplet of the first fluid within the second fluid is thereby generated. In embodiments, the first fluid comprises an aqueous liquid, which may comprise water, and / or a reaction mixture, and / or a sample, wherein the sample may comprise at least one target oligonucleotide. In embodiments, the second fluid comprises an organic liquid, which may comprise an oil.

[0069] The term ‘liquid droplet’ as used herein, refers to a droplet comprising the first fluid. The first fluid and liquid droplet comprising the first fluid is immiscible with the second fluid, which means that the droplet and the second phase (e.g. an oil) do not mix to attain homogeneity. The liquid droplets are isolated from one another by the second phase and encapsulated (i.e. enclosed / surrounded) by the second fluid.

[0070] Forthe purposes of this invention, an ‘oil’ may be any liquid compound or mixture of liquid compounds that is immiscible with water and that has a high content of carbon. In embodiments, an oil may also have a high content of hydrogen, fluorine, silicon, oxygen, or any combination thereof, among others. The oil may comprise a fluorinated base oil which may additionally be stabilized by combination with a fluorinated surfactant such as a perfluorinated polyether. In embodiments, the base oil comprises one or more of a HFE 7500, FC-40, FC-43, FC-70, or another common fluorinated oil. In embodiments, the oil comprises an anionic fluorosurfactant. In embodiments, the anionic fluorosurfactant is ammonium Krytox (Krytox-AS), the ammonium salt of Krytox FSH, or a morpholino derivative of Krytox FSH. Krytox-AS may be present at a concentration of about 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 .0%, 2.0%, 3.0%, or 4.0% (w / w). In embodiments, the concentration of Krytox-AS is about 1 .8%. In embodiments, the concentration of Krytox-AS is about 1 .62%. Morpholino derivative of Krytox FSH may be present at a concentration of about 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w / w). In embodiments, the concentration of morpholino derivative of Krytox FSH is about 1 .8%. In embodiments, the concentration of morpholino derivative of Krytox FSH is about 1 .62%. In embodiments, the oil may further comprise an additive for tuning the oil properties, such as vapor pressure, viscosity, or surface tension. Non-limiting examples include perfluorooctanol and 1 H,1 H,2H,2H-perfluorodecanol. In embodiments, 1 H,1 H,2H,2H- Perfluorodecanol is added to a concentration of about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0% (w / w). In embodiments, 1 H,1 H,2H,2H-perfluorodecanol is added to a concentration of about 0.18% (w / w). In embodiments, the emulsion is formulated to produce highly monodisperse droplets having a liquid-like interfacial film that can be converted by heating into emulsion droplets having a solid-like interfacial film; such emulsion droplets may behave as bioreactors able to retain their contents through an incubation period (see, e.g., U.S. Patent No. 10,378,048, which is incorporated herein in its entirety). The conversion to microcapsule form may occur upon heating. For example, such conversion may occur at a temperature of greater than about 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 95°C. During the heating process, a fluid or mineral oil overlay may be used to prevent evaporation. Excess continuous phase oil may or may not be removed prior to heating. The biocompatible capsules may be resistant to coalescence and / or flocculation across a wide range of thermal and mechanical processing. Following conversion, the emulsion droplets may be stored at about -70°C, -20°C, 0°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C.

[0071] In embodiments, the oil has a density of between about 0.2 to 3 g / mL, between about 0.4 to about 2.5 g / mL, between about 0.6 to about 2 g / mL, or between about 0.8 to about 1 .63 g / mL. In embodiments, the oil has a viscosity of between about 0.2 to 7 mPa-s, between about 0.4 to about 6.5 mPa-s, between about 0.6 to about 6 mPa-s, between about 0.8 to about 5.5, or between about 1 .0 and about 5.0 mPa-s.

[0072] In embodiments, the method comprises extrusion of the liquid droplet from the pipettor; optionally wherein the liquid droplet is extruded from a nozzle tip of the pipettor. In embodiments, the first location from which the at least one liquid droplet is extruded is spaced vertically above the second location.

[0073] In embodiments, the pipettor has a frequency of liquid droplet drive, wherein the frequency of liquid droplet drive is between about 500 to about 100,000 droplets per second, between about 1 ,000 to about 50,000 droplets per second, between about 2,000 to about 25,000 droplets per second, between about 3,000 to about 15,000 droplets per second, or between about 4,000 to about 7,500 droplets per second, e.g. about 5,000 droplets per second. In embodiments, the liquid droplet has an initial velocity of between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s.

[0074] Liquid droplets that are generated by the pipettor and / or pipettor nozzle may be substantially uniform in shape and / or size. For example, in embodiments, the liquid droplets are substantially uniform in average diameter. In embodiments, the liquid droplets that are generated have an average diameter of about 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05 microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5 microns, about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 300 microns, about 400 microns, about 500 microns, about 600 microns, about 700 microns, about 800 microns, about 900 microns, or about 1 ,000 microns. In embodiments, the liquid droplets that are generated have an average diameter of less than about 1 ,000 microns, less than about 900 microns, less than about 800 microns, less than about 700 microns, less than about 600 microns, less than about 500 microns, less than about 400 microns, less than about 300 microns, less than about 200 microns, less than about 100 microns, less than about 50 microns, or less than about 25 microns.

[0075] In embodiments, the diameter of the liquid droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, between about 30 to about 50 pm, e.g. about 40 pm.

[0076] In embodiments, the liquid droplets that are generated may be non-uniform in shape and / or size. Generated liquid droplets may, for example, vary in size by a standard deviation that is less than about plus or minus 100%, 50%, 20%, 10%, 5%, 2%, or 1 % of the average droplet size.

[0077] In embodiments, the liquid droplets that are generated are substantially uniform in volume. For example, in embodiments, the liquid droplets that are generated have an average volume of about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about

[0078] 1 .5 nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about

[0079] 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about

[0080] 9.5 nL, about 10 nL, about 1 1 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL.

[0081] In embodiments, the volume of the liquid droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL. In embodiments, the liquid droplets that are generated may be non-uniform in volume. Generated liquid droplets may, for example, vary in volume by a standard deviation that is less than about plus or minus 100%, 50%, 20%, 10%, 5%, 2%, or 1 % of the average volume size.

[0082] In embodiments, the at least one liquid droplet is extruded from a nozzle tip of a pipettor at the first location, wherein the pipettor is defined according to an apparatus of the invention.

[0083] In embodiments, the liquid droplet has a direction of travel, and the second fluid may have a direction of travel, optionally wherein the second fluid is provided as a continuous stream, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect at the second location. In embodiments, the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect in the main channel of the apparatus according to an apparatus of the invention. In embodiments, the second location comprises the intersection between the direction of travel of the liquid droplet and the direction of travel of the second fluid in the main channel of an apparatus defined according to the invention. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the direction of travel of the liquid droplet and the direction of travel of the second fluid.

[0084] In embodiments, the direction of travel of the liquid droplet and the direction of travel of the second fluid intersects at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect at an angle of between about 10° and 90°, between about 1 1 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0085] In embodiments, the direction of travel of the liquid droplet may be against the direction of travel of the second fluid.

[0086] In embodiments, the direction of travel of the liquid droplet and the second fluid intersect, optionally wherein the second fluid is provided as a stagnant body, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the direction of travel of the liquid droplet and the second fluid. In embodiments, the second location comprises the intersection between the direction of travel of the liquid droplet and the second fluid. In embodiments, the direction of travel of the liquid droplet and the second fluid intersect at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0087] In embodiments, the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv), and the second fluid has a speed and direction of travel defining a second velocity vector (sv). In embodiments, the first velocity vector (Iv) has a speed between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s. In embodiments, the second velocity vector (sv) has a speed between about 0.05 to about 3 m / s, between about 0.1 to about 2 m / s, between about 0.15 to about 1 .5 m / s, between about 0.2 to about 1 m / s, or between about 0.25 to about 0.75 m / s., e.g. about 0.6 m / s.

[0088] In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect, optionally wherein the second fluid is provided as a continuous stream. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv), wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect in the main channel of an apparatus according to the invention. In embodiments, the second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv) in the main channel of an apparatus defined according to the invention.

[0089] In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 175°, between about 1 1 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0090] In embodiments, the direction of travel of the first velocity vector (Iv) may be against the direction of travel of the second velocity vector (sv).

[0091] In embodiments, the first velocity vector (Iv) and the second fluid intersect, optionally wherein the second fluid is provided as a stagnant body. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the first velocity vector (Iv) and the second fluid. In embodiments, wherein the second location comprises the intersection between the first velocity vector (Iv) and the second fluid. In embodiments, the first velocity vector (Iv) and the second fluid intersect at an angle of between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0092] In embodiments, the second fluid may be provided as a continuous stream, wherein the second location is at a surface of the continuous stream of the second fluid. In embodiments, the second fluid may be provided as a stagnant body, wherein the second location is at a surface of the stagnant body of the second liquid. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises contacting the at least one liquid droplet with the continuous stream of the second fluid, or contacting the at least one liquid droplet with the stagnant body of the second liquid. In embodiments, the continuous stream of the second fluid has a flow rate of between about 0.5 to about 100 pL / min, between about 1 to about 50 pL / min, between about 1 .5 to about 20 pL / min, between about 1 .75 to about 15 pL / min or between about 2 to about 10 pL / min.

[0093] In embodiments, transferring the at least one emulsion droplet to a third location comprises transferring the at least one emulsion droplet from the second location to the outlet via a region of the main channel, and from the outlet to the third location by the continuous stream of second fluid. In embodiments, the third location comprises an amplification vessel, and / or comprises a monolayer, and / or comprises an imaging chip.

[0094] In embodiments, the at least one emulsion droplet comprises a partitioned sample, and / or comprises a reaction mixture.

[0095] Methods and compositions for delivering reagents to one or more partitions include microfluidic methods as known in the art; droplet or microcapsule merging, coalescing, fusing, bursting, or degrading (e.g., as described in U.S. 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151 ,776); and combinations thereof, each of which is incorporated in its entirety by reference herein. In embodiments, the components of a reaction mixture may be combined by the methods described above.

[0096] In embodiments, the emulsion droplets may be relatively stable and have minimal coalescence between two or more droplets. In embodiments, less than 0.0001 %, 0.0005%, 0.001 %, 0.005%, 0.01 %, 0.05%, 0.1 %, 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of droplets generated from a sample coalesce with other droplets. The emulsions can also have limited flocculation, a process by which the dispersed phase, comes out of suspension in aggregates. Methods of emulsion formation are described, for example, in published patent applications WO 201 1 / 109546 and WO 2012 / 061444, each of which is incorporated in its entirety by reference herein.

[0097] Accordingly, the emulsion droplets can be incubated at a very high density (e.g., number of partitions per unit volume). In embodiments, greater than 100,000, 500,000, 1 ,000,000, 1 ,500,000, 2,000,000, 2,500,000, 5,000,000, 10,000,000, 20,000,000, 30,000,000 or40,000,000 partitions may be incubated per mL. In embodiments, the number of partitions that may be incubated per mL is between about 1 ,000,000 to about 50,000,000, between about 10,000,000 to about 40,000,000, between about 15,000,000 to about 30,000,000, or between about 17,000,000 to about 20,000,000, e.g. about 17,500,000.

[0098] In some embodiments, a sample-probe incubation may occur in a single well, e.g., a well of a microtiter plate, without inter-mixing between partitions. The emulsion droplets may also contain other components necessary for the incubation. In embodiments, a sample is partitioned into at least 500 partitions, at least 1 ,000 partitions, at least 2,000 partitions, at least 3,000 partitions, at least 4,000 partitions, at least 5,000 partitions, at least 6,000 partitions, at least 7000 partitions, at least 8000 partitions, at least 10,000 partitions, at least 15,000 partitions, at least 20,000 partitions, at least 30,000 partitions, at least 40,000 partitions, at least 50,000 partitions, at least 60,000 partitions, at least 70,000 partitions, at least 80,000 partitions, at least 90,000 partitions, at least 100,000 partitions, at least 200,000 partitions, at least 300,000 partitions, at least 400,000 partitions, at least 500,000 partitions, at least 600,000 partitions, at least 700,000 partitions, at least 800,000 partitions, at least 900,000 partitions, at least 1 ,000,000 partitions, at least 2,000,000 partitions, at least 3,000,000 partitions, at least 4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000 partitions, at least 20,000,000 partitions, at least 30,000,000 partitions, at least 40,000,000 partitions, at least 50,000,000 partitions, at least 60,000,000 partitions, at least 70,000,000 partitions, at least 80,000,000 partitions, at least 90,000,000 partitions, at least 100,000,000 partitions, at least 150,000,000 partitions, or at least 200,000,000 partitions.

[0099] The emulsion droplets may resist coalescence, particularly at high temperatures. Any emulsion disclosed herein may be a heat-stable emulsion. A heat-stable emulsion may be any emulsion that resists coalescence when heated to at least 50 °C. A heat-stable emulsion may be a PCR-stable emulsion, which may be an emulsion that resists coalescence throughout the thermal cycling of PCR (e.g., to permit performance of digital PCR). Accordingly, a PCR-stable emulsion may be resistant to coalescence when heated to at least 80 °C or 90 °C, among others. Due to heat stability, a PCR- stable emulsion, in contrast to a standard emulsion, enables PCR assays to be performed in droplets that remain substantially monodisperse throughout thermal cycling. Beneficially, digital PCR assays with PCR-stable emulsions may be substantially more quantitative than with standard emulsions. An emulsion may be formulated as PCR stable by, for example, proper selection of carrier fluid and surfactants, among others. An exemplary oil formulation to generate PCR-stable emulsions for flow- through assays may comprise the following reagents: Dow Corning 5225C Formulation Aid (10% active ingredient in decamethylcyclopentasiloxane) - 20% w / w, 2% w / w final concentration active ingredient, Dow Corning 749 Fluid (50% active ingredient in decamethylcyclopentasiloxane) - 5% w / w, 2.5% w / w active ingredient, and Poly(dimethylsiloxane) Dow Corning 200® fluid, viscosity 5.0 cSt (25 °C) - 75% w / w. An exemplary oil formulation to generate PCR-stable emulsions for batch assays may comprise the following reagents: Dow Corning 5225C Formulation Aid (10% active ingredient in decamethylcyclopentasiloxane) - 20% w / w, 2% w / w final concentration active ingredient, Dow Corning 749 Fluid (50% active ingredient in decamethylcyclopentasiloxane) - 60% w / w, 30% w / w active ingredient, and Poly(dimethylsiloxane) Dow Corning 200® fluid, viscosity 5.0 cSt (25 °C) - 20% w / w. Emulsion droplets that are generated by the pipettor and / or pipettor nozzle may be substantially uniform in shape and / or size. For example, in embodiments, the emulsion droplets are substantially uniform in average diameter. In embodiments, the emulsion droplets that are generated have an average diameter of about 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05 microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5 microns, about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 300 microns, about 400 microns, about 500 microns, about 600 microns, about 700 microns, about 800 microns, about 900 microns, or about 1 ,000 microns. In embodiments, the emulsion droplets that are generated have an average diameter of less than about 1 ,000 microns, less than about 900 microns, less than about 800 microns, less than about 700 microns, less than about 600 microns, less than about 500 microns, less than about 400 microns, less than about 300 microns, less than about 200 microns, less than about 100 microns, less than about 50 microns, or less than about 25 microns.

[0100] In embodiments, the diameter of the emulsion droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, between about 30 to about 50 pm, e.g. about 40 pm.

[0101] In embodiments, the emulsion droplets that are generated may be non-uniform in shape and / or size. Generated emulsion droplets may, for example, vary in size by a standard deviation that is less than about plus or minus 100%, 50%, 20%, 10%, 5%, 2%, or 1 % of the average droplet size.

[0102] In embodiments, the emulsion droplets that are generated are substantially uniform in volume. For example, in embodiments, the emulsion droplets that are generated have an average volume of about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about

[0103] 1 .5 nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about

[0104] 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about

[0105] 9.5 nL, about 10 nL, about 1 1 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL.

[0106] In embodiments, the volume of the emulsion droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL. In embodiments, the emulsion droplets that are generated may be non-uniform in volume. Generated emulsion droplets may, for example, vary in volume by a standard deviation that is less than about plus or minus 100%, 50%, 20%, 10%, 5%, 2%, or 1 % of the average volume size.

[0107] Suitable samples and target oligonucleotides

[0108] In embodiments, the first fluid comprises a sample. In embodiments, the sample comprises at least one target oligonucleotide. In embodiments, the methods and systems disclosed herein may be performed on any suitable oligonucleotide derived from a sample wherein a target oligonucleotide to be analysed is present. The disclosed methods and systems may be carried out on any one or more suitable oligonucleotides derived from samples that are known to contain or suspected to contain one or more target oligonucleotides. Alternatively, the invention may be carried out on one or more suitable oligonucleotides derived from samples to confirm the presence of one or more target oligonucleotides whose presence in the sample is unknown.

[0109] The term ‘oligonucleotide’ as used herein, is a macromolecule comprising at least two or more covalently-linked nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds, such as a nucleic acid. Oligonucleotides that may be applied are as follows. The oligonucleotide may be natural or synthetic. For the purposes of the present invention such oligonucleotides may constitute nucleic acids. Typical nucleic acids include DNA and RNA. The oligonucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. An oligonucleotide may be linear and single stranded and may be derived from an oligonucleotide in linear or circular conformation, and in either single- or double-stranded conformation, or comprise one or more double stranded regions and one or more single regions. The terms ‘oligonucleotide’, and ‘polynucleotide’ are used interchangeably. Oligonucleotides are comprised of nucleotides.

[0110] The term ‘nucleotide’ as used herein, typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases include but are not limited to purines and pyrimidines and more specifically include but are not limited to, adenine, guanine, thymine, uracil and cytosine. The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide. For the purposes of this invention, the terms ‘nucleotide’ and ‘base’ are used interchangeably.

[0111] The oligonucleotide may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in any oligonucleotide of the invention may be modified, comprising one or more chemical modifications of a nucleotide. Modified nucleotides may occur singularly or in a plurality with other nucleotide modifications that are the same modification or a different modification, which may be contiguous or non-contiguous with other nucleotide modifications that are the same modification or a different modification. One or more nucleotides in any oligonucleotide of the invention may be non-natural or synthetic nucleotides. Non-natural nucleotides may include analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (e.g. phosphorothioate backbones). The oligonucleotides of the invention may also comprise any synthetic nucleic acid known in the art, including but not limited to peptide nucleic acids (PNA), glycerol nucleic acids (GNA), threose nucleic acids (TNA), locked nucleic acids (LNA) or other synthetic polymers with nucleotide side chains. One or more nucleotides in the oligonucleotides may be modified with a label or a tag. The oligonucleotides may comprise one or more spacers. Modifications can also include 3' and 5' modifications including but not limited to capping with a fluorophore (e.g., quantum dot) or another moiety.

[0112] The term ‘target oligonucleotide’ refers to any oligonucleotide originating from a sample suitable for use in the method. In embodiments, a target oligonucleotide may be derived from single stranded oligonucleotides, or a double stranded oligonucleotide, and may be linear or circular. In embodiments, the target oligonucleotide may be comprised of DNA, cDNA, or RNA, wherein the RNA may comprise a poly(A) tail such as an mRNA and / or may be a ncRNA such as a microRNA (miRNA), a small interfering RNA (siRNAs), or a piwi-interacting RNAs (piRNAs).

[0113] The target oligonucleotide can be any length, wherein the length corresponds to the number of nucleotides constituting an oligonucleotide. In embodiments, the target oligonucleotide has a length of about 25, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1 ,000, or about 1 ,500 nucleotides. In embodiments, the target single stranded oligonucleotide has a length between about 25 and 1 ,500, between about 50 and 500, between about 50 and 300, or between about 100 and 200 nucleotides.

[0114] The sample from which an oligonucleotide is derived may be a biological sample. The methods and systems disclosed herein may be carried out on an oligonucleotide derived from a sample obtained from or extracted from any organism. The organism or microorganism may be prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera, and protista. The methods and systems disclosed herein may be carried out on an oligonucleotide derived from a sample obtained from or extracted from any virus.

[0115] The sample is typically a fluid sample. Alternatively, where the sample is solid or semi-solid in origin, it may subsequently be treated to provide a fluid sample. Examples of such samples are faecal, skin, tissue, hair, bone and muscle. The sample may comprise a body fluid of a patient. The sample may be chosen for example from urine, blood, plasma, serum, lymph, saliva, interstitial fluid, tears, mucus or amniotic fluid. Typically, the sample is human in origin, but alternatively it may be from another animal, particularly a mammal, such as a mouse model organism, or from commercially farmed animals, such as horses, cattle, sheep, or pigs; or may alternatively be from pets such as cats or dogs. The sample may be a non-biological sample. The non-biological sample may be a fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and industrial samples such as reagents for laboratory tests, or samples obtained from the synthesis of a polymer reagent.

[0116] The term ‘expression’ as used herein relates to the process by which the information encoded in a sequence of nucleotides which may comprise a gene or a pseudogene is transcribed to produce functional gene products. Expression of a given gene may be detectable using techniques such as qPCR or dPCR, wherein the expression of a gene may manifest in the number of detected molecules or partitions which contain a target oligonucleotide originating from a given RNA produced by transcription of a gene or pseudogene.

[0117] Reactions and reaction mixtures

[0118] In embodiments, the first fluid comprises a reaction mixture. In embodiments, the methods and systems disclosed herein are suitable for performing one or more reactions comprising a reaction mixture, wherein the reactions may comprise a droplet-based assay.

[0119] Forthe purposes of this invention, a ‘reaction’ may referto any chemical reaction, wherein any suitable reaction may be performed in a droplet-based assay as disclosed herein e.g. a binding interaction, a phenotypic change, or a combination thereof, which generally provides a detectable signal (e.g., a fluorescence signal) indicating occurrence and / or an extent of occurrence of the reaction. An exemplary reaction is an enzyme reaction that involves an enzyme-catalysed conversion of a substrate to a product. For example, the reactions may be catalyzed by a kinase, nuclease, nucleotide cyclase, nucleotide ligase, nucleotide phosphodiesterase, polymerase (DNA or RNA), prenyl transferase, pyrophospatase, reporter enzyme (e.g., alkaline phosphatase, beta-galactosidase, chloramphenicol acetyl transferse, glucuronidase, horse radish peroxidase, luciferase, etc.), reverse transcriptase, topoisomerase, etc.

[0120] The term ‘amplification reaction’ for the purposes of this invention refers to any in vitro method for multiplying the copies of a target nucleic acid sequence in a linear or non-linear manner, such as an exponential manner, producing at least one or more amplicons.

[0121] The term ‘amplicon’ forthe purposes of this invention refers to an oligonucleotide product synthesised during an amplification reaction which corresponds to any suitable segment or entire length of any target oligonucleotide. An amplicon may be single- stranded or double-stranded, or a combination thereof.

[0122] Amplification reaction methods include but are not limited to two-primer methods such as polymerase chain reaction (PCR); ligase methods such as DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et at., eds, 1990)) (LCR); QBeta, RNA replicase and RNA transcription-based amplification reactions (e.g., amplification that involves T7, T3, or 5P6 primed RNA polymerization), such as the transcription amplification system (TAS), nucleic acid sequence based amplification (NASBA), and self-sustained sequence replication (3 SR), as well as others known to those of skill in the art. An amplification reaction may also comprise an isothermal amplification reaction, such as single-primer isothermal amplification (SPIA), branched-probe DNA assays, cascade-RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nicking enzyme amplification reaction (NEAR), PAN- AC, Q-beta replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, strand-displacement amplification, as well as others known to those of skill in the art. An amplification reaction may utilise a linear or circular template. For the purposes of this invention an amplification reaction may comprise a droplet-based assay.

[0123] For the purposes of this invention, ‘PCR’ may refer to an amplification reaction comprising alternating cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication, resulting in a generally exponential increase in the number of amplicons generated over successive thermal cycles. PCR may be performed by thermal cycling between two or more temperature set points, such as a higher melting (denaturation) temperature and a lower annealing / extension temperature, or among three or more temperature set points, such as a higher melting temperature, a lower annealing temperature, and an intermediate extension temperature, among others.

[0124] Components of an amplification reaction include, but are not limited to: primers, an oligonucleotide template, a polymerase, and nucleotides.

[0125] A ‘primer’ refers to an oligonucleotide sequence that anneals / hybridizes to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths, typically less than 60 nucleotides in length, for example between about 12 and 35 nucleotides in length. The length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art, see, e.g., Innis et al., eds, 1990. Primers can be DNA, RNA, or a chimera of DNA and RNA portions. In some cases, primers can include one or more modified or nonnatural nucleotide bases. In some cases, primers are labelled.

[0126] As used herein, the term ‘oligonucleotide probe’ refers to an oligonucleotide conjugated to a moiety that may emit a detectable signal under desired circumstances. For example, the probe moiety may change signal status depending on whether the probe anneals to a target nucleic acid. The oligonucleotide probe may further be extended during an amplification reaction such as PCR, as described herein. Suitable probes include fluorescent probes, such as Taqman™ probes. Such probes may require hydrolysis by a 5' to 3' exonuclease activity of a DNA polymerase to separate a fluorescent moiety of the probe from a quenching moiety in the structure of the probe, in order to allow generation of a fluorescent signal during amplification. Any suitable probe may be used. For example, suitable fluorescent probes include molecular beacons, which comprise a stem-loop structure increasing the binding specificity of the probe to its target. During an annealing step of an amplification reaction, the loop portion of the molecular beacon probe may anneal to a target sequence, thereby denaturing the stem and separating a fluorescent moiety from a quenching moiety, resulting in detectable fluorescence from the fluorescent moiety.

[0127] Fluorophores or fluorescent agents can include a variety of organic and / or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof. Fluorophores and quenchers are reported in the literature and thus known to those skilled in the art, and many are readily available from commercial suppliers to the biotechnology industry. Literature sources for fluorophores include Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988); Dexter, D.L., J. of Chemical Physics 21 : 836- 850 (1953); Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40: 83- 114 (1971); Stryer, L. Ann. Rev. Biochem., 47: 819-846 (1978); Wang et al., Tetrahedron Letters 31 : 6493-6496 (1990); Wang et al., Anal. Chem. 67: 1197-1203 (1995), which are hereby incorporated by reference in their entirety. Non-limiting examples of fluorophores include cyanines, fluoresceins (e.g., 5' carboxyfluorescein (FAM), Oregon Green, and Alexa 488), HEX, rhodamines (e.g., N,N,N',N- tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC)), eosin, coumarins, pyrenes, tetra pyrroles, arylmethines, oxazines, polymer dots, and quantum dots. In embodiments, the probe may comprise a blocked 3' end preventing a polymerase from extending the annealed probe.

[0128] A ‘polymerase’ refers to an enzyme that performs template-directed synthesis of oligonucleotides. The term encompasses both the full-length polypeptide and a domain that has polymerase activity. DNA polymerases are well-known to those skilled in the art. DNA polymerases for use in the compositions and methods disclosed herein can be any polymerase capable of replicating a DNA molecule. The DNA polymerase may be a thermostable polymerase. Thermostable DNA polymerases are known in the art and are commercially available. The DNA polymerase may be Taq, Tbr, Tfl, Tru, Tth, Tij, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENTTm, DEEPVENTTm, or an active mutant, variant, or derivative thereof, as known in the art. In aspects and embodiments, the DNA polymerase may be Taq DNA polymerase. In other aspects and embodiments, the DNA polymerase may be a high-fidelity DNA polymerase (e.g., iProofrm High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA polymerase, Q5 High-Fidelity DNA polymerase, Platinum® Taq High Fidelity DNA polymerase, Accura® High-Fidelity Polymerase). The DNA polymerase may be a faststart or hot-start polymerase. Polymerases also include a family of polymerases known as ‘reverse transcriptases’, which are typically used to synthesise the complementary strand of a template RNA oligonucleotide, but can also synthesise the complementary strand of a DNA or cDNA oligonucleotide (see Gerard G. F., and D' Alessio J. M., Chapter 6 (73-93), Methods in Molecular Biology, Vol.16: Enzymes of Molecular Biology Edited by: M. M. Burell 1993 Humana Press Inc. Totowa, NJ). Reverse transcriptases are well-known to those skilled in the art. In embodiments, suitable reverse transcriptases for use in the methods, systems and compositions disclosed herein can be any polymerase capable of synthesising a complementary strand. In embodiments, the reverse transcriptase exhibits terminal nucleotide transferase activity, which comprises the non-templated addition of one or more terminal nucleotides to the 3’ end of a reverse transcription synthesised oligonucleotide sequence complementary to a template sequence.

[0129] Reaction characteristics of an amplification reaction, such as amplification rate, or reaction outputs, such as a detectable number of molecules, may be detected and measured. These amplification methods may comprise a detection reagent or a detectable label which can be detected using any of a variety of detector devices. Exemplary detection methods include optical detection (e.g., fluorescence, or chemiluminescence) as well as others known to those of skill in the art. As a nonlimiting example, a fluorescent label can be detected using a detector device equipped with a module to generate excitation light that can be absorbed by a fluorophore, as well as a module to detect light emitted by the fluorophore.

[0130] The term ‘amplification vessel’ for the purposes of this invention refers to any vessel suitable for use in an amplification reaction known in the art, which may have high thermal conductivity, reduced thermal mass, maximum surface area to volume area and thin outer walls, and wherein the amplification reaction is constructed to survive many repeated cycles of thermocycling. In embodiments, the amplification vessel may comprise a monolayer. In embodiments, the amplification vessel may comprise an imaging chip.

[0131] Any suitable PCR methodology or combination of methodologies may be utilized in the droplet-based assays disclosed herein, such as allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, endpoint PCR, hot-start PCR, in situ PCR, intersequence-specific PCR, inverse PCR, linear after exponential PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap- extension PCR, polymerase cycling assembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermal asymmetric interlaced PCR, touchdown PCR, or universal fast walking PCR, among others.

[0132] In embodiments, the amplification reaction may comprise a dPCR reaction. For the purposes of this invention, a dPCR reaction may be performed on portions of a sample to determine the presence / absence, concentration, and / or copy number of a nucleic acid target in the sample, based on how many of the sample portions support amplification of the target. dPCR may be performed as endpoint PCR, wherein amplicon formation is measured afterthe completion of thermal cycling. dPCR may be performed as real-time PCR for each of the partitions, in which amplicon formation may be measured during the reaction, for example after completion of one or more thermal cycles prior to the final thermal cycle of the reaction. In embodiments, digital PCR may qualitative, performed by determining whether a packet of droplets contains at least a predefined percentage of positive droplets (a positive sample) or not (a negative sample).

[0133] In embodiments, dPCR may be performed in droplets, comprising droplet digital PCR (ddPCR). Methods for performing PCR in droplets are described, for example, in US 2014 / 0162266, US 2014 / 0302503, and US 2015 / 0031034, the contents of each of which is incorporated by reference. In embodiments, the QX200, QX600, or QX One Droplet Digital PCR (ddPCR) System (Bio-Rad) may be used.

[0134] In embodiments, dPCR may be extended to other types of analytes, besides nucleic acids. In particular, a signal amplification reaction may be utilized to permit detection of a single copy of a molecule of the analyte in individual droplets, to permit data analysis of droplet signals for other analytes in the manner described in Section VII (e.g., using an algorithm based on Poisson statistics). Exemplary signal amplification reactions that permit detection of single copies of other types of analytes in droplets include enzyme reactions.

[0135] An amplification reaction may be performed, or tested for its occurrence, in a reaction mixture. For the purposes of this invention, the term ‘reaction mixture’ refers to any aqueous composition capable of generating multiple copies of a target oligonucleotide, if present, in the composition during an amplification reaction. In embodiments, the reaction mixture may comprise any combination of at least one primer or primer pair, at least one probe, at least one replication enzyme (e.g., at least one polymerase, such as at least one DNA and / or RNA polymerase), and nucleotides (e.g. dNTPs and / or NTPs).

[0136] The reaction mixture may comprise, but is not limited to, enzymes, aqueous buffers, salts, and nucleoside triphosphates. The reaction mixture may comprise nucleotides. Nucleotides for use in the method described herein can be any nucleotide useful in the polymerization of a nucleic acid. Nucleotides can be naturally occurring, modified, derivative, or artificial. In embodiments, nucleotides will typically be unlabelled. In embodiments, the reaction mixture may comprise one or more buffers or salts which optimise reaction efficiency and specificity. A wide variety of buffers and salt solutions and modified buffers are known in the art. For example, in embodiments, the buffer is TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, or CAPS. In embodiments, the salt is potassium acetate, potassium sulfate, potassium chloride, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, or lithium acetate. In embodiments, the reaction mixture may comprise a salt (e.g., potassium chloride) at a concentration of about 10 mM to about 100 mM.

[0137] The reaction mixture may comprise one or more stabilizers to optimize reaction efficiency and specificity. Stabilizers for use in the methods described herein include, but are not limited to, a polyol (glycerol, threitol, etc.), a polyether including cyclic polyethers, polyethylene glycol, organic or inorganic salts, such as ammonium sulfate, sodium sulfate, sodium molybdate, sodium tungstate, organic sulfonate, etc., sugars, polyalcohols, amino acids, peptides or carboxylic acids, a quencher and / or scavenger such, as mannitol, glycerol, reduced glutathione, superoxide dismutase, bovine serum albumin (BSA) or gelatine, spermidine, dithiothreitol (or mercaptoethanol) and / or detergents such as TRITON® X-100 [Octophenol(ethyleneglycolether)], THESIT® [Polyoxyethylene 9 lauryl ether (Polidocanol C12 E9)], TWEEN® (Polyoxyethylenesorbitan monolaurate 20, NP40) and BRIJO-35 (Polyoxyethylene 23 lauryl ether).

[0138] In embodiments, the reaction mixture comprises reagents and oligonucleotides suitable for an amplification reaction. In embodiments, the reaction mixture comprises any combination of: at least one oligonucleotide, and / or at least one amplification primer, and / or at least one enzyme, optionally wherein the enzyme is a polymerase. In embodiments, the reaction mixture comprises reagents and oligonucleotides suitable for the preparation of target oligonucleotides for an amplification reaction.

[0139] The term ‘preparation of oligonucleotides for an amplification reaction’ for the purposes of this invention relates to any suitable process or reaction for producing oligonucleotides suitable for an amplification reaction, which may comprise, but is not limited to, nucleotide extraction, reverse transcription, template switching reactions, second strand synthesis, ligation, end repair, and the like.

[0140] Once formed, the reaction mixture may be submitted to conditions to allow generation of a suitable target single stranded oligonucleotide, such as a denaturing step. The reaction mixture may be submitted to conditions for primer annealing, such as a first oligonucleotide or second oligonucleotide, and extension using one or more primers that anneal to a target oligonucleotide to be detected. The reaction mixture may be submitted to primer extension conditions, which can be, but is not limited to, conditions suitable for PCR, allowing a primer to anneal to the target oligonucleotide or prepared oligonucleotide, if present, and be extended by a polymerase in a template specific manner, which may be repeated for one or more cycles. The amplification reaction can be monitored for oligonucleotide probe signals at an endpoint or in real time, i.e., continuously or every cycle. In embodiments, an emulsion droplet may comprise a reaction mixture, or may comprise a partial reaction mixture comprising some but not all of the necessary reagents and components required to perform the method, or may be empty.

[0141] Apparatus for generating and dispensing emulsion droplets

[0142] For the purposes of this invention the term ‘channel’ refers to a passage for fluid travel. A channel generally includes at least one inlet, where fluid enters the channel, and at least one outlet, where fluid exits the channel. A channel may include walls that define and enclose the passage between the inlet and the outlet. A channel may, for example, be formed by a tubular body (e.g., a capillary tube), in or on a planar structure (e.g., a chip), or a combination thereof, among others. A channel may branch. A channel may be linear or nonlinear. Exemplary nonlinear channels include a channel extending along a planar flow path (e.g., a serpentine channel) a nonplanar flow path (e.g., a helical channel to provide a helical flow path). Any of the channels disclosed herein may be a microfluidic channel, which is a channel having a characteristic transverse dimension (e.g., the channel's average diameter) of less than about one millimeter. Channels also may include one or more venting mechanisms to allow fluid to enter / exit without the need for an open outlet. Examples of venting mechanisms include but are not limited to hydrophobic vent openings or the use of porous materials to either make up a portion of the channel or to block an outlet if present. A channel may or may not be elongate.

[0143] For the purposes of this invention the term ‘manifold’ refers to an assembly for manipulating fluid, generally by transferring fluid between compartments of the assembly and / or by driving flow of fluid along and / or through one or more flow paths defined by the assembly. A fluidics network may include any suitable structure, such as one or more channels, chambers, reservoirs, valves, pumps, thermal control devices (e.g., heaters / coolers), sensors (e.g., for measuring temperature, pressure, flow, etc.), or any combination thereof, among others.

[0144] In aspects of the invention, an apparatus for generating emulsion droplets is provided, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body; and a pipettor comprising a nozzle having a nozzle tip arranged within the side port, the nozzle tip positioned at a first location spaced from the main channel of the manifold, and wherein the nozzle of the pipettor is arranged at an angle relative to the elongate body.

[0145] In embodiments, an apparatus of the invention may further comprise a pipettor comprising a nozzle having a nozzle tip, and wherein the nozzle tip is located within the side port.

[0146] In embodiments, an apparatus of the invention may further comprise a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, wherein the nozzle tip of the pipettor is located within the side port.

[0147] In embodiments, wherein the pipettor is configured to dispense a liquid droplet from the nozzle tip, wherein the nozzle tip is separated from the main channel by a distance ‘E’, wherein distance E defines an air gap between the channel and nozzle tip, wherein the distance E may be a vertical distance. In embodiments, the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, or between about 1 mm to about 5 mm.

[0148] In embodiments, the distance E has a length of at least about 1.0 mm, about 1.1 mm, 1 .2 mm, 1.3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm. In embodiments, an apparatus of the invention may further comprise a pump to drive the flow of the second fluid, wherein the pump pushes the second fluid through the manifold or draws the second fluid through the manifold.

[0149] In embodiments, the main channel may be substantially linear and has an elongate axis (ex) defined between the inlet and the outlet. In embodiments, the pipettor has a proximal end comprising the nozzle and the nozzle tip, and a distal end, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0150] In embodiments, the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body, wherein the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port. In embodiments, the side port and the elongate body of the manifold intersect at an angle, wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the proximal end portion of the main channel and the side port is between about 10° and 175°, between about 1 1 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the proximal end portion of the main channel and the side port is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°. In embodiments the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0151] In embodiments, the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex) intersect.

[0152] In embodiments, the pipettor is directed axially-downwardly, wherein downwardly relates to a vertical direction, and comprises a piezoelectric membrane. In embodiments, the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

[0153] In embodiments, the main channel comprises a channel region of reduced diameter and / or cross- sectional area, wherein the aperture in the wall of the elongate body may directly communicate with the channel region of reduced diameter and / or cross-sectional area. In embodiments, the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel extending proximal and distal of the aperture in the wall of the elongate body at which the side port communicates. In embodiments, the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the distal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port. In embodiments, the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the proximal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port. In embodiments, the channel region of reduced diameter and / or cross- sectional area has a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm. In embodiments, the channel region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm.

[0154] In embodiments, the main channel comprises a channel region of increased diameter and / or cross- sectional area, wherein the channel region of increased diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800 pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm. In embodiments, the channel region of increased diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, between about 150 to about 400 mm, or between about 200 to about 300 mm.

[0155] In embodiments, the main channel has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400 mm. In embodiments, the main channel comprises one or more stream flow diverters.

[0156] In embodiments, the main channel comprises one or more narrowing channel regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more narrowing channel regions is gradually decreased relative to an adjacent region of the main channel. In embodiments, a narrowing channel region has a tapering cross-sectional diameter which connects to a narrowed channel region, wherein the one or more narrowing channel regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0157] In embodiments, the channel comprises one or more widening channel regions of increasing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more widening channel regions is gradually increased relative to an adjacent region of the channel. In embodiments, a widening channel region has an inverse-tapering cross-sectional diameter which connects to a widened channel region, wherein the one or more widening channel regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0158] In embodiments, the main channel outlet communicates with a third location, wherein the third location may comprise an amplification vessel, and / or comprises a monolayer, and / or comprises an imaging chip.

[0159] In aspects of the invention, an apparatus for generating emulsion droplets is provided, the apparatus comprising: a manifold, the manifold comprising a fluid flow path extending between an inlet and an outlet, and a liquid droplet inlet arranged to communicate with the fluid flow path at a location between the inlet and the outlet; and a pipettor configured to generate a liquid droplet, the pipettor comprising a nozzle having a nozzle tip from which a liquid droplet can be dispensed, the pipettor configured such that the nozzle tip is arranged to dispense a liquid droplet comprising a first fluid through the liquid droplet inlet of the manifold, in use, to contact a second fluid flowing along the fluid flow path.

[0160] In embodiments, the fluid flow path is defined by a channel through the manifold, which may be substantially linear, wherein the channel is configured to accept a continuous stream of the second fluid. In embodiments, the apparatus further comprises a pump to drive the flow of the second fluid, wherein the pump may push the second fluid through the manifold, or may draw the second fluid through the manifold.

[0161] In embodiments, the nozzle tip of the pipettor is located within the liquid droplet inlet, and may be separated from the fluid flow path by a distance ‘E’, wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, or between about 1 mm to about 5 mm, which may define an air gap between the fluid flow path and the nozzle tip.

[0162] In embodiments, the nozzle tip of the pipettor is located within the liquid droplet inlet, and may be separated from the fluid flow path by a distance ‘E’, wherein the distance E has a length of at least about 1 .0 mm, about 1.1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.

[0163] In embodiments, the fluid flow path has an elongate axis (ex) defined between the inlet and the outlet of the manifold. In embodiments, the pipettor has a proximal end comprising the nozzle and the nozzle tip and a distal end, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the manifold inlet is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the manifold inlet is between about 10° and 90°, between about 1 1 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0164] In embodiments, the liquid droplet inlet has a proximal end connected to the wall of the manifold and a distal end directed away from the manifold. In embodiments, the liquid droplet inlet has a longitudinal axis (px) extending between an aperture in the wall of the manifold at the proximal end of the liquid droplet inlet to the distal end of the liquid droplet inlet and running parallel with the liquid droplet inlet. In embodiments, the liquid droplet inlet and the manifold intersect at an angle, wherein the angle is defined between the longitudinal axis (lx) of the liquid droplet inlet and the elongate axis (ex) defined between the inlet and the outlet of manifold. In embodiments, the longitudinal axis (lx) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the manifold inlet and the liquid droplet inlet, and wherein the angle is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (lx) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the manifold inlet and the liquid droplet inlet, and wherein the angle is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0165] In embodiments, the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex).

[0166] In embodiments, the pipettor is directed axially-downwardly, wherein downwardly relates to a vertical direction, and may comprise a piezoelectric membrane. In embodiments, the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

[0167] In embodiments, the manifold comprises a fluid flow path region of reduced diameter and / or cross- sectional area, wherein the region of the manifold comprising the liquid droplet inlet may directly communicate with the fluid flow path region of reduced diameter and / or cross-sectional area. In embodiments, the fluid flow path region of reduced diameter and / or cross-sectional area has a cross- sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm. In embodiments, the fluid flow path region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm.

[0168] In embodiments, the manifold comprises a fluid flow path region of increased diameter and / or cross- sectional area. In embodiments, the fluid flow path region of increased diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800 pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm. In embodiments, the fluid flow path region of increased diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 and about 500 mm, or between about 200 to about 400 mm.

[0169] In embodiments, the fluid flow path has a length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400 mm, and may comprise one or more stream flow diverters. In embodiments, the fluid flow path comprises one or more narrowing fluid flow path regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more narrowing fluid flow path regions is gradually decreased relative to an adjacent region of the fluid flow path. In embodiments, a narrowing fluid flow path region has a tapering cross-sectional diameter which connects to a fluid flow path region of reduced diameter and / or cross-sectional area, which may be configured to facilitate mixing of the liquid droplet and the second fluid.

[0170] In embodiments, the fluid flow path comprises one or more widening fluid flow path regions of increasing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the widening fluid flow path regions is gradually increased relative to an adjacent region of the fluid flow path. In embodiments, a widening fluid flow path region has an inverse-tapering cross-sectional diameter which connects to a widened fluid flow path region, which may be configured to facilitate mixing of the liquid droplet and the second fluid.

[0171] In embodiments, the outlet communicates with a third location, which may comprise an amplification vessel, and / or comprise a monolayer, and / or comprise an imaging chip.

[0172] Systems for generating and dispensing emulsion droplets

[0173] In aspects of the invention, a system for generating emulsion droplets is provided, the system comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid entering the main channel, and a distal end portion comprising an outlet for fluid exiting the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and a pipettor configured to generate and extrude at least one liquid droplet, the pipettor comprising: a nozzle having a nozzle tip from which the at least one liquid droplet is extruded; and wherein the pipettor is arranged such that the nozzle tip is located within the side port of the manifold at a first location spaced from the main channel of the manifold.

[0174] In embodiments, the nozzle tip is spaced from the main channel of the manifold by a distance ‘E’, which may be a vertical distance and may define an air gap between the channel and nozzle tip. In embodiments, the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, or between about 1 mm to about 5 mm.

[0175] In embodiments, the distance E has a length of at least about 1.0 mm, about 1.1 mm, 1 .2 mm, 1.3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm. In embodiments, the pipettor is arranged to generate and extrude at least one liquid droplet comprising a first fluid at a first location, and wherein the at least one liquid droplet passes through the air gap between the first location and the main channel before contacting a second fluid flowing through the main channel at a second location. In embodiments, the first fluid is immiscible in the second fluid, and wherein the at least one liquid droplet is immersed in the second fluid, at least one emulsion droplet of the first fluid within the second fluid is thereby generated. In embodiments, the at least one emulsion droplet is transferred to a third location.

[0176] In embodiments, the first fluid comprises an aqueous liquid, which may comprise water, and / or a reaction mixture, and / or a sample, wherein the sample may comprise at least one target oligonucleotide. In embodiments, the second fluid comprises an organic liquid, which may comprise an oil.

[0177] In embodiments, the system may further comprise a pump to drive the flow of the second fluid, wherein the pump may push the second fluid through the manifold, or may draw the second fluid through the manifold.

[0178] In embodiments, the main channel may be substantially linear, and may have an elongate axis (ex) defined between the inlet and the outlet, and the pipettor may have a proximal end defined by the nozzle and the nozzle tip, and a distal end, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end of the pipettor. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end of the proximal end portion of the main channel is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0179] In embodiments, the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body.

[0180] In embodiments, the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port. In embodiments, the side port and the elongate body of the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the proximal end portion of the main channel and the side port, and wherein the angle is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the proximal end portion of the main channel and the side port is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0181] In embodiments, the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex) intersect.

[0182] In embodiments, the system comprises extrusion of the liquid droplet from the nozzle tip of the pipettor. In embodiments, the at least one liquid droplet may be extruded from a nozzle tip of a pipettor at the first location. In embodiments, nozzle tip of a pipettor comprises the first location.

[0183] In embodiments, the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv), wherein the first velocity vector (Iv) has a speed between about 0.001 to about 20m / s, between about 0.0025 to about 10m / s, between about 0.005 to about 5m / s, between about 0.0075 to about 2m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s, or between about 0.01 to about 0.1 m / s. In embodiments, the second fluid has a speed and direction of travel defining a second velocity vector (sv), wherein the second velocity vector (sv) has a speed between about 0.05 to about 3m / s, between about 0.1 to about 2m / s, between about 0.15 to about 1.5m / s, between about 0.2 to about 1 m / s, or between about 0.25 to about 0.75m / s., e.g. about 0.6m / s.

[0184] In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect, wherein contacting the at least one liquid droplet with a second fluid at a second location is at the intersection between the first velocity vector (Iv) and the second velocity vector (sv). In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect in the main channel of an apparatus defined according to the invention. In embodiments, the second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv) in the main channel of an apparatus defined according to the invention.

[0185] In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 175°, between about 1 1 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°. In embodiments, the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0186] In embodiments, the second fluid may be provided as a continuous stream, wherein the continuous stream of the second fluid flows along the fluid flow path. In embodiments, the continuous stream of the second fluid has a flow rate of between about 0.5 to about 100 pL / min, between about 1 to about 50 pL / min, between about 1 .5 to about 20 pL / min, between about 1 .75 to about 15 pL / min or between about 2 to about 10 pL / min.

[0187] In embodiments, the second location is at a surface of the continuous stream of the second fluid. In embodiments, contacting the at least one liquid droplet with a second fluid at a second location comprises contacting the at least one liquid droplet with the continuous stream of the second fluid.

[0188] In embodiments, the pipettor may be directed axially-downwardly, wherein downwardly relates to a vertical direction, and may comprise a piezoelectric membrane. In embodiments, the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz. In embodiments, the main channel may comprise a channel region of reduced diameter and / or cross- sectional area, wherein the aperture in the wall of the elongate body may directly communicate with the channel region of reduced diameter and / or cross-sectional area. In embodiments, the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel extending proximal and distal of the aperture in the wall of the elongate body at which the side port communicates. In embodiments, the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the distal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port. In embodiments, the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the proximal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port. In embodiments, the channel region of reduced diameter and / or cross- sectional area may have a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm. In embodiments, the channel region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm.

[0189] In embodiments, the main channel comprises a channel region of increased diameter and / or cross- sectional area, wherein the channel region of increased diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800 pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm. In embodiments, the channel region of increased diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, between about 150 to about 400 mm, or between about 200 to about 300 mm.

[0190] In embodiments, the main channel has an axial length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400 mm, and may comprise one or more stream flow diverters.

[0191] In embodiments, the main channel comprises one or more narrowing channel regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more narrowing channel regions is gradually decreased relative to an adjacent region of the main channel. In embodiments, a narrowing channel region has a tapering cross-sectional diameter which connects to a channel region of reduced diameter, and which may be configured to facilitate mixing of the liquid droplet and the second fluid. In embodiments, the channel comprises one or more widening channel regions of increasing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more widening channel regions is gradually increased relative to an adjacent region of the channel. In embodiments, a widening channel region has an inverse-tapering cross-sectional diameter which connects to a channel region of increased diameter, and which may be configured to facilitate mixing of the liquid droplet and the second fluid.

[0192] In embodiments, the first fluid comprises an aqueous liquid, for example, water. In embodiments, the second fluid comprises an organic liquid, for example, an oil, wherein the oil may have a density of between about 0.2 to 3 g / mL, between about 0.4 to about 2.5 g / mL, between about 0.6 to about 2 g / mL, or between about 0.8 to about 1.63 g / mL. In embodiments, the oil may have a viscosity of between about 0.2 to 7 mPa-s, between about 0.4 to about 6.5 mPa-s, between about 0.6 to about 6 mPa-s, between about 0.8 to about 5.5 mPa-s, or between about 1 .0 and about 5.0 mPa-s.

[0193] In embodiments, the first fluid comprises a sample, wherein the sample may comprise at least one target oligonucleotide. In embodiments, the first fluid comprises a reaction mixture, wherein the reaction mixture comprises reagents and oligonucleotides suitable for the preparation of target oligonucleotides for an amplification reaction. In embodiments, the reaction mixture comprises reagents and oligonucleotides suitable for an amplification reaction. In embodiments, the reaction mixture comprises any combination of: at least one oligonucleotide, and / or at least one amplification primer, and / or at least one enzyme, optionally wherein the enzyme is a polymerase.

[0194] In embodiments, the first location from which the at least one liquid droplet is extruded is spaced vertically above the second location. In embodiments, the pipettor has a frequency of liquid droplet drive, wherein the frequency of liquid droplet drive is between about 500 to about 100,000 droplets per second, between about 1 ,000 to about 50,000 droplets per second, between about 2,000 to about 25,000 droplets per second, between about 3,000 to about 15,000 droplets per second, or between about 4,000 to about 7,500 droplets per second, e.g. about 5,000 droplets per second.

[0195] In embodiments, the diameter of the liquid droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, or between about 30 to about 50 pm, e.g. about 40 pm. In embodiments, the volume of the liquid droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250 pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL.

[0196] In embodiments, the main channel outlet communicates with a third location, wherein the third location may comprise an amplification vessel, and / or comprise a monolayer, and / or comprise an imaging chip. In embodiments, the at least one emulsion droplet is transferred to a third location. In embodiments, transferring the at least one emulsion droplet to a third location may comprise transferring the at least one emulsion droplet from the second location to the outlet via a region of the main channel, and from the outlet to the third location by the continuous stream of second fluid. In embodiments, the at least one emulsion droplet comprises a partitioned sample, and / or comprises the reaction mixture.

[0197] Examples

[0198] The invention will now be further illustrated by way of the following non-limiting examples. Unless otherwise indicated, standard techniques in molecular biology, chemistry, biochemistry, and fluid dynamics were used.

[0199] The following examples further illustrate aspects and embodiments of the methods, systems, and apparatuses of the present disclosure.

[0200] Example 1

[0201] An example emulsion droplet generation and dispensing apparatus

[0202] Figure 1 displays a perspective side view of an example emulsion droplet generation and dispensing apparatus according to embodiments of the invention. In embodiments, the apparatus comprises a manifold, the manifold comprising: an elongate body (10) having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion (52) comprising an inlet (54) for fluid to enter the main channel, and a distal end portion (51) comprising an outlet (53) for fluid to exit the main channel, the main channel extending between the inlet (52) and the outlet (51) to provide a fluid flow path therebetween; and a side port (40) connecting with the elongate body (20) and arranged between the inlet (54) and the outlet (53) of the elongate body.

[0203] Example 2

[0204] Droplet generation at the intersection between the first channel and second channel of the emulsion droplet generation and dispensing apparatus

[0205] Figure 2 displays a cross sectional view of an example apparatus for droplet generation and dispensation, In embodiments, the side port (40) is configured for fluid communication with the main channel (33 and 34) of the elongate body through an aperture in the wall of the elongate body (35); and a pipettor (61) comprising a nozzle (62) having a nozzle tip (63) arranged within the side port (40), the nozzle tip positioned at a first location spaced from the main channel (33 and 34) of the manifold, and wherein the nozzle (63) of the pipettor (61) is arranged at an angle relative to the elongate body.

[0206] Example 3

[0207] Emulsion droplets generated by dispensing a liquid droplet into an oil

[0208] Figure 3 demonstrates an example of a plurality of generally uniformly sized emulsion droplets (small dark dots) in oil (surrounding liquid) generated by dispensing liquid droplets comprising a dark liquid into a stagnant body of a translucent oil. Example 4

[0209] A schematic of an example emulsion droplet generation and dispensing instrument

[0210] Figure 4 is a schematic of an example emulsion droplet generation and dispensing instrument, comprising a sample tube (A) which accepts a liquid sample, connected to a vibrating membrane (B), optionally a piezoelectric membrane, wherein a liquid droplet is extruded from a pipettor nozzle (C), optionally a pipettor tip, into the manifold (D) described herein, which is connected to a pump (P) which drives the movement of a second fluid, optionally an oil, through the manifold, wherein one or more emulsion droplets are generated by contacting the liquid droplet with the second fluid, wherein the one or more emulsion droplets subsequently flow from the manifold outlet to an amplification vessel (E), optionally an imaging chip, facilitated by the action of the pump.

[0211] Example 5

[0212] Second fluid flow simulations

[0213] Figure 5 is a visualisation of second fluid (e.g. oil) flow simulations in the main channel of an emulsion droplet generation and dispensing apparatus, wherein the velocity of the second fluid is indicated by a corresponding colour gradient ranging from blue to green (comparatively low speeds) at the wider inlet and outlet regions, to orange to red (comparatively higher speeds) through the narrower central channel. Figure 5 demonstrates acceleration of the second fluid from the proximal end portion (52) to the narrowed section (31 and 32) of the main channel, wherein the second fluid has a higher velocity, e.g. in the depicted embodiment, up to about 0.6 m / s, in the narrowed section of the main channel relative to the velocity of the second fluid in the proximal end portion of the main channel. Figure 5 further demonstrates de-acceleration of the second fluid from the narrowed section (31 and 32) to the distal end portion (51) of the main channel, wherein the second fluid has a higher velocity in the narrowed section of the main channel relative to the velocity of the second fluid in the distal end portion of the main channel.

[0214] Example 6

[0215] Trajectory and velocity of liquid droplets

[0216] Figure 6 is a visualisation of liquid droplet trajectory and velocity (blue) from the pipettor tip (right hand side) into the second fluid (pale green) and within the second fluid stream (right to left). Figure 7 is a visualisation of liquid droplet trajectory and velocity from the pipettor tip (pink - top, right), wherein the velocity of the liquid droplet is indicated by a corresponding colour gradient ranging from blue to green (comparatively low speeds - bottom of vertical axis scale) to yellow to orange to red (comparatively higher speeds - top of vertical axis scale).

[0217] Clauses Expressions of the inventive concept are set out in the following clauses:

[0218] Methods (M):

[0219] M1a: A method for generating emulsion droplets, the method comprising:

[0220] (a) forming at least one liquid droplet comprising a first fluid;

[0221] (b) extruding the at least one liquid droplet from a first location;

[0222] (c) passing the at least one liquid droplet through an air gap; and

[0223] (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; and wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally

[0224] (e) transferring the at least one emulsion droplet to a third location.

[0225] M1 b: A method for generating emulsion droplets, the method comprising:

[0226] (a) forming at least one liquid droplet comprising a first fluid;

[0227] (b) extruding the at least one liquid droplet from a first location;

[0228] (c) passing the at least one liquid droplet through an air gap; and

[0229] (d) contacting the at least one liquid droplet with a second fluid at a second location,; wherein the first fluid is immiscible in the second fluid; wherein the second fluid is provided as a stagnant body; wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally

[0230] (e) transferring the at least one emulsion droplet to a third location.

[0231] M1c: A method for generating emulsion droplets, the method comprising:

[0232] (a) forming at least one liquid droplet comprising a first fluid;

[0233] (b) extruding the at least one liquid droplet from a first location;

[0234] (c) passing the at least one liquid droplet through an air gap; and

[0235] (d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; wherein the second fluid is provided as a stream; wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet, and

[0236] (e) transferring the at least one emulsion droplet to a third location.

[0237] M2: The method according to any of the preceding clauses, wherein the first fluid comprises an aqueous liquid.

[0238] M3: The method according to clause M2, wherein the first fluid comprises water. M4: The method according to any of the preceding clauses, wherein the second fluid comprises an organic liquid.

[0239] M5: The method according to any of the preceding clauses, wherein the second fluid comprises an oil.

[0240] M6: The method according to clause M5, wherein the oil has a density of between about 0.2 to 3 g / mL, between about 0.4 to about 2.5 g / mL, between about 0.5 to about 2 g / mL, between about 0.8 to about 1 g / mL, or about 1 .6, e.g. 1 .63 g / mL.

[0241] M7: The method according to clause M5 or M6, wherein the oil has a viscosity of between about

[0242] 0.2 to 10 mPa-s, between about 0.4 to about 8 mPa-s, between about 0.6 to about 6 mPa-s, between about 0.8 to about 5.5, or between about 1 .0 and about 5.0 mPa-s.

[0243] M8: The method according to any of the preceding clauses, wherein the first fluid comprises a sample.

[0244] M9: The method according to clause M8, wherein the sample comprises at least one target oligonucleotide.

[0245] M10: The method according to any of the preceding clauses, wherein the first fluid comprises a reaction mixture.

[0246] M11 : The method according to clause M10, wherein the reaction mixture comprises reagents and oligonucleotides suitable for the preparation of target oligonucleotides for an amplification reaction.

[0247] M12: The method according to clause M10 or M11 , wherein the reaction mixture comprises reagents and oligonucleotides suitable for an amplification reaction.

[0248] M13: The method according to any of clauses M10 to M12, wherein the reaction mixture comprises any combination of: at least one oligonucleotide, and / or at least one amplification primer, and / or at least one enzyme, optionally wherein the enzyme is a polymerase.

[0249] M14: The method according to any of the preceding clauses, comprising extrusion of the liquid droplet from the pipettor; optionally wherein the pipettor is defined according to any of clauses A1 a, or A1 c to A50, optionally wherein the liquid droplet is extruded from a nozzle tip of the pipettor.

[0250] M15: The method according to any of the preceding clauses, wherein the first location from which the at least one liquid droplet is extruded is spaced vertically above the second location. M16: The method according to clause M14 or M15, wherein the pipettor has a frequency of liquid droplet drive, wherein the frequency of liquid droplet drive is between about 500 to about 100,000 droplets per second, between about 1 ,000 to about 50,000 droplets per second, between about 2,000 to about 25,000 droplets per second, between about 3,000 to about 15,000 droplets per second, or between about 4,000 to about 7,500 droplets per second, e.g. about 5,000 droplets per second.

[0251] M17: The method according to any of the preceding clauses, wherein the liquid droplet has an initial velocity of between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s.

[0252] M18: The method according to any of the preceding clauses, wherein the diameter of the liquid droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, between about 30 to about 50 pm, e.g. about 40 pm.

[0253] M19: The method according to any of the preceding clauses, wherein the volume of the liquid droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL.

[0254] M20: The method according to any of the preceding clauses, wherein the at least one liquid droplet is extruded from a nozzle tip of a pipettor at the first location.

[0255] M21 : The method according to clause M20, wherein the pipettor is according to any of clauses A1 a, or A1 c to A50.

[0256] M22: The method according to any of the preceding clauses, wherein the liquid droplet has a direction of travel.

[0257] M22a: The method according to any of the preceding clauses, wherein the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv).

[0258] M22b: The method according to clause M22a, wherein the first velocity vector (Iv) has a speed between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s.

[0259] M23: The method according to any of the preceding clauses, wherein the second fluid has a direction of travel. M23a: The method according to any of the preceding clauses, wherein the second fluid has a speed and direction of travel defining a second velocity vector (sv).

[0260] M23b: The method according to clause M23, wherein the second velocity vector (sv) has a speed between about 0.05 to about 3 m / s, between about 0.15 to about 2 m / s, between about 0.1 to about 1.5 m / s, between about 0.2 to about 1 m / s, or between about 0.25 to about 0.75 m / s, e.g. about 0.6 m / s.

[0261] M23c: The method according to any of the preceding clauses, wherein the first velocity vector (Iv) and the second velocity vector (sv) are directly proportional to each other.

[0262] M24: The method according to any of clauses M23 to M23c, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect at the second location.

[0263] M24a: The method according to clause M23a or M23c, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect.

[0264] M25: The method according to any of clauses M22 to M22b, wherein the direction of travel of the liquid droplet and the second fluid intersect.

[0265] M25a: The method according to clauses M22a or M22b, wherein the first velocity vector (Iv) and the second fluid intersect.

[0266] M26: The method according to clause M24, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the direction of travel of the liquid droplet and the direction of travel of the second fluid.

[0267] M26a: The method according to clause M24a, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv).

[0268] M27: The method according to clause M25, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the direction of travel of the liquid droplet and the second fluid.

[0269] M27a: The method according to clause M25a, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises the intersection between the first velocity vector (Iv) and the second fluid. M28: The method according to any of clauses M24, M26 or M27, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect in the main channel of the apparatus according to any of clauses A1 a to A50.

[0270] M28a: The method according to any of clauses M24a, M26a or M27a, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect in the main channel of the apparatus according to any of clauses A1 a to A50.

[0271] M29: The method according to clause M28, wherein the second location comprises the intersection between the direction of travel of the liquid droplet and the direction of travel of the second fluid in the main channel of the apparatus according to any of clauses A1 a to A50.

[0272] M29a: The method according to clause M28a, wherein the second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv) in the main channel of the apparatus according to any of clauses A1 a to A50.

[0273] M29b: The method according to clause M27, wherein the second location comprises the intersection between the direction of travel of the liquid droplet and the second fluid.

[0274] M29c: The method according to clause M27a, wherein the second location comprises the intersection between the first velocity vector (Iv) and the second fluid.

[0275] M30: The method according to any of clauses M24, or M26 to M29, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersects at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0276] M30a: The method according to any of clauses M24a, M26a, M27a, M28a, or M29a, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 175°, between about 1 1 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0277] M31 : The method according to any of clauses M24, or M26 to M29, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersect at an angle of between about 10° and 90°, between about 1 1 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0278] M31 a: The method according to any of clauses M24a, M26a, M27a, M28a, or M29a, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0279] M32: The method according to clause M25 or M27, wherein the direction of travel of the liquid droplet and the second fluid intersect at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0280] M32a: The method according to any of the clauses M25a, M27a, M29b, or M29c, wherein the first velocity vector (Iv) and the second fluid intersect at an angle of between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0281] M33: The method according to any of the clauses M1 a, or M1 c to M24a, M26, M26a, or M30 to M31 a, wherein the second fluid is provided as a continuous stream.

[0282] M34: The method according to any of the clauses M1 b to M21 , M22, M22a, M25, M25a, M27, M27a, M29b, M29c, M32, M32a, wherein the second fluid is provided as a stagnant body.

[0283] M35: The method according to clause M33, wherein the second location is at a surface of the continuous stream of the second fluid.

[0284] M36: The method according to clause M34, wherein the second location is at a surface of the stagnant body of the second liquid.

[0285] M36a: The method according to any of the clauses M1 b to M21 , M22, M22a, M25, M25a, M27, M27a, M29b, M29c, M32, M32a, M34, or M36, wherein transferring the at least one emulsion droplet to a third location comprises applying a motive force to the stagnant body of second fluid to generate a continuous stream of second fluid.

[0286] M37: The method according to clause M35, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises contacting the at least one liquid droplet with the continuous stream of the second fluid. M38: The method according to any of clauses M33, M35, or M37, wherein the continuous stream of the second fluid has a flow rate of between about 0.5 to about 100 pL / min, between about 1 to about 50 pL / min, between about 1.5 to about 20 pL / min, between about 1.75 to about 15 pL / min, or between about 2 to about 10 pL / min.

[0287] M38a: The method according to any of the preceding clauses, wherein the first velocity vector (Iv) and the continuous stream of the second fluid are directly proportional to each other.

[0288] M39: The method according to any of the preceding clauses, wherein the at least one emulsion droplet comprises a partitioned sample.

[0289] M40: The method according to any of the preceding clauses, wherein the at least one emulsion droplet comprises a reaction mixture.

[0290] M41 : The method according to any of clauses M33, M35, or M36a to M40, wherein transferring the at least one emulsion droplet to a third location comprises transferring the at least one emulsion droplet from the second location to the outlet via a region of the main channel, and from the outlet to the third location by the continuous stream of second fluid.

[0291] M42: The method according to any of the preceding clauses, wherein the third location comprises an amplification vessel.

[0292] M43: The method according to any of the preceding clauses, wherein the third location comprises a monolayer.

[0293] M44: The method according to any of the preceding clauses, wherein the third location comprises an imaging chip.

[0294] Systems (S):

[0295] S1 : A system for generating emulsion droplets, the system comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid entering the main channel, and a distal end portion comprising an outlet for fluid exiting the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and a pipettor configured to generate and extrude at least one liquid droplet, the pipettor comprising: a nozzle having a nozzle tip from which the at least one liquid droplet is extruded; and wherein the pipettor is arranged such that the nozzle tip is located within the side port of the manifold at a first location spaced from the main channel of the manifold.

[0296] S2: The system according to clause S1 , wherein the nozzle tip is spaced from the main channel of the manifold by a distance ‘E’.

[0297] S3: The system according to clause S2, wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, or between about 1 mm to about 5 mm.

[0298] S3a: The system according to any of clauses S2 to S3, wherein the distance E has a length of at least about 1 .0 mm, about 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.

[0299] S4: The system according to any of clauses S2 to S3a, wherein distance E defines an air gap between the channel and nozzle tip.

[0300] S4a. The apparatus according to any of clauses S2 to S4, wherein the distance E is a vertical distance.

[0301] S5: The system of clauses S4 or S4a, wherein, in use, the pipettor is arranged to generate and extrude at least one liquid droplet comprising a first fluid at a first location, and wherein the at least one liquid droplet passes through the air gap between the first location and the main channel before contacting a second fluid flowing through the main channel at a second location.

[0302] 56. The system of any of the preceding clauses, wherein the first fluid is immiscible in the second fluid, and wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet of the first fluid within the second fluid.

[0303] 57. The system of any of the preceding clauses, wherein the at least one emulsion droplet is transferred to a third location.

[0304] S8: The system according to any of the preceding clauses, further comprising a pump to drive the flow of the second fluid. S9: The system according to clause S8, wherein the pump pushes the second fluid through the manifold.

[0305] S10: The system according to clause S8, wherein the pump draws the second fluid through the manifold.

[0306] S11 : The system according to any of the preceding clauses, wherein the main channel is substantially linear.

[0307] 512. The system according to any of the preceding clauses, wherein the main channel has an elongate axis (ex) defined between the inlet and the outlet.

[0308] 513. The system according to any of the preceding clauses, wherein the pipettor has a proximal end defined by the nozzle and the nozzle tip, and a distal end.

[0309] 514. The system according to clause S13, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end of the pipettor.

[0310] S15: The system according to clause S14, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0311] S16: The system according to clause S14, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end of the proximal end portion of the main channel is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0312] 517. The system according to any of the preceding clauses, wherein the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body.

[0313] S17a. The system according to clause S17, wherein the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port.

[0314] 518. The system according to any of the preceding clauses, wherein the side port and the elongate body of the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold.

[0315] S19: The system according to clauses S17a or S18, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0316] S20: The system according to clauses S17a or S18, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0317] S21 : The system according to clauses S17a or S18, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the proximal end portion of the main channel and the side port, and wherein the angle is between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0318] S22: The system according to clauses S17a or S18, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the proximal end portion of the main channel and the side port is between about 10° and 90°, between about 1 1 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0319] S23: The system according to clauses S17a or S18, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0320] S24: The system according to clauses S17a or S18, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0321] S25: The system according to any of clauses S18 to S24, wherein the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex) intersect.

[0322] S26: The system according to clause S5 to S25, comprising extrusion of the liquid droplet from the nozzle tip of the pipettor.

[0323] S27: The system according to any of the preceding clauses, wherein the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv).

[0324] S28: The system according to clause S27, wherein the first velocity vector (Iv) has a speed between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s.

[0325] S29: The system according to any of the preceding clauses, wherein the second fluid has a speed and direction of travel defining a second velocity vector (sv).

[0326] S30: The system according to clause S29, wherein the second velocity vector (sv) has a speed between about 0.05 to about 3 m / s, between about 0.1 to about 2 m / s, between about 0.15 to about 1 .5 m / s, between about 0.2 to about 1 m / s, or between about 0.25 to about 0.75 m / s., e.g. about 0.6 m / s.

[0327] S31 : The system according to clause S29 or S30, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect.

[0328] S32: The system according to clause S31 , wherein contacting the at least one liquid droplet with a second fluid at a second location is at the intersection between the first velocity vector (Iv) and the second velocity vector (sv).

[0329] S33: The system according to clause S31 or S32, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect in the main channel of an apparatus according to any of clauses A1 a to A50. S34: The system according to any of clauses S31 to S33, wherein the second location comprises the intersection between the first velocity vector (Iv) and the second velocity vector (sv) in the main channel of the apparatus according to any of clauses A1 a to A50.

[0330] S35: The system according to any of clauses S31 to S34, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0331] S36: The system according to any of clauses S31 to S34, wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect at an angle of between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0332] S37: The system according to any of the preceding clauses, wherein the second fluid is provided as a continuous stream.

[0333] S38: The system according to S37, wherein the continuous stream of the second fluid flows along the fluid flow path.

[0334] S39: The system according to S38, wherein the second velocity vector (sv) has a direction parallel to the fluid flow path.

[0335] S40: The system according to any of clauses S37 to S39, wherein the continuous stream of the second fluid has a flow rate of between about 0.5 to about 100 pL / min, between about 1 to about 50 pL / min, between about 1 .5 to about 20 pL / min, between about 1 .75 to about 15 pL / min or between about 2 to about 10 pL / min.

[0336] S41 : The system according to clause S5 or any of clauses S6 to S40 when dependent on clause S5, wherein the at least one liquid droplet is extruded from a nozzle tip of a pipettor at the first location.

[0337] S42: The system according to clause S5 or any of clauses S6 to S41 when dependent on clause S5, wherein the second location is at a surface of the continuous stream of the second fluid.

[0338] S43: The system according to clause S42, wherein contacting the at least one liquid droplet with a second fluid at a second location comprises contacting the at least one liquid droplet with the continuous stream of the second fluid. S44: The system according to any of the preceding clauses, wherein the pipettor is directed axially- downwardly, wherein downwardly relates to a vertical direction.

[0339] S45: The system according to any of the preceding clauses, wherein the pipettor comprises a piezoelectric membrane.

[0340] S46: The system according to clause S45, wherein the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

[0341] S47: The system according to any of the preceding clauses, wherein the main channel comprises a channel region of reduced diameter and / or cross-sectional area.

[0342] S48: The system according to clause S47, wherein the aperture in the wall of the elongate body directly communicates with the channel region of reduced diameter and / or cross-sectional area.

[0343] S49: The system according to clause S48, wherein the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel extending proximal and distal of the aperture in the wall of the elongate body at which the side port communicates.

[0344] S50: The system according to any of the clauses S47 to S49, wherein the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the distal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port.

[0345] S51 : The system according to any of the clauses S47 to S50, wherein the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the proximal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port.

[0346] S52: The system according to any of the clauses S47 to S51 , wherein the channel region of reduced diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm.

[0347] S53: The system according to any of the clauses S47 to S52, wherein the channel region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm. S54: The system according to any of the preceding clauses, wherein the main channel comprises a channel region of increased diameter and / or cross-sectional area.

[0348] S55: The system according to clause S54, wherein the channel region of increased diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800 pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm.

[0349] S56: The system according to clause S54 or S55, wherein the channel region of increased diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, between about 150 to about 400 mm, or between about 200 to about 300 mm.

[0350] S57: The system according to any of clauses S54 to S56, wherein the main channel has an axial length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400 mm.

[0351] S58: The system according to any of the preceding clauses, wherein the main channel comprises one or more stream flow diverters.

[0352] S59: The system according to any of the clauses S47 to S58, wherein the main channel comprises one or more narrowing channel regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more narrowing channel regions is gradually decreased relative to an adjacent region of the main channel.

[0353] S60: The system according to clause S59, wherein a narrowing channel region has a tapering cross-sectional diameter which connects to a channel region of reduced diameter.

[0354] S61 : The system according to clause S59 or S60, wherein the one or more narrowing channel regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0355] S62: The system according to any of clauses S54 to S61 , wherein the channel comprises one or more widening channel regions of increasing diameter and / or cross-sectional area, wherein the cross- sectional diameter of the one or more widening channel regions is gradually increased relative to an adjacent region of the channel.

[0356] S63: The system according to clause S62, wherein a widening channel region has an inversetapering cross-sectional diameter which connects to a channel region of increased diameter. S64: The system according to clause S62 or S63, wherein the one or more widening channel regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0357] S65: The system according to any of the preceding clauses, wherein the main channel outlet communicates with a third location.

[0358] S66: The system according to clause S65, wherein the third location comprises an amplification vessel.

[0359] S67: The system according to clause S65 or S66, wherein the third location comprises an imaging chip.

[0360] S68: The system according to any of the clauses S5 to S67, wherein the first fluid comprises an aqueous liquid.

[0361] S69: The system according to clause S68, wherein the first fluid comprises water.

[0362] S70: The system according to any of the clauses S5 to S69, wherein the second fluid comprises an organic liquid.

[0363] S71 : The system according to any of clauses S5 to S70, wherein the second fluid comprises an oil.

[0364] S72: The system according to clause S71 , wherein the oil has a density of between about 0.2 to 3 g / mL, between about 0.4 to about 2.5 g / mL, between about 0.6 to about 2 g / mL, or between about 0.8 to about 1 .63 g / mL.

[0365] S73: The system according to clause S71 or S72, wherein the oil has a viscosity of between about 0.2 to 7 mPa-s, between about 0.4 to about 6.5 mPa-s, between about 0.6 to about 6 mPa-s, between about 0.8 to about 5.5 mPa-s, or between about 1 .0 and about 5.0 mPa-s.

[0366] S74: The system according to any of the clauses S5 to S73, wherein the first fluid comprises a sample.

[0367] S75: The system according to clause S74, wherein the sample comprises at least one target oligonucleotide.

[0368] S76: The system according to any of the clauses S5 to S75, wherein the first fluid comprises a reaction mixture.

[0369] S77: The system according to clause S76, wherein the reaction mixture comprises reagents and oligonucleotides suitable for the preparation of target oligonucleotides for an amplification reaction. S78: The system according to clause S76 or S77, wherein the reaction mixture comprises reagents and oligonucleotides suitable for an amplification reaction.

[0370] S79: The system according to any of the clauses S76 to S78, wherein the reaction mixture comprises any combination of: at least one oligonucleotide, and / or at least one amplification primer, and / or at least one enzyme, optionally wherein the enzyme is a polymerase.

[0371] S80: The system according to any of the clauses S5 to S79, wherein the first location from which the at least one liquid droplet is extruded is spaced vertically above the second location.

[0372] S81 : The system according to any of the clauses S5 to S80, wherein the pipettor has a frequency of liquid droplet drive, wherein the frequency of liquid droplet drive is between about 500 to about 100,000 droplets per second, between about 1 ,000 to about 50,000 droplets per second, between about 2,000 to about 25,000 droplets per second, between about 3,000 to about 15,000 droplets per second, or between about 4,000 to about 7,500 droplets per second, e.g. about 5,000 droplets per second.

[0373] S82: The system according to any of clauses S5 to S81 , wherein the diameter of the liquid droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, or between about 30 to about 50 pm, e.g. about 40 pm.

[0374] S83: The system according to any of clauses S5 to S82, wherein the volume of the liquid droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250 pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL.

[0375] S84: The system according to any of clauses S5 to S83, wherein the at least one emulsion droplet comprises a partitioned sample.

[0376] S85: The system according to any of clauses S5 to S84, wherein the at least one emulsion droplet comprises the reaction mixture.

[0377] S86: The system according to any of clauses S65 to S85, wherein transferring the at least one emulsion droplet to a third location comprises transferring the at least one emulsion droplet from the second location to the outlet via a region of the main channel, and from the outlet to the third location by the continuous stream of second fluid.

[0378] Apparatus clauses (A):

[0379] A1 a: An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and an aperture through the wall of the elongate body and located between the inlet and the outlet; a pipettor comprising a nozzle having a nozzle tip, the nozzle tip arranged at a first location spaced from the main channel of the manifold and vertically above the aperture, and wherein the nozzle of the pipettor is arranged at an angle relative to the elongate body.

[0380] A1 b: An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and wherein the side port is arranged at an angle relative to the elongate body.

[0381] A1 c: An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body; and a pipettor comprising a nozzle having a nozzle tip arranged within the side port, the nozzle tip positioned at a first location spaced from the main channel of the manifold, and wherein the nozzle of the pipettor is arranged at an angle relative to the elongate body.

[0382] A2: The apparatus according to clause A1 a, further comprising a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body.

[0383] A3: The apparatus according to clause A2, wherein the nozzle tip of the pipettor is located within the side port.

[0384] A4: The apparatus according to clause A1 b, further comprising a pipettor comprising a nozzle having a nozzle tip, and wherein the nozzle tip is located within the side port.

[0385] A5: The apparatus according to any of clauses A1 a, or A1 c, to A4, wherein the pipettor is configured to dispense a liquid droplet from the nozzle tip.

[0386] A6: The apparatus according to any of clauses A1 a or A1 c to A5, wherein the nozzle tip is separated from the main channel by a distance ‘E’.

[0387] A7: The method according to clause A6, wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, or between about 1 mm to about 5 mm.

[0388] A7a: The method according to clause A6, wherein the distance E has a length of at least about 1 .0 mm, about 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.

[0389] A8: The apparatus according to any of clauses A6 to A7a, wherein distance E defines an air gap between the channel and nozzle tip.

[0390] A9. The apparatus according to any of clauses A6 to A8, wherein the distance E is a vertical distance.

[0391] A10: The apparatus according to any of the preceding clauses, further comprising a pump to drive the flow of the second fluid.

[0392] A11 : The apparatus according to clause A10, wherein the pump pushes the second fluid through the manifold. A12: The apparatus according to clause A10, wherein the pump draws the second fluid through the manifold.

[0393] A13: The apparatus according to any of the preceding clauses, wherein the main channel is substantially linear.

[0394] A14. The apparatus according to any of the preceding clauses, wherein the main channel has an elongate axis (ex) defined between the inlet and the outlet.

[0395] A14a. The apparatus according to any of clauses A1 a, or A1 c to A14, wherein the pipettor has a proximal end comprising the nozzle and the nozzle tip, and a distal end.

[0396] A15. The apparatus according to clause A14a, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end.

[0397] A16: The apparatus according to clause A15, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 1 10°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0398] A17: The apparatus according to clause A15, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0399] A18. The apparatus according to any of clauses A1 b to A17, wherein the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body.

[0400] A18a. The apparatus according to clause A18, wherein the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port.

[0401] A19. The apparatus according to any of clauses A1 b to A18a, wherein the side port and the elongate body of the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold. A20: The apparatus according to clause A18a or A19, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0402] A21 : The apparatus according to clause A18a or A19, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0403] A22: The apparatus according to clause A18a or A19, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the proximal end portion of the main channel and the side port is between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0404] A23: The apparatus according to clause A18a or A19, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the proximal end portion of the main channel and the side port is between about 10° and 90°, between about 1 1 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0405] A24: The apparatus according to clause A18a or A19, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0406] A25: The apparatus according to clause A18a or A19, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the distal end portion of the main channel and the side port is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0407] A26: The apparatus according to any of clauses A18a to A25, wherein the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex) intersect.

[0408] A27: The apparatus according to clause A1 a or A1 c to A26, wherein the pipettor is directed axially- downwardly, wherein downwardly relates to a vertical direction.

[0409] A28: The apparatus according to clause A1 a or A1 c to A27, wherein the pipettor comprises a piezoelectric membrane.

[0410] A29: The apparatus according to clause A28, wherein the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

[0411] A30: The apparatus according to any of the preceding, wherein the main channel comprises a channel region of reduced diameter and / or cross-sectional area.

[0412] A31 : The apparatus according to clause A30, wherein the aperture in the wall of the elongate body directly communicates with the channel region of reduced diameter and / or cross-sectional area.

[0413] A32: The apparatus according to clause A30 or A31 , wherein the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel extending proximal and distal of the aperture in the wall of the elongate body at which the side port communicates.

[0414] A33: The apparatus according to any of clauses A30 to A32, wherein the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the distal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port.

[0415] A34: The apparatus according to any of clauses A30 to A33, wherein the channel region of reduced diameter and / or cross-sectional area encompasses a region of the main channel towards the proximal end of the elongate body from the aperture in the wall of the elongate body connecting with the side port.

[0416] A35: The apparatus according to any of the clauses A30 to A34, wherein the channel region of reduced diameter and / or cross-sectional area has a cross-sectional diameter or width of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm.

[0417] A36: The apparatus according to any of the clauses A30 to A35, wherein the channel region of reduced diameter and / or cross-sectional area has an axial length of up to about 1 ,000 mm, up to about 500 mm, up to about 400 mm, e.g. between about 0.3 and about 400 mm, between about 0.4 and about 350 mm, or between about 0.5 and about 300 mm.

[0418] A37: The apparatus according to any of the preceding clauses, wherein the main channel comprises a channel region of increased diameter and / or cross-sectional area.

[0419] A38: The apparatus according to clause A37, wherein the channel region of increased diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800 pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm..

[0420] A39: The apparatus according to clause A37 or A38, wherein the channel region of increased diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, between about 150 to about 400 mm, or between about 200 to about 300 mm.

[0421] A40: The apparatus according to any of the preceding clauses, wherein the main channel has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400 mm.

[0422] A41 : The apparatus according to any of the preceding clauses, wherein the main channel comprises one or more stream flow diverters.

[0423] A42: The apparatus according to any of the clauses A30 to A41 , wherein the main channel comprises one or more narrowing channel regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more narrowing channel regions is gradually decreased relative to an adjacent region of the main channel.

[0424] A43: The apparatus according to clause A42, wherein a narrowing channel region has a tapering cross-sectional diameter which connects to a narrowed channel region.

[0425] A44: The apparatus according to clause A42 or A43, wherein the one or more narrowing channel regions are configured to facilitate mixing of the liquid droplet and the second fluid. A45: The apparatus according to any of the clauses A37 to A44, wherein the channel comprises one or more widening channel regions of increasing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one or more widening channel regions is gradually increased relative to an adjacent region of the channel.

[0426] A46: The apparatus according to any of the clauses A37 to A45, wherein a widening channel region has an inverse-tapering cross-sectional diameter which connects to a widened channel region.

[0427] A47: The apparatus according to clause A45 or A46, wherein the one or more widening channel regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0428] A48: The apparatus according to any of the preceding clauses, wherein the main channel outlet communicates with a third location.

[0429] A49: The apparatus according to clause A48, wherein the third location comprises an amplification vessel.

[0430] A50: The apparatus according to clause A48 or A49, wherein the third location comprises an imaging chip.

[0431] Apparatus clauses (T):

[0432] T1 : An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising a fluid flow path extending between an inlet and an outlet, and a liquid droplet inlet arranged to communicate with the fluid flow path at a location between the inlet and the outlet; and a pipettor configured to generate a liquid droplet, the pipettor comprising a nozzle having a nozzle tip from which a liquid droplet can be dispensed, the pipettor configured such that the nozzle tip is arranged to dispense a liquid droplet comprising a first fluid through the liquid droplet inlet of the manifold, in use, to contact a second fluid flowing along the fluid flow path.

[0433] T2: The apparatus according to clause T1 , wherein the fluid flow path is defined by a channel through the manifold, and wherein the channel is configured to accept a continuous stream of the second fluid.

[0434] T3: The apparatus according to clause T1 or T2, further comprising a pump to drive the flow of the second fluid.

[0435] T4: The apparatus according to clause T3, wherein the pump pushes the second fluid through the manifold. T5: The apparatus according to clause T3, wherein the pump draws the second fluid through the manifold.

[0436] T6: The apparatus according to any of the preceding clauses, wherein the nozzle tip of the pipettor is located within the liquid droplet inlet.

[0437] T7: The apparatus according to any of the preceding clauses, wherein the nozzle tip is separated from the fluid flow path by a distance ‘E’.

[0438] T8: The method according to clause T7, wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, or between about 1 mm to about 5 mm.

[0439] T8a: The method according to clause T7, wherein the distance E has a length of at least about 1 .9 mm, about 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.

[0440] T9: The apparatus according to any of clauses T7 to T8a, wherein the distance E defines an air gap between the fluid flow path and the nozzle tip.

[0441] T10: The apparatus according to any of the preceding clauses, wherein the fluid flow path is substantially linear.

[0442] T1 1 : The apparatus according to any of the preceding clauses, wherein the fluid flow path has an elongate axis (ex) defined between the inlet and the outlet of the manifold.

[0443] T12: The apparatus according to any of the preceding clauses, wherein the pipettor has a proximal end comprising the nozzle and the nozzle tip and a distal end.

[0444] T13: The apparatus according to any of the preceding clauses, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end.

[0445] T14: The apparatus according to clause T13, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the manifold inlet is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0446] T15: The apparatus according to clause T13, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the manifold inlet is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0447] T16: The apparatus according to any of the preceding clauses, wherein the liquid droplet inlet has a proximal end connected to the wall of the manifold and a distal end directed away from the manifold.

[0448] T16a: The apparatus according to clause T16, wherein the liquid droplet inlet has a longitudinal axis (px) extending between an aperture in the wall of the manifold at the proximal end of the liquid droplet inlet to the distal end of the liquid droplet inlet and running parallel with the liquid droplet inlet.

[0449] T17: The apparatus according to clause T16 or T16a when dependent on clause T11 , wherein the liquid droplet inlet and the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (lx) of the liquid droplet inlet and the elongate axis (ex) defined between the inlet and the outlet of manifold.

[0450] T18: The apparatus according to clause T16a or T17, wherein the longitudinal axis (lx) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the manifold inlet and the liquid droplet inlet, and wherein the angle is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

[0451] T19: The apparatus according to clause T16a or T17, wherein the longitudinal axis (lx) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the manifold inlet and the liquid droplet inlet, and wherein the angle is between about 10° and 90°, between about 11 ° and 87°, between about 12° and 84°, between about 13° and 81 °, between about 14° and 78°, between about 15° and 75°, between about 16° and 72°, between about 17° and 69°, between about 18° and 66°, between about 19° and 63°, or between about 20° to about 60°.

[0452] T19a: The apparatus according to any of clauses T16a to T19, wherein the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex).

[0453] T20: The apparatus according to any of the preceding clauses, wherein the pipettor is directed axially-downwardly, wherein downwardly relates to a vertical direction.

[0454] T21 : The apparatus according to any of the preceding clauses, wherein the pipettor comprises a piezoelectric membrane. T22: The apparatus according to clause T21 , wherein the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

[0455] T23: The apparatus according to any of the preceding clauses, wherein the manifold comprises a fluid flow path region of reduced diameter and / or cross-sectional area.

[0456] T24: The apparatus according to clause T24, wherein the region of the manifold comprising the liquid droplet inlet directly communicates with the fluid flow path region of reduced diameter and / or cross-sectional area.

[0457] T25: The apparatus according to clause T23 or T24, wherein the fluid flow path region of reduced diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm.

[0458] T26: The apparatus according to any of clauses T23 to T25, wherein the fluid flow path region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm.

[0459] T27: The apparatus according to any of the preceding clauses, wherein the manifold comprises a fluid flow path region of increased diameter and / or cross-sectional area.

[0460] T28: The apparatus according to clause T27, wherein the fluid flow path region of increased diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 3,000 pm, 20 to about 2,000 pm, between about 30 to about 1 ,500 pm, between about 40 to about 1 ,000 pm, between about 50 to about 800pm, between about 100 to about 600 pm, between about 150 to about 550 pm, or between about 200 to about 500 pm.

[0461] T29: The apparatus according to clause T27 or T28, wherein the fluid flow path region of increased diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 and about 500 mm, or between about 200 to about 400 mm.

[0462] T30: The apparatus according to any of clauses T27 to T29, wherein the fluid flow path has a length of between about 5 to 10,000 mm, between about 10 and about 5,000 mm, between about 50 to about 1 ,000 mm, between about 100 to about 500 mm, or between about 200 to about 400mm. T31 : The apparatus according to any of the preceding clauses, wherein the fluid flow path comprises one or more stream flow diverters.

[0463] T32: The apparatus according to any of clauses T23 to T31 , wherein the fluid flow path comprises one or more narrowing fluid flow path regions of reducing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the one ore more narrowing fluid flow path regions is gradually decreased relative to an adjacent region of the fluid flow path.

[0464] T33: The apparatus according to any of clauses T23 to T32, wherein a narrowing fluid flow path region has a tapering cross-sectional diameter which connects to a fluid flow path region of reduced diameter and / or cross-sectional area.

[0465] T34: The apparatus according to any of clauses T23 to T33, wherein the one or more narrowing fluid flow path regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0466] T35: The apparatus according to any of clauses T27 to T34, wherein the fluid flow path comprises one or more widening fluid flow path regions of increasing diameter and / or cross-sectional area, wherein the cross-sectional diameter of the widening fluid flow path regions is gradually increased relative to an adjacent region of the fluid flow path.

[0467] T36: The apparatus according to any of clauses T27 to T35, wherein a widening fluid flow path region has an inverse-tapering cross-sectional diameter which connects to a widened fluid flow path region.

[0468] T37: The apparatus according to any of clauses T27 to T36, wherein the one or more widening fluid flow path regions are configured to facilitate mixing of the liquid droplet and the second fluid.

[0469] T38: The apparatus according to any of the preceding clauses, wherein the outlet communicates with a third location.

[0470] T39: The apparatus according to clause T38, wherein the third location comprises an amplification vessel.

[0471] T40: The apparatus according to clause T38 or T39, wherein the third location comprises an imaging chip.

Claims

Claims1 . A method for generating emulsion droplets, the method comprising:(a) forming at least one liquid droplet comprising a first fluid;(b) extruding the at least one liquid droplet from a first location;(c) passing the at least one liquid droplet through an air gap; and(d) contacting the at least one liquid droplet with a second fluid at a second location; wherein the first fluid is immiscible in the second fluid; and wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet; and optionally(e) transferring the at least one emulsion droplet to a third location.

2. The method of Claim 1 , wherein the second fluid is provided as a stagnant body.

3. The method of Claim 1 , wherein the second fluid is provided as a stream.

4. The method according to any of the preceding claims, wherein the first fluid comprises an aqueous liquid and the second fluid comprises an oil.

5. The method according to any of the preceding claims, wherein the first fluid comprises a sample, wherein the sample comprises at least one target oligonucleotide.

6. The method according to any of the preceding claims, wherein the first fluid comprises a reaction mixture, optionally wherein the reaction mixture is suitable for preparation of target oligonucleotides for an amplification reaction and / or comprises reagents and oligonucleotides suitable for an amplification reaction of target oligonucleotides.

7. The method of any of the preceding claims, wherein the first comprises any combination of: at least one oligonucleotide, and / or at least one amplification primer, and / or at least one enzyme, optionally wherein the enzyme is a polymerase.

8. The method according to any of the preceding claims, wherein the at least one liquid droplet is extruded from a nozzle tip of a pipettor tip at the first location, and the first location is spaced vertically above the second location.

9. The method according to Claim 8, wherein the pipettor has a frequency of liquid droplet drive, wherein the frequency of liquid droplet drive is between about 500 to about 100,000 droplets per second, between about 1 ,000 to about 50,000 droplets per second, between about 2,000 to about 25,000 droplets per second, between about 3,000 to about 15,000 droplets per second, or between about 4,000 to about 7,500 droplets per second, e.g. about 5,000 droplets per second.

10. The method according to any of the preceding claims, wherein the liquid droplet has an initial velocity of between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s.

11. The method according to any of the preceding claims, wherein the diameter of the liquid droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, between about 30 to about 50 pm, e.g. about 40 pm.

12. The method according to any ofthe preceding claims, wherein the volume of the liquid droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL.

13. The method according to any of the preceding claims, wherein the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv).

14. The method according to Claim 13, wherein the first velocity vector (Iv) has a speed between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s.

15. The method according to any of the preceding claims, wherein the liquid droplet has a direction of travel and wherein the direction of travel of the liquid droplet and the second fluid intersect at the second location.

16. The method according to Claim 1 or Claim 3, or any of Claims 4 to 15 when dependent on Claim 1 or Claim 3, wherein the second fluid has a speed and direction of travel defining a second velocity vector (sv).

17. The method according to Claim 16, wherein the second velocity vector (sv) has a speed between about 0.05 to about 3 m / s, between about 0.15 to about 2 m / s, between about 0.1 to about 1.5 m / s, between about 0.2 to about 1 m / s, or between about 0.25 to about 0.75 m / s, e.g. about 0.6 m / s.

18. The method according to Claim 1 or Claim 3, or any of Claims 4 to 17 when dependent on Claim 1 or Claim 3, wherein the second fluid has a direction of travel, and wherein the direction of travel ofthe liquid droplet and the direction of travel ofthe second fluid intersect at the second location.

19. The method according to any of the preceding claims, wherein the second location is at a surface of the continuous stream of the second fluid, or at a surface of the stagnant body of the second liquid.

20. The method according to Claim 18 or Claims 19, wherein the direction of travel of the liquid droplet and the direction of travel of the second fluid intersects at an angle of between about 10° and 175°, between about 1 1 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

21. The method according to Claim 1 or Claim 3, or any of Claims 4 to 20 when dependent on Claim 1 or Claim 3, wherein the second fluid is provided as a continuous stream.

22. The method according to Claim 22, wherein the continuous stream of the second fluid has a flow rate of between about 0.5 to about 100 pL / min, between about 1 to about 50 pL / min, between about 1.5 to about 20 pL / min, between about 1.75 to about 15 pL / min, or between about 2 to about 10 pL / min.

23. The method according to any of the preceding claims, wherein the third location comprises an amplification vessel, and optionally an imaging chip.

24. A system for generating emulsion droplets, the system comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid entering the main channel, and a distal end portion comprising an outlet for fluid exiting the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and a pipettor configured to generate and extrude at least one liquid droplet, the pipettor comprising: a nozzle having a nozzle tip from which the at least one liquid droplet is extruded; and wherein the pipettor is arranged such that the nozzle tip is located within the side port of the manifold at a first location spaced from the main channel of the manifold.

25. The system according to Claim 24, wherein the nozzle tip is spaced from the main channel of the manifold by a distance ‘E’ which defines an air gap; optionally wherein the distance E is a vertical distance.

26. The system according to Claim 25, wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, between about 1 mm to about 5 mm, at least about 1 .0 mm, about 1.1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.

27. The system of any of Claims 24 to 26, wherein the at least one emulsion droplet is transferred to a third location.

28. The system according to any of Claims 24 to 27, further comprising a pump to drive the flow of the second fluid, or to push the second fluid through the manifold.

29. The system according to any of Claims 24 to 28, wherein, in use, the pipettor is arranged to generate and extrude at least one liquid droplet comprising a first fluid at a first location, and wherein the at least one liquid droplet passes through the air gap between the first location and the main channel before contacting a second fluid flowing through the main channel at a second location.

30. The system according to Claim 29, wherein the first fluid is immiscible in the second fluid, and wherein the at least one liquid droplet is immersed in the second fluid, thereby generating at least one emulsion droplet of the first fluid within the second fluid.31 . The system according to any of Claims 24 to 30, wherein: the main channel has an elongate axis (ex) defined between the inlet and the outlet; the pipettor has a proximal end defined by the nozzle and the nozzle tip, and a distal end; and wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end of the pipettor.

32. The system according to Claim 31 , wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

33. The system according to any of Claims 24 to 32, wherein the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body.

34. The system according to Claim 33, wherein the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port.

35. The system according to any of Claims 24 to 34, wherein the side port and the elongate body of the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold.

36. The system according to Claims 34 or Claim 35, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

37. The system according to any of Claims 24 to 36, wherein the pipettor is directed axially- downwardly, wherein downwardly relates to a vertical direction.

38. The system according to any of Claims 24 to 37, wherein the liquid droplet has a speed and direction of travel defining a first velocity vector (Iv), and the second fluid has a speed and direction of travel defining a second velocity vector (sv), wherein the first velocity vector (Iv) and the second velocity vector (sv) intersect.39 The system according to Claim 38, wherein the first velocity vector (Iv) has a speed between about 0.001 to about 20 m / s, between about 0.0025 to about 10 m / s, between about 0.005 to about 5 m / s, between about 0.0075 to about 2 m / s, between about 0.0075 to about 1 m / s, between about 0.0075 to about 0.5 m / s, or between about 0.01 to about 0.1 m / s; and / or wherein the second velocity vector (sv) has a speed between about 0.05 to about 3 m / s, between about 0.1 to about 2 m / s, between about 0.15 to about 1.5 m / s, between about 0.2 to about 1 m / s, or between about 0.25 to about 0.75 m / s., e.g. about 0.6 m / s.40 The system according to Claim 38 or Claim 39, wherein contacting the at least one liquid droplet with a second fluid at a second location is at the intersection between the first velocity vector (Iv) and the second velocity vector (sv).

41. The system according to any of Claims 24 to 40, wherein the second fluid is provided as a continuous stream, and wherein the continuous stream of the second fluid flows along the fluid flow path.

42. The system according to Claim 41 , wherein the continuous stream of the second fluid has a flow rate of between about 0.5 to about 100 pL / min, between about 1 to about 50 pL / min, between about 1 .5 to about 20 pL / min, between about 1 .75 to about 15 pL / min or between about 2 to about 10 pL / min.

43. The system according to any of Claims 24 to 42, wherein the pipettor comprises a piezoelectric membrane; optionally wherein the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

44. The system according to any of Claims 24 to 43, wherein the main channel comprises a channel region of reduced diameter and / or cross-sectional area.

45. The system according to Claim 44, wherein the aperture in the wall of the elongate body directly communicates with the channel region of reduced diameter and / or cross-sectional area.

46. The system according to Claim 44 or Claim 45, wherein the channel region of reduced diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm; and optionally wherein the channel region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm.

47. The system according to any of Claims 24 to 46, wherein the main channel outlet communicates with a third location; optionally wherein the third location comprises an amplification vessel or an imaging chip.

48. The system according to any of Claims 24 to 47, wherein the first fluid comprises an aqueous liquid and the second fluid comprises an oil.

49. The system according to any of Claims 24 to 48, wherein the first fluid comprises a sample, wherein the sample comprises at least one target oligonucleotide.

50. The system according to any of Claims 24 to 49, wherein the first fluid comprises a reaction mixture, optionally wherein the reaction mixture is suitable for preparation of target oligonucleotides for an amplification reaction and / or comprises reagents and oligonucleotides suitable for an amplification reaction of target oligonucleotides.51 . The system according to any of Claims 24 to 50, wherein the first comprises any combination of:at least one oligonucleotide, and / or at least one amplification primer, and / or at least one enzyme, optionally wherein the enzyme is a polymerase.

52. The system according to any of Claims 24 to 51 , wherein the second fluid comprises an organic liquid, such as an oil.

53. The system according to any of Claims 24 to 52, wherein the diameter of the liquid droplet is between about 1 to about 200 pm, between about 5 to about 150 pm, between about 10 to about 100 pm, or between about 30 to about 50 pm, e.g. about 40 pm.

54. The system according to any of Claims 24 to 53, wherein the volume of the liquid droplet is between about 1 to about 1 ,000 pL, between about 5 to about 500 pL, between about 10 to about 250 pL, between about 20 to about 100 pL, or between about 25 to about 50 pL, e.g. about 33.5 pL.

55. An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, and a distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body, and wherein the side port is arranged at an angle relative to the elongate body.

56. The apparatus according to Claim 55, further comprising a pipettor comprising a nozzle having a nozzle tip, and wherein the nozzle tip is located within the side port.57: The apparatus according to Claim 56, wherein the pipettor is configured to dispense a liquid droplet from the nozzle tip.

58. An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising: an elongate body having a wall defining a main channel through the manifold, the elongate body having: a proximal end portion comprising an inlet for fluid to enter the main channel, anda distal end portion comprising an outlet for fluid to exit the main channel, the main channel extending between the inlet and the outlet to provide a fluid flow path therebetween; and a side port connecting with the elongate body and arranged between the inlet and the outlet of the elongate body, the side port in fluid communication with the main channel of the elongate body through an aperture in the wall of the elongate body; and a pipettor comprising a nozzle having a nozzle tip arranged within the side port, the nozzle tip positioned at a first location spaced from the main channel of the manifold, and wherein the nozzle of the pipettor is arranged at an angle relative to the elongate body.

59. The apparatus according to any of Claims 56 to 58, wherein the nozzle tip of the pipettor is separated from the main channel by a distance ‘E’.

60. The apparatus according to Claim 59, wherein distance E defines an air gap between the channel and nozzle tip.

61. The apparatus according to Claim 59 or Claim 60, wherein the distance E is a vertical distance.

62. The method according to any of Claims 59 to 61 , wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, between about 1 mm to about 5 mm, at least about 1 .0 mm, about 1.1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.

63. The apparatus according to any of Claims 55 to 62, further comprising a pump to drive the flow of the second fluid, or to push the second fluid through the manifold.

64. The apparatus according to any of Claims 55 to 63, wherein the main channel is substantially linear, and wherein the main channel has an elongate axis (ex) defined between the inlet and the outlet.

65. The apparatus according to any of Claims 55 to 64, wherein the side port has a proximal end connected to the wall of the elongate body and a distal end directed away from the elongate body.

66. The apparatus according to Claim 65, wherein the side port has a longitudinal axis (px) extending between the aperture in the wall of the elongate body at the proximal end of the side port to the distal end of the side port and running parallel with the side port.

67. The apparatus according to any of Claims 55 to 66, wherein the side port and the elongate body of the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (px) of the side port and the elongate axis (ex) through the proximal end of the elongate body of the manifold.

68. The apparatus according to Claim 66 or Claim 67, wherein the longitudinal axis (px) and the elongate axis (ex) intersect at an angle between about and the proximal end portion of the main channel is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

69. The apparatus according to any of Claims 56 to 58 or any of Claims 59 to 68 when dependent on any of Claims 56 to 58, wherein the pipettor comprises a piezoelectric membrane.

70. The apparatus according to Claim 69, wherein the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

71. The apparatus according to any of Claims 55 to 70, wherein the main channel comprises a channel region of reduced diameter and / or cross-sectional area.

72. The apparatus according to Claim 71 , wherein the aperture in the wall of the elongate body directly communicates with the channel region of reduced diameter and / or cross-sectional area.

73. The apparatus according to Claim 71 or Claim 72, wherein the channel region of reduced diameter and / or cross-sectional area has a cross-sectional diameter or width of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm; and / or wherein the channel region of reduced diameter and / or cross-sectional area has an axial length of up to about 1 ,000 mm, up to about 500 mm, up to about 400 mm, e.g. between about 0.3 and about 400 mm, between about 0.4 and about 350 mm, or between about 0.5 and about 300 mm.

74. The apparatus according to any of Claims 55 to 73, wherein the main channel outlet communicates with a third location; optionally wherein the third location comprises an amplification vessel or an imaging chip.

75. An apparatus for generating emulsion droplets, the apparatus comprising: a manifold, the manifold comprising a fluid flow path extending between an inlet and an outlet, and a liquid droplet inlet arranged to communicate with the fluid flow path at a location between the inlet and the outlet; and a pipettor configured to generate a liquid droplet, the pipettor comprising a nozzle having a nozzle tip from which a liquid droplet can be dispensed, the pipettor configured such that the nozzle tip is arranged to dispense a liquid droplet comprising a first fluid through the liquid droplet inlet of the manifold, in use, to contact a second fluid flowing along the fluid flow path.

76. The apparatus according to Claim 75, wherein the fluid flow path is defined by a channel through the manifold, and wherein the channel is configured to accept a continuous stream of the second fluid.

77. The apparatus according to Claim 75 or Claim 76, further comprising a pump to drive the flow of the second fluid or to push the second fluid through the manifold.

78. The apparatus according to any of Claims 75 to 77, wherein the nozzle tip of the pipettor is located within the liquid droplet inlet.

79. The apparatus according to any of Claims 75 to 78, wherein the nozzle tip is separated from the fluid flow path by a distance ‘E’; optionally wherein the distance E defines an air gap between the fluid flow path and the nozzle tip.

80. The method according to Claim 79, wherein the distance E has a length between about 1 mm to about 100 mm, 1 mm to about 75 mm, between about 1 mm to about 50 mm, between about 1 mm to about 25 mm, between about 1 mm to about 10 mm, between about 1 mm to about 5 mm, at least about 1 .9 mm, about 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, or about 2.0 mm.81 . The apparatus according to any of Claims 75 to 80, wherein the fluid flow path is substantially linear, and wherein the fluid flow path has an elongate axis (ex) defined between the inlet and the outlet of the manifold.

82. The apparatus according to any of Claims 75 to 81 , wherein the pipettor has a proximal end comprising the nozzle and the nozzle tip and a distal end.

83. The apparatus according to any of Claims 75 to 82, wherein the pipettor and nozzle tip have a longitudinal axis (tx) defined between the proximal end and the distal end.

84. The apparatus according to Claim 83, wherein the longitudinal axis (tx) and the elongate axis (ex) intersect at an angle, wherein the angle defined between the pipettor and the manifold inlet is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

85. The apparatus according to any of Claims 75 to 84, wherein the liquid droplet inlet has a proximal end connected to the wall of the manifold and a distal end directed away from the manifold.

86. The apparatus according to Claim 85, wherein the liquid droplet inlet has a longitudinal axis (px) extending between an aperture in the wall of the manifold at the proximal end of the liquid droplet inlet to the distal end of the liquid droplet inlet and running parallel with the liquid droplet inlet.

87. The apparatus according to Claim 85 or Claim 86 when dependent on Claim 81 , wherein the liquid droplet inlet and the manifold intersect at an angle, and wherein the angle is defined between the longitudinal axis (lx) of the liquid droplet inlet and the elongate axis (ex) defined between the inlet and the outlet of manifold.

88. The apparatus according to Claim 86 or Claim 87, wherein the longitudinal axis (lx) and the elongate axis (ex) intersect at an angle, wherein the angle is formed between the manifold inlet and the liquid droplet inlet, and wherein the angle is between about 10° and 175°, between about 11 ° and 160°, between about 12° and 145°, between about 13° and 130°, between about 14° and 120°, between about 15° and 110°, between about 16° and 100°, between about 17° and 90°, between about 18° and 80°, between about 19° and 70°, or between about 20° to about 60°.

89. The apparatus according to any of Claims 86 to 88, wherein the angle of intersection between the longitudinal axis (tx) and the elongate axis (ex) is the same as the angle of intersection between the longitudinal axis (px) and the elongate axis (ex).

90. The apparatus according to any of Claims 75 to 89, wherein the pipettor is directed axially- downwardly, wherein downwardly relates to a vertical direction.

91. The apparatus according to any of Claims 75 to 90, wherein the pipettor comprises a piezoelectric membrane; optionally wherein the piezoelectric membrane has a frequency of oscillation of between about 100 to about 50,000 Hz, between about 1 ,000 to about 25,000 Hz, between about 5,000 to about 20,000 Hz, or between about 10,000 to about 15,000 Hz, e.g. about 13,000 Hz.

92. The apparatus according to any of Claims 75 to 91 , wherein the manifold comprises a fluid flow path region of reduced diameter and / or cross-sectional area.

93. The apparatus according to Claim 92, wherein the region of the manifold comprising the liquid droplet inlet directly communicates with the fluid flow path region of reduced diameter and / or cross- sectional area.

94. The apparatus according to Claim 92 and Claim 93, wherein the fluid flow path region of reduced diameter and / or cross-sectional area has a cross-sectional diameter of between about 10 to about 500 pm, between about 25 to about 250 pm, or between about 75 to about 150 pm, e.g. about 100 pm; and / or wherein the fluid flow path region of reduced diameter and / or cross-sectional area has an axial length of between about 5 to 10,000 mm, between about 10 to about 5,000 mm, between about 20 to about 1 ,000 mm, between about 50 to about 500 mm, or between about 100 to about 250 mm.

95. The apparatus according to any of Claims 75 to 94, wherein the outlet communicates with a third location; optionally wherein the third location comprises an amplification vessel or an imaging chip.

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