阅读说明：本技术 微流控芯片中优化样品加载的孔 (Well for optimized sample loading in microfluidic chip ) 是由 尼古拉斯·费尔南德斯 艾蒂安·弗拉代 雷米·当格拉 于 2019-11-27 设计创作，主要内容包括：本发明涉及一种加载孔(320),其包括位于平行于基础平面(x/y)的横截面中的侧壁部分(3211)和/或包括至少一个倾斜底部(32121)的底壁部分。本发明还涉及一种包括加载孔的微流控芯片；包括加载孔的系统,该系统被配置为减少待加载到微流控芯片中的样品液滴的死体积和/或将液滴捕获在限定位置；另外还涉及使用加载孔的方法。(The invention relates to a loading aperture (320) comprising a side wall portion (3211) lying in a cross-section parallel to a base plane (x/y) and/or a bottom wall portion comprising at least one inclined bottom (32121). The invention also relates to a microfluidic chip comprising a loading well; a system comprising a loading well configured to reduce the dead volume of a sample droplet to be loaded into a microfluidic chip and/or to capture the droplet at a defined location; also, methods of using the loading wells.)

1. A loading well (320) configured to receive a droplet of sample (313),

wherein the loading aperture (320) is an open cavity (324) comprising a wall (321) and a loading opening (325),

wherein the wall (321) comprises a bottom wall portion (3212) extending integrally according to a bottom plane (wbp) parallel to a base plane (x/y) and a side wall portion (3211) extending along an aperture transverse direction (wld) arranged according to an angle 0 ° < α <180 ° with respect to the bottom plane (wbp),

wherein the loading opening (325) is defined by a free end of the side wall portion (3211) opposite the bottom plane (wbp), and

wherein:

-the bottom wall portion (3212) comprises at least one inclined bottom (32121),

-the inlet (330) is located off-centre in the bottom wall portion (3212).

2. A loading well (320) configured to receive a droplet of sample (313),

wherein the loading aperture (320) is an open cavity (324) comprising a wall (321) and a loading opening (325),

wherein the wall (321) comprises a bottom wall portion (3212) extending integrally according to a bottom plane (wbp) parallel to a base plane (x/y) and a side wall portion (3211) extending along a bore transverse direction (wld) arranged according to an angle 0 ° < α <180 ° with respect to the bottom plane (wbp), and wherein the loading bore (320) is at least partially filled with oil,

wherein the loading opening (325) is defined by a free end of the sidewall portion (23211) opposite the bottom plane (wbp), and

wherein:

-said bottom wall portion (3212) comprises at least one inclined bottom (32121).

3. The loading aperture (320) of any of claims 1 or 2, wherein the angle a has a value ranging from about 80 ° to about 105 ° relative to the bottom plane.

4. The loading aperture (320) according to any of claims 2 and 3, wherein the relative density of the oil is greater than 1.01, preferably greater than 1.05, preferably greater than 1.1, preferably greater than 1.5.

5. The loading aperture (320) of any of claims 1-4, wherein the inlet (330) is located at:

-a distance d in the base plane (x/y) from the curvature of the side wall portion (3211)_inlet-wallWherein the distance ranges from about 0.5mm to about 2.5 mm; and/or

-a distance in the base plane (x/y) from a deepest depth (d) of the bottom wall portion (3212) relative to the loading opening (325), the distance ranging from about 1mm to about 5 mm.

6. The loading aperture (2) of claim 5, wherein the curved portion of the sidewall portion (3211) has a radius of curvature ranging from about 0.5mm to about 2.5mm, preferably about d_inlet-wallThe radius of curvature of (a).

7. A microfluidic chip (300) comprising a loading well (320) according to any one of claims 1 to 6.

8. The microfluidic chip (300) of claim 7, further comprising a continuous phase (312), preferably wherein the continuous phase (312) partially or completely fills a microfluidic network of the microfluidic chip (300), more preferably wherein the microfluidic network of the microfluidic chip (300) comprises at least a droplet generator (340) and a droplet chamber (350).

9. A system for reducing the dead volume of a sample droplet (313) to be loaded into a microfluidic chip (300), the system comprising a loading well (320) according to any one of claims 1 to 6, wherein the system is configured to position the sample droplet (313) at a distance in the base plane (x/y) in the range of 0 to 1 times the diameter of the sample droplet (313).

10. A method for reducing the dead volume of a sample droplet (313) to be loaded into a microfluidic chip (300) comprising an inlet (330), the method comprising:

-providing a microfluidic chip (300) according to claim 7 or 8,

-placing a sample droplet (313) into the loading well (320),

such that the sample droplet (313) is located at a distance from the inlet (330) in the range of 0 to 1 times the diameter of the sample droplet (313).

11. A system for capturing and/or moving a sample droplet (313) in a well, the system comprising a loading well (320) according to any one of claims 1 to 6, wherein the system is configured to allow the sample droplet (313) to be captured and/or moved to a defined position of the loading well (320).

12. A method for capturing and/or moving a sample droplet (313) in a well, the method comprising:

-providing a loading hole (320) according to any of claims 1 to 6,

-placing a sample droplet (313) into the loading well (320),

thereby causing droplets of the dispersed phase to be captured and/or moved to a defined location of the loading aperture (320).

Background

Microfluidic processes typically use emulsions comprising droplets of a dispersed liquid phase surrounded by an immiscible continuous liquid phase. The droplets may be used as reaction vessels, storage vessels for chemical or biological reactions, and/or methods of separating and partitioning molecules (e.g., chemical or biological elements). The droplets may be "stabilised" by the use of a suitable chemical, such as a surfactant on the surface of the droplets, which means that they do not substantially mix and coalesce when in contact with each other. This stability allows one to create clusters or libraries of droplets of different chemical or biological components that can be stored in approximately the same volume of space without mixing or contamination between the components of one droplet and another.

One problem with the prior art is that, at present, the sample is lost when loaded into the microfluidic device. In some cases, it may be advantageous to design a specific geometry for the loading aperture. However, the existing geometry does not prevent the sample from being wasted and dead volume of the sample may remain in the well. This is a major problem with detection accuracy and reproducibility. This is also a major problem when dealing with rare samples (i.e. only available in very small quantities).

The present disclosure addresses these issues.

Disclosure of Invention

The present disclosure relates to a loading well configured to receive a sample droplet, wherein the loading well is an open cavity comprising a wall and a loading opening, wherein the wall comprises a bottom wall portion extending integrally according to a bottom plane parallel to a base plane (x/y) and a side wall portion extending along an aperture transverse direction arranged according to an angle 0 ° < α <180 ° with respect to the bottom plane, wherein the loading opening is defined by a free end of the side wall portion opposite to the bottom plane, and

wherein:

-the bottom wall portion comprises at least one inclined bottom,

-the inlet is located off-centre in the bottom wall portion.

The present disclosure also relates to a loading well configured to receive a droplet of sample,

wherein the loading aperture is an open cavity comprising a wall and a loading opening,

wherein the wall comprises a bottom wall portion extending integrally according to a bottom plane parallel to a base plane (x/y) and a side wall portion extending along a bore lateral direction arranged according to an angle 0 ° < α <180 ° with respect to the bottom plane, and wherein the loading bore is at least partially filled with oil,

wherein the loading opening is defined by a free end of the side wall portion opposite the bottom plane, and wherein the bottom wall portion includes at least one sloped bottom.

In other possible modifications, the following embodiments may be combined with the above two configurations.

In some embodiments, the angle a has a value ranging from about 80 ° to about 105 ° relative to the bottom plane.

In some embodiments, the relative density of the oil is greater than 1.01, preferably greater than 1.05, preferably greater than 1.1, preferably greater than 1.5.

In some embodiments, the inlet is located at:

-a distance d in the base plane (x/y) from the curvature of the side wall part_inlet-wallWherein the distance ranges from about 0.5mm to about 2.5 mm; and/or

-a distance in the base plane (x/y) from a deepest depth relative to the loading opening in the bottom wall portion, said distance ranging from about 1mm to about 5 mm.

In some embodiments, the curved portion of the sidewall portion has a radius of curvature ranging from about 0.5mm to about 2.5mm, preferably about d_inlet-wallThe radius of curvature of (a).

The present disclosure also relates to a microfluidic chip comprising a loading well according to the present invention.

In some embodiments, the microfluidic chip further comprises a continuous phase, preferably wherein the continuous phase partially or completely fills a microfluidic network of the microfluidic chip, more preferably wherein the microfluidic network of the microfluidic chip comprises at least a droplet generator and a droplet chamber.

The present disclosure also relates to a system for reducing the dead volume of a sample droplet to be loaded into a microfluidic chip, the system comprising a loading well according to the present invention, wherein the system is configured to position the sample droplet at a distance in the base plane (x/y) in the range of 0 to 1 times the diameter of the sample droplet.

The present disclosure also relates to a method for reducing the dead volume of a sample droplet to be loaded into a microfluidic chip comprising an inlet, the method comprising:

-providing a microfluidic chip according to the present disclosure,

-placing a sample droplet into the loading well,

such that the sample droplet is located at a distance from the inlet, the distance ranging from 0 to 1 times the diameter of the sample droplet.

The present disclosure also relates to a system for capturing and/or moving a sample droplet in a well, the system comprising a loading well according to the present disclosure, wherein the system is configured to allow the sample droplet to be captured and/or moved to a defined position of the loading well.

The present disclosure also relates to a method for capturing and/or moving a sample droplet in a well, the method comprising:

-providing a loading hole according to the present disclosure,

-placing a sample droplet into the loading well,

thereby causing droplets of the dispersed phase to be captured and/or moved to a defined location of the loading aperture.

The microfluidic devices disclosed herein have a number of advantages over other methods of forming and collecting droplets.

These advantages include:

(1) optimizing (i.e., increasing) the ability to load samples into the microfluidic chip, thereby reducing sample waste, for example, by reducing the dead volume of the dispersed phase;

(2) the ability to flow the continuous phase in the microfluidic chip without compromising the integrity of the droplet population;

(3) the ability to homogenize a locally static continuous phase throughout the droplet loading or generation process in a microfluidic chip;

(4) the ability to produce stable droplet populations without refreshing the continuous phase;

(5) the ability to optimize the reproducibility and accuracy of the detection, particularly the number of droplets produced per detection;

(6) the ability to increase the droplet/surface ratio in the droplet chamber;

(7) the ability to prevent warping of sensitive microfluidic channels in close proximity to loading holes in the microfluidic channels, and/or

(8) Optimizing the capacity of the microfluidic element for space occupation.

Drawings

Fig. 1 is a flow chart listing exemplary steps that may be performed in a method of sample analysis by droplet-based detection, according to some aspects of the present disclosure.

Fig. 2A is a perspective top view of an exemplary embodiment of a microfluidic chip 300.

Fig. 2B is a perspective bottom view of an exemplary embodiment of a microfluidic chip 300.

Fig. 3A is a perspective view of an exemplary embodiment of a system 100 for performing droplet-based detection, wherein the system 100 includes an instrument 200 and three microfluidic chips 300.

Fig. 3B is a detailed view of the receiving area 210 of the instrument 200 shown in fig. 2A, including three microfluidic chips 300.

Fig. 4 is a perspective view of an exemplary embodiment of a system 100 for performing droplet-based detection, wherein the system 100 includes an instrument 200 and three microfluidic chips 300.

Fig. 5 is a top view of an exemplary embodiment of a microfluidic chip 300 incorporating an array of microfluidic cells 301.

Fig. 6 is a side view of an exemplary embodiment of a microfluidic chip 300 incorporating an array of microfluidic cells.

Fig. 7 is a bottom view of an exemplary embodiment of a microfluidic chip 300 incorporating an array of microfluidic cells 301.

Fig. 8 is a side cross-sectional view of the loading aperture 320 according to plane B-B' in fig. 10.

Fig. 9 is a side cross-sectional view of the loading aperture 320 according to plane C-C' in fig. 10.

Fig. 10 is a top view of the loading holes 320 in the region indicated by "a" in fig. 5, shown in an enlarged manner.

Fig. 11 is a bottom view of one microfluidic cell in the area indicated by "D" in fig. 7, shown in an enlarged manner, specifically including a droplet generator 340, a droplet chamber 350, an air tank 360, and a chamber column 370.

Fig. 12 is a bottom plan view of one of the drop generators in the area indicated by "E" in fig. 11 shown in an enlarged manner.

Fig. 13 is a view of the bonding pad 341 in the region indicated by "F" in fig. 11 to 12 shown in an enlarged manner.

Fig. 14 is a bottom plan view of the ejector 343 in the region indicated by "G" in fig. 11 to 12 shown in an enlarged manner.

Fig. 15 is a side sectional view of a portion of the microfluidic chip 300 according to plane H-H' in fig. 5 or 7.

Fig. 16 is a side sectional view of the inlet 330 in the region indicated by "J" in fig. 15, shown in an enlarged manner.

Fig. 17 is a side cross-sectional view of a portion of the microfluidic chip 300 according to plane I-I' in fig. 5 or 7.

Fig. 18 is a side sectional view of the distribution passage 342 and the ejector 343 in the region indicated by "K" in fig. 17, shown in an enlarged manner.

Fig. 19 is a side sectional view of the ejector 343 in the region indicated by "L" in fig. 17 and the inclined region 344 operatively coupled to the droplet chamber 350, shown in an enlarged manner.

Fig. 20 is a side sectional view of the air tank 360 shown in an enlarged manner and taken according to the plane M-M' in fig. 11.

Fig. 21 is a side sectional view of the air tank 360 shown in an enlarged manner and taken according to the plane N-N' in fig. 11.

Fig. 22 is a side sectional view of the output channel 361 operably coupling the air tank and the droplet chamber 350 in the region indicated by "P" in fig. 21 shown in an enlarged manner.

Fig. 23 is a bottom view of the cell column 370 in the region indicated by "Q" in fig. 11, shown in an enlarged manner.

Fig. 24 is a schematic cross-sectional view of an exemplary embodiment of a loading well 320 filled with a continuous phase 312 and including a sample droplet 313 at a first location in the loading well according to the present disclosure.

Fig. 25 is a schematic cross-sectional view of an exemplary embodiment of a loading well 320 according to the present disclosure filled with a continuous phase 312 and including a sample droplet 313 at a second location in the loading well 320.

Fig. 26 is a schematic cross-sectional view of an exemplary embodiment of a loading well 320 filled with a continuous phase 312 and including a sample droplet 313 proximate to an inlet 330 according to the present disclosure.

Fig. 27-32 are schematic cross-sectional views of exemplary embodiments of loading wells 320 filled with continuous phase 312 and including the illustrated sample droplet 313 at different locations in loading well 320 according to the present disclosure.

Fig. 33 is a schematic illustration of the lattice of droplets 314.

Fig. 34 is an offset of three photographs showing the lattice of droplets 314 stored in droplet chamber 350 comprising circular cross-section (a and B) or elliptical cross-section (C) chamber pillars 370.

Fig. 35 is a schematic illustration of a lattice of droplets 314 in a droplet chamber 350 including diamond-shaped cross-section chamber pillars 370.

Fig. 36 is a photograph showing a lattice of droplets 314 stored in a droplet chamber 350 including diamond-shaped cross-sectional chamber pillars 370.

Fig. 37 is a schematic view of a sidewall portion of a loading aperture exhibiting sink marks at the bottom of the sidewall portion.

Fig. 38 is a side cross-sectional view of a microfluidic chip design including a narrow loading well.

Fig. 39 is a side cross-sectional view of a microfluidic chip design including a wide loading well 320.

Fig. 40 is a superposition of the top view of fig. 5 and the bottom view of fig. 7 with a transparent display according to the present disclosure, showing a microfluidic chip 300.

Fig. 41 is a schematic diagram of a portion of an exemplary microfluidic chip design including a droplet chamber operatively coupled to an inlet microchannel and an output channel by a capillary trap.

Fig. 42 is a schematic diagram of the exemplary microfluidic chip of fig. 41 during droplet loading.

Fig. 43 is a schematic of opposing drops and continuous phase flow in a drop chamber.

Fig. 44 is a schematic diagram of the exemplary microfluidic chip of fig. 41 after droplet loading is complete.

Fig. 45 is a schematic diagram of an exemplary microfluidic chip design including a droplet chamber operatively coupled to an inlet microchannel and an output channel by a capillary well, step a showing operation of the exemplary microfluidic chip design during droplet loading, and step B showing operation of the exemplary microfluidic chip design after droplet loading is complete.

Fig. 46 is a schematic diagram of an exemplary microfluidic chip design including a droplet chamber operatively coupled to an inlet microchannel by a droplet generator and to an output channel by a capillary trap, step a showing operation of the exemplary microfluidic chip design during droplet loading and step B showing operation of the exemplary microfluidic chip design after droplet loading is complete.

Fig. 47 is a schematic diagram of an exemplary microfluidic chip design including a droplet chamber operatively coupled to a plurality of inlet microchannels by a droplet generator and to an output channel by a capillary trap, step a showing operation of the exemplary microfluidic chip design during droplet loading and step B showing operation of the exemplary microfluidic chip design after droplet loading is complete.

Fig. 48 is a schematic diagram of an exemplary microfluidic chip design including a droplet chamber operatively coupled to a plurality of inlet microchannels by a droplet generator and to a plurality of output channels by a capillary trap, step a showing operation of the exemplary microfluidic chip design during droplet loading and step B showing operation of the exemplary microfluidic chip design after droplet loading is complete.

Fig. 49 is a perspective view of an exemplary microfluidic chip design including a droplet chamber operably coupled to a droplet generator including multiple ejectors and two air tanks through an output channel including a capillary trap.

Fig. 50 is a bottom plan view of the exemplary microfluidic chip design of fig. 49.

Fig. 51 is a schematic diagram of the exemplary microfluidic chip of fig. 49-50 in operation, with step a showing operation during droplet loading and step B showing operation after droplet loading is complete.

Fig. 52 is a bottom view of the microfluidic cell in operation in the region indicated by "D" in fig. 7, shown in an enlarged manner. Step a shows the operation during droplet loading and step B shows the operation after droplet loading is complete.

Detailed Description

The present disclosure provides components, devices, systems, devices and methods for performing droplet-based detection using microfluidic chips. For example, these may relate to, for example, preparing a sample for analysis (e.g., a clinical or environmental sample); separating the sample components by separating the components into droplets or other partitions, each droplet or partition containing only one or a few components (e.g., a single copy of a nucleic acid target or other analyte of interest); amplifying or otherwise reacting the components within the droplets or partitions; detecting the components or characteristics of the amplification or reaction; and/or analyzing data generated by the detection. In this way, a complex sample can be converted into a plurality of simpler, more easily analyzed samples, while reducing background and analysis time.

The following detailed description will be better understood when read in conjunction with the appended drawings. For purposes of illustration, devices, systems, and apparatuses are shown in the presently contemplated embodiments. It should be understood, however, that the present disclosure is not limited to the particular arrangements, structures, features, embodiments, and aspects shown. The drawings are not necessarily to scale and are not intended to limit the scope of the claims to the depicted embodiments.

It is, therefore, to be understood that where the features mentioned in the appended claims are followed by reference signs, these reference signs have been included for the sole purpose of increasing the intelligibility of the claims and shall not be intended to limit the scope of the claims in any manner.

Fig. 1 shows exemplary steps that may be performed in a sample analysis method by droplet-based detection. Briefly, droplet-based detection may include one or more of the following steps: sample preparation, droplet generation, reactions (e.g., amplification), detection, and data analysis. The assay can be used, for example, to perform digital Polymerase Chain Reaction (PCR) analysis.

More specifically, sample preparation can involve collecting or providing a sample (e.g., a clinical or environmental sample), processing the sample to release the relevant nucleic acid and/or forming a reaction mixture comprising the nucleic acid (e.g., for amplification of a target nucleic acid).

Droplet generation can involve encapsulating nucleic acids in droplets, e.g., each droplet having one or several copies of each target nucleic acid, where the droplets are suspended in a continuous phase (e.g., oil) to form an emulsion.

The reaction may involve subjecting the droplets to a suitable reaction, such as thermal cycling to induce PCR amplification, so that the target nucleic acid (if any) within the droplets is amplified to form additional copies.

Detection may involve detecting some signal from the droplet indicating whether amplification was achieved.

Finally, data analysis can involve estimating the concentration of the target nucleic acid in the sample based on the percentage of droplets in which amplification occurred.

These and other aspects of the apparatus, systems, devices, and methods are described below. In particular, provided herein are various aspects for microfluidic devices such as microfluidic chips, including but not limited to for reducing the dead volume of a dispersed phase to be injected into a microfluidic chip; for optimizing the space occupation of the microfluidic element by preventing warpage of the precision microfluidic channel; a two-dimensional (2D) lattice pattern for optimizing a layer of dispersed phase droplets in the droplet chamber; for increasing the dispersed phase droplet/surface ratio in the droplet chamber; a component and method for controlling a fluid carrier.

Definition of

In the present disclosure, the following terms have the following meanings:

the term "about" as used herein before a number or value refers to the margin or error of the number or value as would be readily understood by a worker skilled in the art. Specifically, the term "about" may refer to a margin of error of 1%, 2%, 5%, or 10%.

The term "amplicon" refers to the product of an amplification reaction. The amplicon may be single stranded or double stranded or a combination thereof. The amplicon corresponds to any suitable fragment or full length of the nucleic acid target.

The term "amplification" refers to a reaction in which replication occurs repeatedly over a period of time to form multiple copies of at least one fragment of a template molecule. Amplification can produce an exponential or linear increase in copy number as amplification proceeds. Typical amplification will increase copy number and/or signal by more than 1,000 fold. Exemplary amplification reactions for droplet-based detection disclosed herein may include Polymerase Chain Reaction (PCR) or ligase chain reaction, each of which is driven by thermal cycling. Droplet-based detection may also or alternatively use other amplification reactions that can be performed isothermally, such as branched-strand probe DNA detection, cascade rolling circle amplification (cascade-RCA), helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid-based amplification (NASBA), Nicking Enzyme Amplification Reaction (NEAR), PAN-AC, Q-beta replicase amplification, Rolling Circle Amplification (RCA), self-sustained sequence replication, strand displacement amplification, and the like. Amplification may utilize linear or circular templates. Amplification may be performed using any suitable reagent. Amplification may be performed or its occurrence detected in an amplification mixture, which is any composition capable of producing multiple copies of a nucleic acid target molecule (if present) in the composition. An "amplification mixture" may comprise any combination of at least one primer or primer pair, at least one probe, at least one replicase (e.g., at least one polymerase, such as at least one DNA and/or RNA polymerase), and deoxynucleotide (and/or nucleotide) triphosphates (dntps and/or NTPs), and the like.

The term "analyte" refers to a component or potential component of a sample that is analyzed in an assay. An "analyte" is a specific object of interest in an assay, in which a "sample" is a generic object of interest. The analyte may be, for example, a nucleic acid, protein, peptide, enzyme, cell, bacteria, spore, virus, organelle, macromolecular assembly, drug candidate, lipid, carbohydrate, metabolite, or any combination thereof, and the like. The presence, activity and/or other characteristics of the analyte in the sample and/or partitions thereof may be detected. The presence of an analyte can relate to an absolute or relative amount, concentration, binary assessment (e.g., presence or absence), etc., of the analyte in the sample or one or more partitions thereof. In some examples, the sample may be partitioned such that copies of the analyte are not present in all partitions, e.g., in partitions at an average concentration of about 0.0001 to 10000, 0.001 to 1000, 0.01 to 100, 0.1 to 10, or one copy per partition.

The term "detecting" refers to a procedure and/or reaction used to characterize a sample, as well as any signals, values, data, and/or results obtained from the procedure and/or reaction. An exemplary droplet-based assay is a biochemical assay using an aqueous assay mixture. More specifically, the droplet-based detection may be an enzyme detection and/or a binding detection, or the like. For example, enzyme detection can determine whether a single droplet contains copies of a substrate molecule (e.g., a nucleic acid target) of an enzyme and/or copies of an enzyme molecule. Based on these detection results, the concentration and/or copy number of the substrate and/or enzyme in the sample can be estimated.

The term "channel" refers to an elongated channel for the movement of a fluid. The channels generally include at least one inlet at which fluid enters the channel and at least one outlet at which fluid exits the channel. The functions of the inlet and outlet may be interchanged (i.e. fluid may flow through the channel in one direction only or in the opposite direction, typically at different times). The channel may include a wall defining and enclosing the channel between the inlet and the outlet. The channels may be formed, for example, by tubes (e.g., capillaries), in or on a planar structure (e.g., a chip), or by a combination thereof. The channel may or may not be branched. The channels may be linear or non-linear. Exemplary non-linear channels include channels that extend along a planar flow path (e.g., a serpentine channel), a non-planar flow path (e.g., a spiral channel that provides a spiral flow path). Any of the channels disclosed herein can be microfluidic channels, which are channels having a characteristic transverse dimension (e.g., average diameter of the channel) of less than about one mm. The channels may also include one or more venting mechanisms or dead ends to allow fluid entry/exit without the need for open outlets. Examples of a drain mechanism include, but are not limited to, a hydrophobic drain opening or the use of a porous material to form a portion of a channel or to block an outlet, if present. Examples of dead ends include, but are not limited to, air tanks.

The term "continuous phase," also referred to as a "carrier phase," "carrier" and/or "background phase," refers to a liquid or semi-liquid material in which immiscible materials (e.g., dispersed phases) are dispersed, e.g., to form an emulsion.

Examples of continuous phases for use in microfluidic systems are well known to those skilled in the art and include, but are not limited to, oils such as fluorinated oils, silicone oils, hydrocarbon oils, and the like.

Examples of suitable fluorinated oils include, but are not limited to, perfluoro-hexane, perfluoro-cyclohexane, perfluoro-decahydronaphthalene, perfluoro-perhydrophenanthrene, polyhexafluoropropylene oxide (e.g., polyhexafluoropropylene oxide having a carboxyl end group), perfluoropolytrimethylene ether, polyperfluoroalkylene oxide, fluorinated amines (e.g., N-bis (perfluorobutyl) -N-trifluoromethylamine, tris (perfluoropentyl) amine, a mixture of perfluorooctylamine and perfluoro-1-oxetanylamine or perfluorotripropylamine), fluorinated ethers (e.g., a mixture of methylnonafluorobutyl ether and perfluorobutyl methyl ether), 3-ethoxy-1, 1,1,2,3,4,4,5,5,6,6, 6-dodecafluoro-2- (trifluoromethyl) hexane, 2,3,3,4, 4-pentafluorotetrahydro-5-methoxy-2, 5-bis [1,2,2, 2-tetrafluoro-1-trifluoromethyl) ethyl ] -furan and mixtures thereof.

In some embodiments, the continuous phase may also include a surfactant, particularly a fluorinated surfactant (i.e., including at least one fluorine atom). Examples of suitable surfactants include, but are not limited to, perfluoro-octanol, 1H, 2H-perfluoro-1-octanol, perfluoro-decanol, 1H, 2H-perfluoro-1-decanol, perfluoro-tetradecane oligoethylene glycol, perfluoropolyether-polyethylene glycol-perfluoropolyether, perfluoropolyether-dimorpholino phosphate, polyhexafluoropropylene oxide carboxylate, polyhexafluoropropylene oxide polyethylene glycol-polyhexafluoropropylene oxide, polyhexafluoropropylene oxide polyether-polyhexafluoropropylene oxide ethylene glycol-polyethylene glycol-polypropylene glycol-polyhexafluoropropylene oxide, and mixtures thereof. Other exemplary surfactants include, but are not limited to, Span80 (sigma), Span80/Tween-20 (sigma), Span80/Triton X-100 (sigma), Abil EM90 (degussa), Abil we09 (degussa), polyglycerol polyricinoleate PGPR90 (Danisc), Tween-85, 749Fluid (Dow Corning), Krytox 157FSL ammonium carboxylate (DuPont), Krytox 157FSM ammonium carboxylate (DuPont), and Krytox 157FSH ammonium carboxylate (DuPont). Exemplary oil formulations to produce PCR stable emulsions for flow-through assays are commercially available and well known to those skilled in the art. An example of such a formulation includes the following mixtures: dow Corning 5225C Formulation Aid (10% active ingredient in decamethylcyclopentasiloxane), 20% w/w, 2% w/w final concentration of active ingredient; dow Corning 749Fluid (50% active ingredient in decamethylcyclopentasiloxane), 5% w/w, 2.5% w/w active ingredient; and poly (dimethylsiloxane) Dow CorningFluid, viscosity 5.0cSt (25 ℃), 75% w/w. Exemplary oil formulations to produce PCR stable emulsions for batch testing are commercially available and well known to those skilled in the art. An example of such a formulation includes the following mixtures: dow Corning 5225C Formulation Aid (10% active ingredient in decamethylcyclopentasiloxane), 20% w/w, 2% w/w final concentration of active ingredient; dow Corning 749Fluid (50% active ingredient in decamethylcyclopentasiloxane), 60% w/w, 30% w/w active ingredient; and poly (dimethylsiloxane) Dow CorningFluid, viscosity 5.0cSt (25 ℃), 20% w/w.

In some embodiments, the surface tension (at room temperature and atmospheric pressure) of the continuous phase/air interface is greater than about 1mn.m^-1About 2mN.m^-1About 5mN.m^-1About 10mN.m^-1About 20mN.m^-1About 30mN.m^-1About 40mN.m^-1About 50mN.m^-1About 75mN.m^-1About 100mN.m^-1About 250mN.m^-1About 500mN.m^-1. In some embodiments, the surface tension (at room temperature and atmospheric pressure) at the continuous phase/air interface ranges from about 1mn.m^-1To about 100mN.m^-1Preferably from about 1mn.m^-1To about 50mN.m^-1More preferably from about 1mn.m^-1To about 25mN.m^-1Even more preferably from about 5mn.m^-1To about 20mN.m^-1。

The term "dead volume" refers to the volume of fluid, i.e. the volume of dispersed phase (e.g. sample), that is not efficiently drained into the microfluidic network upon loading and is therefore left in the loading well. Dead volumes of dispersed phase are often encountered when handling small volumes of fluid, which may not be entirely drained into the microfluidic network and thus lost or wasted.

The term "digital PCR" or "dPCR" refers to a PCR assay performed on a portion 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 sample portions support target amplification. Digital PCRs may (or may not) be performed as endpoint PCRs. For each partition, the digital PCR may (or may not) be performed as a real-time PCR. PCR theoretically results in exponential amplification of the nucleic acid sequence (analyte) from the sample. By measuring the number of amplification cycles required to reach an amplification threshold level (as in real-time PCR), the initial concentration of nucleic acid can theoretically be calculated. However, in practice, there are many factors that make the PCR process non-exponential, such as different amplification efficiencies, low copy numbers of the starting nucleic acids, and competition with background contaminant nucleic acids. Digital PCR is generally insensitive to these factors because it does not rely on the assumption that the PCR process is exponential. In digital PCR, a single nucleic acid molecule is separated from an initial sample into multiple partitions, which are then amplified to detectable levels. Each partition then provides digital information about the presence or absence of each individual nucleic acid molecule within each partition. When sufficient partitions are measured using this technique, the numerical information can be combined to make a statistically relevant measurement of the initial concentration of the nucleic acid target (analyte) in the sample. The concept of digital PCR can be extended to other types of analytes besides nucleic acids. In particular, a signal amplification reaction may be utilized to allow detection of a single copy of an analyte molecule in a single droplet to allow data analysis of droplet signals of other analytes (e.g., using an algorithm based on poisson statistics). Exemplary signal amplification reactions that allow detection of single copies of other types of analytes in a droplet include enzymatic reactions.

The term "droplet" refers to a small volume of liquid (e.g., a dispersed phase), typically having a spherical shape, encapsulated by an immiscible fluid (e.g., a continuous phase). The volume of the droplets and/or the average volume of the population of droplets can be, for example, less than about 1 μ L (and thus referred to as "microdroplets"), less than about 1nL, or less than about 1 pL. The diameter (or average diameter) of the droplet (or population of droplets) may be less than about 1000 μm, about 100 μm, about 10 μm; or in the range of from about 10 μm to about 1000 μm. The droplets may be spherical or aspherical. The droplets may be simple droplets or complex droplets (i.e., droplets encapsulating at least one droplet). Droplets of emulsion in the continuous phaseMay have any uniform or non-uniform distribution. If not uniform, the concentration of droplets may be varied to provide one or more regions of higher droplet density and one or more regions of lower droplet density in the continuous phase. For example, the droplets may sink or float in the continuous phase, may collect in one or more packets along a channel or in a reservoir, may collect toward the center or periphery of the flow stream, and so forth. In some embodiments of the invention, the diameter (or average diameter) of the droplets ranges from about 10 μm to about 150 μm, preferably from about 25 μm to about 125 μm, more preferably from about 50 μm to about 100 μm, and even more preferably from about 65 μm to about 80 μm. In some embodiments of the invention, the diameter (or mean diameter) of the droplets is about 10 μm + -5 μm, 20 μm + -5 μm, 30 μm + -5 μm, 40 μm + -5 μm, 50 μm + -5 μm, 60 μm + -5 μm, 70 μm + -5 μm, 80 μm + -5 μm, 90 μm + -5 μm, 100 μm + -5 μm, 110 μm + -5 μm, 120 μm + -5 μm, 130 μm + -5 μm, 140 μm + -5 μm, 150 μm + -5 μm. In some embodiments of the invention, the diameter (or average diameter) of the droplets is about 75 μm 5 μm. The diameter of a droplet can also be defined mathematically as a function of its volume, as follows:in some embodiments of the invention, the volume (or average volume) of the droplets ranges from about 1pL to about 1nL, preferably from about 50pL to about 750pL, more preferably from about 100pL to about 500pL, and even more preferably from about 150pL to about 250 pL. In some embodiments of the invention, the volume (or average volume) of the droplet is 1pL, 10pL, 25pL, 50pL, 75pL, 100pL, 125pL, 150pL, 175pL, 200pL, 225pL, 250pL, 275pL, 300pL, 400pL, 500pL, 600pL, 700pL, 800pL, 900pL, 1 nL. In some embodiments of the invention, the volume (or average volume) of the droplets is 220pL ± 20 pL. Those skilled in the art will readily appreciate that such diameters and/or volumes may have a substantial margin of error.

The term "emulsion" refers to a composition comprising at least one droplet, particularly a population of droplets, disposed in an immiscible carrier fluid, which is also a liquid. The carrier fluid, also referred to as the background fluid, forms the "continuous phase". The droplets are formed from at least one droplet fluid (typically the sample), also referred to as the foreground fluid, which is a liquid that forms the "dispersed phase". The dispersed phase is immiscible with the continuous phase, which means that the dispersed and continuous phases are not homogeneous by mixing. In some embodiments, the density of the dispersed phase is at least about 1% less than the density of the continuous phase, preferably at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 150%, about 200% less. The droplets are isolated from each other by and encapsulated (i.e., enclosed or surrounded) by the continuous phase. Any of the emulsions disclosed herein may be monodisperse, i.e., composed of populations of droplets that are at least substantially uniform in size, or may be polydisperse, i.e., composed of populations of droplets that differ in size. If monodisperse, the droplets of the emulsion may, for example, vary in volume with a standard deviation of less than about plus or minus 100%, 50%, 20%, 10%, 5%, 2%, or 1% of the average droplet volume. The droplets produced from the orifice or from the droplet generator may be monodisperse or polydisperse. The emulsion may have any suitable composition. Emulsions can be characterized by the predominant liquid compound or type of liquid compound in each phase. The main liquid compounds in the emulsion may be water and oil. For example, any of the emulsions disclosed herein can be a water-in-oil (W/O) emulsion (i.e., water droplets in a continuous oil phase). Any other suitable ingredient may be present in any emulsion phase (dispersed and/or continuous), such as at least one surfactant, reagent, sample (i.e., partitions thereof), other additive, label, particle, or any combination thereof. Standard emulsions in an encapsulated state (e.g., each droplet is near an adjacent droplet) can become unstable when heated (e.g., to temperatures above 60 ℃), as heat typically reduces interfacial tension, which can lead to droplet coalescence. Thus, standard encapsulated emulsions cannot maintain their integrity during high temperature reactions (e.g., PCR) unless the emulsion droplets do not contact each other or additives (e.g., other oil bases, surfactants, etc.) are used to alter stability conditions (e.g., interfacial tension, viscosity, steric hindrance, etc.). For example, the droplets may be arranged in a single column and spaced apart from each other along the channel to allow thermal cycling to perform PCR. However, following this approach using standard emulsions does not allow for high density droplets, thereby significantly limiting the throughput of droplet-based detection. Any of the emulsions disclosed herein can be a heat stable emulsion. A "thermally stable emulsion" is any emulsion that resists coalescence when heated to at least 50 ℃. The thermostable emulsion can be a PCR stable emulsion, which is an emulsion that is resistant to coalescence throughout PCR thermocycling (e.g., allowing for digital PCR to be performed). Thus, the PCR stabilized emulsion can resist coalescence when heated to at least 80 ℃ or 90 ℃ or the like. Due to the thermal stability, the PCR stable emulsion enables PCR detection in droplets that do not coalesce during thermal cycling, compared to standard emulsions. Thus, the quantification of digital PCR analysis using PCR stabilized emulsions is significantly greater compared to using standard emulsions. For example, the emulsion can be tailored for PCR stabilization by proper selection of carrier streams and surfactants, among others.

The term "end-point PCR" refers to a PCR-based assay in which amplicon formation is measured after completion of thermal cycling.

The term "interface," when referring to an interface between a continuous phase and a dispersed phase, between a continuous phase and an air phase (simply referred to as air), or between a dispersed phase and an air phase, describes a surface that forms a common boundary between two adjacent immiscible or partially immiscible phases.

The term "tag" refers to an identifying and/or distinguishing mark or identifier attached to or incorporated into any entity, such as a compound, biological particle (e.g., cell, bacteria, spore, virus, or organelle), or droplet. For example, the label may be a dye that makes the entity optically detectable and/or optically distinguishable. Exemplary dyes for labeling are fluorescent dyes (fluorophores) and fluorescence quenchers.

The term "microfluidic channel" refers to a confined channel disposed within or on a substrate, wherein at least one cross-sectional dimension of the channel is in the range of about 0.1 μm to about 1 mm. In particular, the term "precision microfluidic channel" as used herein refers to a microfluidic channel having a level of ± 5% precision in its smallest dimension ranging from about 0.1 μm to about 200 μm.

The term "microfluidic chip" refers to a substrate comprising microfluidic channels, wherein within a microfluidic channel of the microfluidic chip, volumes as small as picoliters (pL) are processed. There are a variety of methods and materials for constructing microfluidic channels and networks thereof, which are well known and understood by those skilled in the art. For example, the microfluidic channel may use a simple tubing configuration, but may further involve sealing the surface of one plate including etched open channels to a second plate. Materials from which microfluidic channels can be formed include silicon, glass, Polydimethylsiloxane (PDMS), and plastics (such as polymethylmethacrylate, cyclo-olefin polymer [ COP ]]Cycloolefin copolymer [ COC ]]Polypropylene, etc.). The same material may also be used for the second sealing plate. The compatible combination of materials used for the two plates depends on the method used to seal them together. The microfluidic channels may be encapsulated in an optically transparent material as required to allow optical excitation (resulting in, for example, fluorescence) or illumination (resulting in, for example, selective absorption) of the sample as required and to allow optical detection of spectral characteristics of light from the sample in the microfluidic chip. Preferred examples of such optically transparent materials exhibiting high optical transparency and low autofluorescence include, but are not limited to, borosilicate glass (e.g., SCHOTT)Glass [ Schott North America, N.Y. ]Amsford]) And Cyclic Olefin Polymers (COPs) (e.g.,[ Zeon Chemicals LP, Louisville, Kentucky])。

The term "microfluidic network" refers to a component for manipulating a fluid, typically by transferring the fluid between compartments of the component and/or by driving the fluid along and/or through one or more flow paths defined by the component. The microfluidic network may include any suitable structure, such as one or more channels, chambers, wells, reservoirs, valves, pumps, thermal control devices (e.g., heaters/coolers), sensors (e.g., for measuring temperature, pressure, flow, etc.), or any combination thereof, and so forth. The microfluidic network may be constructed using simple tubing, but may further involve sealing the surface of one plate including an etched open structure as defined above to a second plate.

The term "nucleic acid" refers to both DNA or RNA, whether it be an amplification product, a synthetic product, an RNA reverse transcription product, or a naturally occurring product. Typically, a nucleic acid is a single-stranded or double-stranded molecule, consisting of naturally occurring nucleotides. Double-stranded nucleic acid molecules may have 3 'or 5' overhangs and thus need not be or are assumed to be completely double-stranded over their entire length. Furthermore, the term nucleic acid may consist of non-naturally occurring nucleotides and/or modifications to naturally occurring nucleotides. Examples are listed herein, but are not limited to, phosphorylation of 5 'or 3' nucleotides to allow ligation or prevent exonuclease degradation/polymerase extension, respectively; amino, thiol, alkyne, or biotin-based modifications for covalent and near-covalent attachment; a fluorophore and a quencher; phosphorothioate, methylphosphonate, phosphoramidate and phosphate linkages between nucleotides to prevent degradation; methylation; and modified bases such as deoxyinosine, 5-bromodU, deoxyuridine, 2-aminopurine, dideoxycytidine, 5-methyl dC, Locked Nucleic Acid (LNA), iso-dC and-dG bases, 2' -O-methyl RNA bases, and fluorine modified bases.

The term "nucleotide" is understood herein to refer to structural variants thereof in relation to, in addition to naturally occurring ribonucleotide or deoxyribonucleotide monomers, including functionally equivalent derivatives and analogs with respect to the particular context in which the nucleotide is used (e.g., hybridizes to a complementary base), unless the context clearly dictates otherwise.

The term "oil" refers to any liquid compound, or mixture of liquid compounds, that is immiscible with water and has a low polarity. In some embodiments, the oil may also have a high content of carbon, hydrogen, fluorine, silicon, oxygen, or any combination thereof, among others. Suitable examples of oils include, but are not limited to, silicone oils, mineral oils, fluorocarbon oils, vegetable oils, combinations thereof, and the like.

The term "operably coupled" is used herein to describe a connection between two or more separate instruments that are part of a system according to the present description. Two or more separate instruments are "operably coupled" if they are arranged such that two or more methods are performed by the two or more separate instruments and the two or more methods appear as a single workflow. Furthermore, two or more separate instruments may also be fully integrated into a third integrated instrument. Another possibility is to integrate the different key features of the various instruments described above into a dedicated integrated device (e.g. a single microfluidic chip containing the areas for microfluidic droplet generation, PCR amplification and droplet readout).

The term "partition" refers to a large volume of a discrete portion. A partition may be a sample partition resulting from the formation of a large volume of a sample (e.g., a prepared sample). The partitions generated from the large volumes may be of substantially uniform size or may be of different sizes (e.g., two or more discrete, uniformly sized groups of partitions). An exemplary partition is a "droplet". The size of the partitions may also vary with a predetermined size distribution or a random size distribution.

The term "PCR" or "polymerase chain reaction" refers to nucleic acid amplification detection that relies on alternating cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature set points (e.g., a higher melting (denaturation) temperature and a lower annealing/extension temperature), or between three or more temperature set points (e.g., a higher melting temperature, a lower annealing temperature, and an intermediate extension temperature, etc.). PCR can be performed with a thermostable polymerase, such as Taq DNA polymerase (e.g., wild-type enzyme, Stoffel fragment, FastStart polymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase, Vent polymerase, or a combination thereof. PCR typically produces an exponential increase in the product amplicon count in successive cycles. Any suitable PCR method or combination of methods can be used for the droplet-based detection disclosed herein, such as allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, end-point PCR, hot-start PCR, in situ PCR, inter-sequence specific PCR, inverse PCR, post-linear exponential PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap-extension PCR, polymerase cycle assembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermal asymmetric staggered PCR, touchdown PCR, or universal walking-fast PCR, among others.

The term "primer" refers to a polynucleotide that is capable of serving as a template-directed nucleic acid synthesis start point when placed under conditions in which polynucleotide extension is initiated (e.g., under conditions in which the necessary nucleoside triphosphates are present (as determined by the replicated template) and a polymerase in a suitable buffer and at a suitable temperature or temperature cycle (e.g., as in a polymerase chain reaction)). To further illustrate, the primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators for de novo RNA synthesis and in vitro transcription related processes (e.g., Nucleic Acid Sequence Based Amplification (NASBA), Transcription Mediated Amplification (TMA), etc.). The primer is typically a single-stranded oligonucleotide (e.g., an oligodeoxyribonucleotide). The appropriate length of the primer depends on the intended use of the primer, but is typically 6 to 40 nucleotides, more typically 15 to 35 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The primer need not reflect the exact sequence of the template, but is usefully complementary enough to hybridize to the template for primer extension to occur. In certain embodiments, the term "primer pair" refers to a set of primers, including a 5 'sense primer (sometimes referred to as "forward") that hybridizes to a complementary sequence at the 5' end of a nucleic acid sequence to be amplified and a 3 'antisense primer (sometimes referred to as "reverse") that hybridizes to the 3' end of the sequence to be amplified (e.g., if the target sequence is expressed as RNA or is RNA). If desired, the primers may be labeled by incorporating a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron dense reagents, enzymes (commonly used in ELISA assays), biotin or haptens, and proteins that may be used with antisera or monoclonal antibodies.

The term "probe" refers to a nucleic acid linked to at least one label (e.g., at least one dye). The probe may be a sequence-specific binding partner of a nucleic acid target and/or amplicon. The probe may be designed to be able to detect target amplification based on Fluorescence Resonance Energy Transfer (FRET). An exemplary probe for use in the detection of nucleic acids disclosed herein comprises one or more nucleic acids linked to a pair of dyes that collectively exhibit Fluorescence Resonance Energy Transfer (FRET) when brought into proximity with one another. The pair of dyes may provide first and second emitters, or one emitter and one quencher, and so on. The fluorescence emission of the pair of dyes may be altered when the dyes are separated from each other, for example by cleaving the probe during primer extension (e.g., 5' nuclease detection, e.g., using a TAQMAN probe), or when the probe hybridizes to an amplicon (e.g., a molecular beacon probe). The nucleic acid portion of the probe may have any suitable structure or origin, e.g., the portion may be a locked nucleic acid, a member of a universal probe library, etc. In other cases, one of the primers in the probe and primer pair can be combined in the same molecule (e.g., AMPLIFLUOR primer or SCORPION primer). For example, a primer-probe molecule may include a primer sequence at its 3 'end and a molecular beacon-type probe at its 5' end. With this arrangement, related primer-probe molecules labeled with different dyes can be used for multiplex detection, where the same reverse primer is used to quantify target sequences that differ by a single nucleotide (single nucleotide polymorphism (SNP)). Another exemplary probe for droplet-based nucleic acid detection is a Plexor primer.

The term "qualitative PCR" refers to a PCR-based assay that determines the presence or absence of a target in a sample, typically without any substantial quantification of the presence of the target. In an exemplary embodiment, qualitative digital PCR can be performed by determining whether a packet of droplets contains at least a predetermined percentage of positive droplets (positive sample) or no positive droplets (negative sample).

The terms "quantitative PCR", "qPCR", "real-time quantitative polymerase chain reaction" or "dynamic polymerase chain reaction" refer to a PCR-based assay that determines the concentration and/or copy number of a target in a sample. This technique uses PCR to simultaneously amplify and quantify a target nucleic acid, where quantification is achieved by an embedded fluorescent dye or sequence-specific probe comprising a fluorescent reporter molecule that is detectable only after hybridization to the target nucleic acid.

The term "reaction" refers to a chemical reaction, binding interaction, phenotypic change, or a combination thereof, which typically provides a detectable signal (e.g., a fluorescent signal) indicative of the occurrence and/or extent of the reaction. An exemplary reaction is an enzymatic reaction involving the enzymatic conversion of a substrate to a product. Any suitable enzymatic reaction may be performed in the droplet-based detection disclosed herein. For example, the reaction may be catalyzed by kinases, nucleases, nucleotide cyclases, nucleotide ligases, nucleotide phosphodiesterases, polymerases (DNA or RNA), prenyltransferases, pyrophosphatases, reporter enzymes (e.g., alkaline phosphatase, β -galactosidase, chloramphenicol acetyltransferase, glucuronidase, horseradish peroxidase, luciferase, etc.), reverse transcriptases, topoisomerases, and the like.

The term "reagent" refers to a compound, a group of compounds, and/or a composition that is combined with a sample to perform a specific assay on the sample. The reagent may be a target-specific reagent, which is any reagent composition that confers specificity to a particular target or analyte in a detection assay. The reagents optionally may include chemical reactants and/or binding partners for detection. For example, the agent may include at least one nucleic acid, protein (e.g., enzyme), cell, virus, organelle, macromolecular assembly, potential drug, lipid, carbohydrate, inorganic substance, or any combination thereof, and may be an aqueous composition, or the like. In exemplary embodiments, the reagents may be amplification reagents, which may include at least one primer or at least one pair of primers for amplifying a nucleic acid target, at least one probe and/or dye that allows detection of amplification, a polymerase, nucleotides (dntps and/or NTPs), divalent magnesium ions, potassium chloride, a buffer, or any combination thereof, and the like.

The term "real-time PCR" refers to a PCR-based assay in which amplicon formation is measured during the reaction, e.g., before the final thermal cycling of the reaction, and after completion of one or more thermal cycles. Real-time PCR generally provides for target quantification based on target amplification kinetics.

The term "relative density" of a substance refers to the ratio of the density (mass per unit volume) of the substance to the density of water as a reference substance under the same conditions of pressure and temperature.

The term "replication" refers to the process of forming copies (i.e., direct copies and/or complementary copies) of a nucleic acid or fragment thereof. Replication typically involves enzymes such as polymerases and/or ligases and the like. The replicated nucleic acids and/or fragments are the templates (and/or targets) for replication.

The term "reporter" refers to a compound or group of compounds that report a condition (e.g., extent of reaction). Exemplary reporter molecules include at least one dye, such as a fluorescent dye or energy transfer pair, and/or at least one oligonucleotide. Exemplary reporters for nucleic acid amplification detection may include probes and/or intercalating dyes (e.g., SYBR Green, ethidium bromide, etc.).

The term "reverse transcription PCR" or "RT-PCR" refers to PCR detection using complementary DNA templates resulting from reverse transcription of RNA. RT-PCR allows analysis of RNA samples by (1) forming complementary DNA copies of the RNA, e.g., using reverse transcriptase, and (2) performing PCR amplification using the complementary DNA as a template. In some embodiments, the same enzyme (e.g., Tth polymerase) may be used for reverse transcription and PCR.

The term "sample" refers to a compound, composition, and/or mixture of interest from any suitable source. A sample is a general object of interest for the detection of an aspect of an analytical sample, for example an aspect related to at least one analyte that may be present in the sample. The sample can be analyzed in its native state, as collected, and/or in an altered state, e.g., after storage, preservation, extraction, lysis, dilution, concentration, purification, filtration, mixing with one or more reagents, pre-amplification (e.g., target enrichment achieved by subjecting the sample to limited-cycle (e.g., <15) PCR prior to PCR), amplicon removal (e.g., treatment with uracil-d-glycosylase (UDG) prior to PCR to eliminate carryover contamination by previously generated amplicons (i.e., amplicons can be digested with UDG because it was generated with dUTP instead of dTTP)), partitions, or any combination thereof, and so forth. Clinical samples may include nasopharyngeal wash, blood, plasma, acellular plasma, buffy coat, saliva, urine, stool, sputum, mucus, wound swab, tissue biopsy, milk, liquid aspirate, swab (e.g., nasopharyngeal swab), and/or tissue, among others. Environmental samples may include water, soil, aerosol, and/or air, among others. Research samples may include cultured cells, primary cells, bacteria, spores, viruses, small organisms, any of the clinical samples listed above, and the like. Other samples may include food, weapon components, biodefense samples to be detected for bio-threat factors, suspected contaminants, and the like. The collected sample may be used for diagnostic purposes (e.g., quantitative measurement of a clinical analyte such as an infectious agent) or for monitoring purposes (e.g., determination that an environmental analyte of interest (such as a bio-threat agent) has exceeded a predetermined threshold).

In some embodiments, the sample may include one or several reagents, such as an amplification mixture.

In some embodiments, the sample droplet has a diameter of about 1mm to about 5mm, preferably about 1mm to about 4.5mm, more preferably about 1mm to about 4mm, even more preferably about 1mm to about 3.5mm, even more preferably about 2mm to about 3 mm. In some embodiments, the sample droplet has a diameter of about 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, 4.5mm, 4.6mm, 4.7mm, 4.8mm, 4.9mm, 5mm, or greater. In some embodiments, the sample droplet has a diameter of about 2.5mm ± 0.2 mm.

In some embodiments, the sample droplet has a volume of about 1 μ L to about 75 μ L, preferably about 1 μ L to about 50 μ L, more preferably about 1 μ L to about 40 μ L, even more preferably about 1 μ L to about 20 μ L, even more preferably about 5 μ L to about 10 μ L. In some embodiments, the sample droplet has a volume of about 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 6 μ L, 7 μ L, 8 μ L, 9 μ L, 10 μ L, 11 μ L, 12 μ L, 13 μ L, 14 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L, 50 μ L, 55 μ L, 60 μ L, 65 μ L, 70 μ L, 75 μ L or more. In some embodiments, the sample droplet has a volume of about 8 μ L ± 2 μ L.

The term "surfactant" refers to a surfactant that is capable of changing the surface tension between two phases. Surfactants can also or alternatively be described as detergents and/or wetting agents, incorporating both hydrophilic and hydrophobic moieties that together impart a dual hydrophilic-lipophilic character to the surfactant. The emulsion disclosed herein and/or any phase thereof may comprise at least one hydrophilic surfactant, at least one lipophilic surfactant, or a combination thereof. Alternatively or additionally, the emulsions disclosed herein and/or any phase thereof may comprise at least one nonionic (and/or ionic) detergent. Further, the emulsions disclosed herein and/or any phase thereof may include surfactants including polyethylene glycol, polypropylene glycol, or tween 20, and the like.

Microfluidic chip architecture

This section describes the architecture of illustrative components, devices, and systems suitable for droplet-based detection in microfluidic chips. The components, devices, and systems described herein can be used alone, in combination with one another, or in any number of different microfluidic chip configurations one such microfluidic chip is shown in fig. 2A and 2B. It should be appreciated that the microfluidic chip 300 of fig. 5-7 is not intended to limit the scope of the embodiments covered by the appended claims. For example, some aspects of the microfluidic chip 300 of fig. 2A and 2B may be used separately from other aspects of the microfluidic chip while using the disclosed configurations.

Fig. 2A and 2B illustrate an exemplary embodiment of a microfluidic chip 300 according to the present disclosure in perspective view.

In the embodiment shown in fig. 2A and 2B, the microfluidic chip 300 comprises an array of sixteen microfluidic cells, each comprising a loading well 320 formed and/or etched in the upper plate 310 leading to an inlet microchannel comprising a droplet generator 340, the droplet generator 340 being operatively coupled to a droplet chamber 350 and an air tank 360. The droplet chamber 350 further includes a chamber column 370.

Fig. 3A, 3B and 4 illustrate exemplary embodiments of suitable systems 100 in the sense of the present disclosure. Such a system may include an instrument 200 and a microfluidic chip 300 received by the instrument. The instrument 200 may be equipped with a receiving area 210 that allows for the placement of at least one or more microfluidic chips 300 into the instrument. In the embodiments shown in fig. 3A, 3B and 4, the microfluidic chip 300 is capped at the loading well level.

The instrument 200 can have an open configuration for receiving one or more microfluidic chips 300 and a closed configuration that restricts the introduction and removal of the microfluidic chips 300 (e.g., during instrument actuation of a loaded microfluidic chip 300). For example, the instrument may include a lid 220, a tray 230, or any other suitable member. In some embodiments, the lid, tray, or any other suitable member may be manually operated, or coupled to a drive mechanism that automatically drives the opening and/or closing of the receiving area 210. In some embodiments, the lid, tray, or any other suitable member may be heated. This is particularly useful for PCR assays where the thermal cycler (typically located below the microfluidic chip 300) and the lid, tray, or any other suitable component are heated to provide a more uniform temperature throughout the sample.

The instrument 200 may also be equipped with a user interface 240 as shown in fig. 3A and 3B. The instrument may be equipped with various other components, such as pressure manifolds, thermocyclers, detectors, pipette and pipette controllers, communication interfaces, control electronics, algorithms, and the like.

In the embodiments shown in fig. 3A, 3B and 4, three microfluidic chips 300 are shown in the receiving area 210.

In some embodiments, the instrument 200 can apply pressure to the microfluidic chip 300 to drive droplet generation. According to some aspects of the present disclosure, in some embodiments, an actuation signal may be input into the instrument 200 to cause the instrument 200 to apply pressure to the microfluidic chip 300 to drive droplet generation.

In some embodiments, the application of pressure may be maintained during the detection. In some embodiments, the application of pressure may be stopped when an endpoint of droplet generation is reached.

In the embodiment of fig. 5-7, the microfluidic chip 300 of fig. 2A and 2B is shown. The microfluidic chip 300 includes an array of sixteen microfluidic cells 301, each of which includes a loading well 320 with an inlet 330 to an inlet microchannel that includes a drop generator 340, the drop generator 340 operably coupled to a drop chamber 350 and an air tank 360. The droplet chamber 350 further includes a chamber column 370.

Specifically, the microfluidic chip 300 shown in fig. 5 to 7 includes an array of sixteen microfluidic cells 301. However, the present disclosure encompasses embodiments wherein the microfluidic chip comprises only one microfluidic cell, and wherein the microfluidic chip comprises several microfluidic cells, e.g. 2,3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more microfluidic units, such as 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 4, 73, 775, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96. In particular, the microfluidic chip may comprise 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88 or 96 microfluidic cells.

In some embodiments, the microfluidic chip 300 may be composed of two overlapping plates glued, bonded, or otherwise attached to each other. In some embodiments, the microfluidic chip comprises an upper plate 310, wherein the bottom of the upper plate 310 is in contact with a lower plate 311. For ease of illustration of the microfluidic chip 300, only the lower plate 311 is shown in fig. 6. In the embodiment shown in fig. 6, the elements of the microfluidic chip 300 shown in transparency are shown in grey dashed lines.

In the embodiment shown in fig. 7, the bottom side of the upper plate 310, i.e. the side of the upper plate 310 in contact with the lower plate 311, is etched in order to define a microfluidic network between the two plates when overlapping. In this regard, the lower plate 311 is flat.

In some embodiments, the lower plate 311 is light transmissive. In some embodiments, the lower plate 311 is optically transparent to be suitable or configured to view the microfluidic network defined by the overlapping upper and lower plates 310, 311 through a transparent member. By "light transmissive" is meant that the lower plate 311 has a light transmittance of greater than about 50%, preferably greater than about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more over a wavelength of light included in the range of at least about 100nm, preferably at least about 200nm, about 300nm, about 400nm, about 500nm, about 600nm or more in the 200-800nm spectrum.

In some embodiments, the lower plate 311 may also be non-fluorescent. By "non-fluorescent" is meant that lower plate 311 emits no or substantially no fluorescence when exposed to light. In some embodiments, "does not emit or substantially does not emit fluorescence" means that the emitted fluorescence is less than about 100AU, preferably less than about 80AU, about 60AU, about 40AU, about 25AU, about 20AU, about 15AU, about 10AU, about 5AU or less at excitation wavelengths included in the range of at least about 50nm, preferably at least about 100nm, about 200nm, about 300nm or more in the 300 and 600nm spectrum.

In some embodiments, lower plate 311 may be, for example, a foil, a film, a microscope slide, a glass slide, a molded polymer part, or any other suitable material.

In some embodiments, the lower plate 311 may be plastic, glass, or any other suitable material.

Examples of materials suitable for the light transmissive and non-fluorescent lower plate 311 are Cyclic Olefin Polymer (COP), Cyclic Olefin Copolymer (COC), polypropylene, polymethyl methacrylate or any other suitable material.

In some embodiments, the upper plate 310 of the microfluidic chip 300 forms a base that supports at least one loading well 320.

In the embodiment shown in fig. 8-10, the loading aperture 320 is an open cavity 324 that includes a loading opening 325.

In some embodiments, the loading well 320 has an x and/or y dimension that is less than about 100 sample droplets 313 in diameter, preferably less than about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 sample droplets 313 in diameter. In some embodiments, loading well 2 has an x and/or y dimension greater than about 1 sample droplet 313 diameter, preferably greater than about 2, about 3, about 4, about 5 sample droplets 313 diameter.

In some embodiments, the loading aperture 320 has a length (on the y-axis) ranging from about 2mm to about 20mm, preferably from about 5mm to about 15mm, more preferably from about 8mm to about 12 mm. In some embodiments, the loading aperture 320 has a length (on the y-axis) of about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, about 11mm, about 12mm, about 13mm, about 14mm, about 15mm, about 16mm, about 17mm, about 18mm, about 19mm, about 20mm, or more. In some embodiments, the loading aperture 320 has a length (in the y-axis) of about 9.3 mm.

In some embodiments, the loading aperture 320 has a width (in the x-axis) ranging from about 1mm to about 15mm, preferably from about 2.5mm to about 12.5mm, more preferably from about 5mm to about 10mm, even more preferably from about 6.5mm to about 8 mm. In some embodiments, the loading aperture 320 has a width (on the x-axis) of about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, about 11mm, about 12mm, about 13mm, about 14mm, about 15mm, or more. In some embodiments, the loading aperture 320 has a width (in the x-axis) of about 7.2 mm.

The loading aperture 320 is defined by a wall 321, the wall 321 including a bottom wall portion 3212 coupled to the side wall portion 3211.

In some embodiments, the bottom wall portion 3212 extends integrally according to a hole bottom plane wbp that is substantially parallel to the base plane (x/y).

In some embodiments, the sidewall portions 3211 extend along an aperture lateral direction wld (in the z-axis) disposed according to an angle α relative to the aperture bottom plane wbp, as shown in fig. 8-9. In some embodiments, angle α has a value ranging from about 80 ° to about 105 °, preferably from about 86 ° to about 100 °, more preferably from about 90 ° to about 96 °. In some embodiments, angle a has a value of about 80 °, about 85 °, about 90 °, about 95 °, about 100 °, about 105 °, or more. In some embodiments, the angle α has a value of about 93 °.

In some embodiments, the sidewall portion 3211 has a thickness (at the level of the aperture bottom plane wbp) ranging from about 0.25mm to about 2.5mm, preferably from about 0.5mm to about 2mm, more preferably from about 0.75mm to about 1.75mm, even more preferably from about 1mm to about 1.5 mm. In some embodiments, sidewall portion 3211 has a thickness (at the level of base plane wbp) of about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2mm, about 2.1mm, about 2.2mm, about 2.3mm, about 2.4mm, about 2.5mm, or more. In some embodiments, the side wall portion 3211 has a thickness (at the level of the bottom plane wbp) of about 1.2 mm.

In some embodiments, the loading opening 325 is defined by the free end of the sidewall portion 3211 opposite the bottom.

In some embodiments, the sidewall portion 3211 has a thickness (at the level of the loading opening 325) ranging from about 0.1mm to about 1.25mm, preferably from about 0.25mm to about 1mm, more preferably from about 0.5mm to about 0.75 mm. In some embodiments, the sidewall portion 3211 has a thickness (at the level of the loading opening 325) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, or more. In some embodiments, the sidewall portions 3211 have a thickness (at the level of the loading opening 325) of about 0.6 mm.

In some embodiments, the inlet 330 may be housed in the side wall portion 3211 or the bottom wall portion 3212 of the wall 321, preferably in the bottom wall portion 3212.

In some embodiments, the inlet 330 has a height (in the z-axis) ranging from about 0.1mm to about 1.25mm, preferably from about 0.25mm to about 1mm, more preferably from about 0.5mm to about 0.75 mm. In some embodiments, the inlet 330 has a height (in the z-axis) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2 mm. In some embodiments, the inlet 330 has a height (in the z-axis) of about 0.6 mm.

In some embodiments, the inlet 330 has a diameter (at the inner edge 3412, on the x/y axis) ranging from about 0.1mm to about 1.5mm, preferably from about 0.25mm to about 1.25mm, more preferably from about 0.5mm to about 1 mm. In some embodiments, the inlet 330 has a diameter (on the x/y axis at the inner edge 3412) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5 mm. In some embodiments, the inlet 330 has a diameter (at the inner edge 3412, in the x/y axis) of about 0.8 mm.

In some embodiments, the inlet 330 may include an inlet plane 323 extending radially outward from the inlet 330 in the base plane (x/y). In some embodiments, the inlet plane 323 is a radial region around the inlet 330 that does not exhibit a slope. In some embodiments, the inlet plane 323 is a radial region around the inlet 330 parallel to the base plane (x/y). In some embodiments, the inlet plane 323 has a diameter ranging from about 0.5mm to about 3mm, preferably from about 1mm to about 2.5mm, more preferably from about 1.5mm to about 2 mm. In some embodiments, the inlet plane 323 has a diameter of about 0.5mm, about 0.75mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2mm, about 2.1mm, about 2.2mm, about 2.3mm, about 2.4mm, about 2.5mm, about 2.75mm, about 3mm, or greater. In some embodiments, the inlet plane 323 has a diameter of about 1.8 mm.

In some embodiments, the inlet 330 is off-center. The off-center location of the inlet 330 allows the maximum possible surface of the bottom wall portion 3212 to be utilized.

"off-center" refers to being away from the center or centroid of the loading aperture 320. In some embodiments, the distance from the center or centroid of the inlet to the center or centroid of the loading aperture 320 is greater than 20%, preferably greater than 30%, 40%, 50% of the maximum distance from the center or centroid of the loading aperture 320 to the side wall portion 3211 of the loading aperture 320. In some embodiments, the inlet 330 is proximate the sidewall portion 3211.

In one embodiment, the inlet 330 is centered relative to the sidewall portion 3211.

In some use conditions, as partially shown in fig. 24, the microfluidic chip 300 is at least partially filled with the continuous phase 312 and the loading wells 320 include a shallow continuous phase 312 that overflows from the microfluidic network (i.e., the continuous phase in the microfluidic network and the continuous phase layer in the loading wells are continuous).

In one embodiment, the continuous phase is an oil, in particular a fluorinated oil. The relative density of the oil is greater than 1.01, preferably greater than 1.05, preferably greater than 1.1, preferably greater than 1.5. When the relative density of the oil is greater than 1.01, droplets (313, which are substantially aqueous) are expected to float on the oil. In fact, the sum of the surface tension between the oil and the air and the surface tension between the oil and the liquid droplets is less than the surface tension between the air and the liquid droplets. Then, a thin layer of oil is formed around the droplets according to the energy minimization principle. The interface surface between the oil and the air then tends to become minimal (competing with buoyancy), which corresponds to the droplets being located above the higher depth d of the loading hole, as shown in fig. 25.

In some embodiments, the continuous phase 312 fills a volume of the microfluidic chip 300 including at least a volume of the inlet microchannel 345 and a volume of the droplet chamber 350. In some embodiments, the continuous phase 312 fills the microfluidic chip 300 including at least the volume of the droplet generator 340 and the volume of the droplet chamber 350. In one embodiment, the continuous phase 312 further fills the volume of the output channel 361. In one embodiment, the continuous phase 312 does not fill the volume of the air tank 360.

In some embodiments, the height (in the z-axis) of continuous phase layer 312 in loading well 320 at deeper position d is less than about 5 times the diameter of sample droplet 313, preferably less than about 4, about 3, about 2, about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 times the diameter of sample droplet 313. In some embodiments, the z-axis height of continuous phase layer 312 in loading well 320 at a deeper position d of loading well 320 is less than about 1 times the diameter of sample droplet 313, preferably less than about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 times the diameter of sample droplet 313. In some embodiments, the height (in the z-axis) of the continuous phase layer 312 in the loading well 320 at the deeper position d is about 0.4 times the diameter of the sample droplet 313.

In some embodiments, the height (in the z-axis) of the continuous phase layer 312 in the loading aperture 320 at the deeper position d is less than about 12.5mm, preferably less than about 10mm, about 7.5mm, about 5mm, about 2.5mm, about 2.25mm, about 2mm, about 1.75mm, about 1.5mm, about 1.25mm, about 1mm, about 0.75mm, about 0.5mm, about 0.25 mm. In some embodiments, the z-axis height of the continuous phase layer 312 in the loading aperture 320 at the deeper position d of the loading aperture 320 is less than about 2.5mm, preferably less than about 2.25mm, about 2mm, about 1.75mm, about 1.5mm, about 1.25mm, about 1mm, about 0.75mm, about 0.5mm, about 0.25 mm. In some embodiments, the height (in the z-axis) of the continuous phase layer 312 in the loading hole 320 at the deeper position d is about 1mm ± 0.2 mm.

In some embodiments, the volume of the continuous phase layer 312 in the loading well 320 is less than about 150 μ L, preferably less than about 100 μ L, about 95 μ L, about 90 μ L, about 85 μ L, about 80 μ L, about 75 μ L, about 70 μ L, about 65 μ L, about 60 μ L, about 55 μ L, about 50 μ L, about 45 μ L, about 40 μ L, about 35 μ L, about 30 μ L, about 25 μ L, about 20 μ L, about 15 μ L, about 10 μ L. In some embodiments, the volume of the continuous phase layer 312 in the loading well 320 is about 35 μ L ± 2.5 μ L.

In some embodiments, the wetting angle (i.e., contact angle θ) between the sidewall portions 3211 of the loading apertures 320 and the continuous phase 312 is less than about 90 °, preferably less than about 80 °, about 70 °, about 60 °, about 50 °, about 40 °, about 30 °, about 20 °, about 10 °, or less. In a preferred embodiment, the wetting angle between the sidewall portions 3211 of the loading apertures 320 and the continuous phase 312 is flat, i.e., about 0 °.

In a first alternative embodiment seen in fig. 8-9, the load well 320 is configured to move and/or capture the sample droplet 313 at a z-position defined within the load well 320.

In some embodiments, the bottom wall portion 3212 is non-planar and includes a sloped bottom 32121. Thus, the bottom wall portion 3212 of the loading aperture 320 may be channel-shaped, cup-shaped, or bowl-shaped. In some embodiments, the depth d of the loading aperture 320 (from the loading opening 325 to the bottom wall portion 3212) is non-uniform. In some embodiments, the sloped bottom 32121 includes at least one slope. In some embodiments, the deeper position d of the inclined bottom 32121 accommodates the loading port 330. In some embodiments, the deeper position d of the inclined bottom 32121 accommodates the entrance plane 323.

In some embodiments, the height of the inclined bottom 32121 (the deeper position from the well bottom plane wbp to the inclined bottom 32121) ranges from about 0.1mm to about 5mm, preferably from about 0.1mm to about 2.5mm, more preferably from about 0.5mm to about 1.5 mm. In some embodiments, the height of the sloped bottom 32121 (the deeper position from the well bottom plane wbp to the sloped bottom 32121) is about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2mm, about 2.5mm, about 3mm, about 3.5mm, about 4mm, about 4.5mm, about 5mm, or greater. Height of the inclined bottom 32121 in some embodiments, the height of the inclined bottom 32121 (the deeper position from the well bottom plane wbp to the inclined bottom 32121) is about 1.01 mm.

In some embodiments, the slope of the inclined bottom 32121 is constant from the side wall portion 3211 to the deeper position of the inclined bottom 32121. In some embodiments, the slope of the inclined bottom 32121 is not constant, i.e., varies from the side wall portion 3211 to the deeper position of the inclined bottom 32121. In the latter embodiment, the average inclination angle may be defined as the depth from the well bottom plane wbp (i.e., at the level of the side wall portion 3211) to the inclined bottom 32121.

In some embodiments, the inclined bottom 32121 includes a primary slope according to the longitudinal axis (in the y-axis) that has an average inclination angle δ (from the well bottom plane wbp to the deeper position of the inclined bottom 32121), as shown in fig. 9. In some embodiments, the average inclination angle δ has a value ranging from about 1 ° to about 45 °, preferably from about 1 ° to about 30 °, more preferably from about 1 ° to about 20 °, even more preferably from about 5 ° to about 15 °, even more preferably from about 5 ° to about 10 °. In some embodiments, the average tilt angle δ has a value of about 1 °, about 2 °, about 3 °, about 4 °, about 5 °, about 6 °, about 7 °, about 8 °, about 9 °, about 10 °, about 11 °, about 12 °, about 13 °, about 14 °, about 15 °, about 16 °, about 17 °, about 18 °, about 19 °, about 20 °, about 21 °, about 22 °, about 23 °, about 24 °, about 25 °, about 30 °, about 35 °, about 40 °, about 45 °. In some embodiments, the average tilt angle δ has a value of about 8.5 ° ± 2 °.

In some embodiments, the sloped bottom 32121 includes a slope according to a first lateral axis (in x)₁On-axis) having an average inclination angle γ (from the hole bottom plane wbp to the deeper position of the inclined bottom 32121), as shown in fig. 8. In some embodiments, the average inclination angle γ has a value ranging from about 1 ° to about 45 °, preferably from about 5 ° to about 35 °, more preferably from about 10 ° to about 25 °, even more preferably from about 15 ° to about 20 °. In some embodiments, the average tilt angle γ has a value of about 1 °,2 °,3 °,4 °,5 °,6 °, 7 °, 8 °, 9 °, 10 °, 11 °, 12 °, 13 °, 14 °, 15 °, 16 °, 17 °, 18 °, 19 °, 20 °, 21 °, 22 °, 23 °, 24 °, 25 °, 30 °, 35 °, 40 °, 45 °. In some embodiments, the average tilt angle γ has a value of about 19.2 ° ± 2 °.

In some embodiments, the sloped bottom 32121 includes a slope according to a second lateral axis (in x)₂On-axis) having an average inclination angle β (from the well bottom plane wbp to the deeper position of the inclined bottom 32121), as shown in fig. 8. In some embodiments, the average inclination angle β has a value ranging from about 1 ° to about 45 °, preferably from about 5 ° to about 35 °, more preferably from about 10 ° to about 25 °, even more preferably from about 15 ° to about 20 °. In some embodiments, the average tilt angle β has about 1 °,2 °,3 °,4 °,5 °,6 °, 7 °, 8 °, 9 °, 10 °, 11 °, 12 °, 13 °, 14 °, 15 °, 16 °, 17 °, 18Values of ° 19 °, 20 °, 21 °, 22 °, 23 °, 24 °, 25 °, 30 °, 35 °, 40 °, 45 °. In some embodiments, the average tilt angle β has a value of about 19.2 ° ± 2 °.

In some embodiments, the inclined bottom portion 32121 of the bottom wall portion 3212 is defined by at least one, and in particular two or three, average inclination angles β, γ, δ relative to the well bottom plane wbp.

In some embodiments, at least two, specifically three, of 1) the main inclined bottom, 2) the first laterally inclined bottom, and 3) the second laterally inclined bottom converge toward a convergence point cp. In some embodiments, convergence point cp is located on bottom wall portion 3212. In some embodiments, the convergence point cp is located at a deeper position d on the bottom wall portion 3212 relative to the loading opening 325.

In a second alternative embodiment seen in fig. 10, the loading well 320 is configured to move and/or capture the sample droplet 313 in a (x and/or y) position within a plane defined within the loading well 320.

In some embodiments, the sidewall portions 3211 of the loading apertures 320 have a variable in-plane local curvature.

In one embodiment, the sidewall portions 3211 of the loading aperture 320 have an elliptical general shape in cross-section parallel to the base plane (x/y).

In some embodiments, the sidewall portions 3211 of the loading apertures 320 have a circular general shape in a cross-section parallel to the base plane (x/y).

As shown in fig. 10, from C to C', the sidewall portion 3211 includes a plurality of segments shown in a cross-sectional plane parallel to the base plane (x/y) of the upper plate 310.

The first straight segment 32111 is coupled to the first curved segment 32112.

The first curved segment 32112 is coupled to the second straight segment 32113.

The second straight segment 32113 is coupled to the second curved segment 32114.

The second curved segment 32114 is coupled to a third straight segment 32115.

The third straight segment 32115 is coupled to a third curved segment 32116.

The third curved segment 32116 is coupled to the fourth straight segment 32117.

The fourth straight segment 32117 is coupled to a fourth curved segment 32118.

Fourth curved segment 32118 is coupled to fifth straight segment 32119.

In some embodiments, the segments 32111-32119 are symmetrical with respect to the major axis MA of the sidewall portion 3211 of the loading aperture 320. As shown in fig. 10, the first and fifth linear segments 32111, 32119 intersect the primary axis MA at their centers.

In some embodiments, first straight segment 32111 has a length (from major axis MA to first curved segment 32112) ranging from about 0.1mm to about 1mm, preferably from about 0.25mm to about 0.75mm, more preferably from about 0.3mm to about 0.7mm, and even more preferably from about 2.2mm to about 2.4 mm. In some embodiments, first straight segment 32111 has a length (from major axis MA to first curved segment 32112) of about 0.5 mm.

In some embodiments, the first curved segment 32112 has a length ranging from about 0.5mm to about 3mm, preferably from about 0.75mm to about 2mm, more preferably from about 1mm to about 1.75mm, even more preferably from about 1.25mm to about 1.5 mm. In some embodiments, the first curved segment 32112 has a length of about 1.4 mm.

In some embodiments, the first curved segment 32112 has a radius of curvature ranging from about 1.5mm to about 3.5mm, preferably from about 1.75mm to about 3.0mm, more preferably from about 2.0mm to about 2.6mm, even more preferably from about 2.2mm to about 2.4 mm. In some embodiments, the first curved segment 32112 has a radius of curvature of about 2.3 mm.

In some embodiments, second straight segment 32113 has a length ranging from about 0.5mm to about 3mm, preferably from about 0.75mm to about 2mm, more preferably from about 1mm to about 1.75mm, even more preferably from about 1.25mm to about 1.5 mm. In some embodiments, second straight segment 32113 has a length of about 1.4 mm.

In some embodiments, the two symmetrical second straight line segments 32113 on each side of the primary axis MA converge toward the primary axis MA at an opening angle s ranging from about 45 ° to about 90 °, preferably from about 50 ° to about 85 °, more preferably from about 60 ° to about 80 °, even more preferably from about 65 ° to about 75 °. In some embodiments, the angle ε has a value of about 45 °, about 50 °, about 55 °, about 60 °, about 65 °, about 70 °, about 75 °, about 80 °, about 85 °, about 90 °. In some embodiments, the opening angle ε has a value of about 70 °.

In some embodiments, second curved segment 32114 has a length ranging from about 1.5mm to about 3.5mm, preferably from about 1.75mm to about 3.0mm, more preferably from about 2.0mm to about 2.6mm, even more preferably from about 2.1mm to about 2.3 mm. In some embodiments, the second curved segment 32114 has a length of about 2.21 mm.

In some embodiments, second curved segment 32114 has a radius of curvature ranging from about 1.5mm to about 3.5mm, preferably from about 1.75mm to about 3.0mm, more preferably from about 2.0mm to about 2.6mm, even more preferably from about 2.2mm to about 2.4 mm. In some embodiments, the second curved segment 32114 has a radius of curvature of about 2.3 mm.

In some embodiments, third straight segment 32115 has a length ranging from about 0.25mm to about 2.5mm, preferably from about 0.5mm to about 2mm, more preferably from about 0.75mm to about 2.5mm, even more preferably from about 1mm to about 1.25 mm. In some embodiments, third straight segment 32115 has a length of about 1.1 mm.

In some embodiments, third curved segment 32116 has a length ranging from about 0.25mm to about 2.5mm, preferably from about 0.5mm to about 2mm, more preferably from about 0.75mm to about 2.5mm, even more preferably from about 1mm to about 1.25 mm. In some embodiments, the third curved segment 32116 has a length of about 1.12 mm.

In some embodiments, third curved segment 32116 has a radius of curvature ranging from about 1.5mm to about 3.5mm, preferably from about 1.75mm to about 3.0mm, more preferably from about 2.0mm to about 2.6mm, even more preferably from about 2.2mm to about 2.4 mm. In some embodiments, the third curved segment 32116 has a radius of curvature of about 2.3 mm.

In some embodiments, fourth straight section 32117 has a length that ranges from about 1mm to about 5mm, preferably from about 2mm to about 4mm, more preferably from about 2.5mm to about 3.5mm, even more preferably from about 2.75mm to about 3.25 mm. In some embodiments, fourth straight section 32117 has a length of about 3.1 mm.

In some embodiments, the two fourth straight line segments 32117 on each side of the primary axis MA converge toward the primary axis MA at an angle λ ranging from about 20 ° to about 90 °, preferably from about 30 ° to about 80 °, more preferably from about 40 ° to about 70 °, even more preferably from about 50 ° to about 60 °. In some embodiments, the angle λ has a value of about 20 °, about 25 °, about 30 °, about 35 °, about 40 °, about 45 °, about 50 °, about 55 °, about 60 °, about 65 °, about 70 °, about 75 °, about 80 °, about 85 °, about 90 °. In some embodiments, the angle λ has a value of about 56 °.

In some embodiments, fourth curved segment 32118 has a length ranging from about 1mm to about 3mm, preferably from about 1.25mm to about 2.5mm, more preferably from about 1.5mm to about 2.25mm, even more preferably from about 1.75mm to about 2 mm. In some embodiments, fourth curved segment 32118 has a length of about 1.95 mm.

In some embodiments, the fourth curved segment 32118 has a radius of curvature ranging from about 1mm to about 2.5mm, preferably from about 1.25mm to about 2.25mm, more preferably from about 1.5mm to about 2.0mm, even more preferably from about 1.7mm to about 1.9 mm. In some embodiments, the fourth curved segment 32118 has a radius of curvature of about 1.8 mm.

In some embodiments, fifth straight segment 32119 has a length (from fourth curved segment 32118 to main axis MA) ranging from about 0.05mm to about 1mm, preferably from about 0.1mm to about 0.75mm, more preferably from about 0.2mm to about 0.5mm, even more preferably from about 0.25mm to about 0.4 mm. In some embodiments, fifth straight segment 32119 has a length (from fourth curved segment 32118 to main axis MA) of about 0.3 mm.

In a third alternative embodiment of the binding feature seen in fig. 8-10, the loading well 320 is configured to move and/or capture the sample droplet 313 within the plane (x and/or y) and z-position defined within the loading well 320.

In this embodiment, as described above, the side wall portions 3211 have a variable in-plane local curvature and the bottom wall portion 3212 is uneven.

In the embodiment shown in fig. 11, the inlet in the loading aperture leads to a droplet generator 340, the droplet generator 340 being operably coupled to a droplet chamber 350 and an air tank 360. The droplet chamber 350 includes a chamber column 370.

The drop generator 340 is further illustrated in the enlarged view of the area indicated by "E" in FIG. 11 shown in FIG. 12. It can be divided into several parts: a bonding pad 341 that opens on a longitudinal (in x-axis) dispensing channel 342, which dispensing channel 342 is connected to at least one (or in some embodiments several) transverse (in y-axis) injectors 343 that open on a longitudinal (in x-axis) inclined area 344. The sloped region 344 is directly connected to the droplet chamber 350.

In the embodiment shown in fig. 13, a close-up view of bond pad 341 is shown. The bond pads have a circular shape, are etched on the bottom side of the upper plate 310, and receive the output of the inlet 330, e.g., at its center, as shown in fig. 16. In the embodiment shown in fig. 13, the etched bond pads 341 are etched to define a ring around the output of the inlet 330, the ring including an outer edge 3411 and an inner edge 3412 that forms a contour of the output of the inlet 330.

In some embodiments, bond pad 341 has an outer diameter (in the x/y axis at outer edge 3411) ranging from about 0.5mm to about 2mm, preferably from about 0.75mm to about 1.5mm, more preferably from about 0.9mm to about 1.25 mm. In some embodiments, bond pad 341 has an outer diameter (on the x/y axis at outer edge 3411) of about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2 mm. In some embodiments, bond pad 341 has an outer diameter (at outer edge 3411, in the x/y axis) of about 1 mm.

In some embodiments, bond pad 341 has an inner diameter (at inner edge 3412, in the x/y axis) ranging from about 0.1mm to about 1.5mm, preferably from about 0.25mm to about 1.25mm, more preferably from about 0.5mm to about 1 mm. In some embodiments, bond pad 341 has an inner diameter (at inner edge 3412, in the x/y axis) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5 mm. In some embodiments, bond pad 341 has an inner diameter (at inner edge 3412, in the x/y axis) of about 0.8 mm.

In some embodiments, bond pad 341 has a height (in the z-axis) ranging from about 0.01mm to about 0.175mm, preferably from about 0.025mm to about 0.15mm, more preferably from about 0.05mm to about 0.125mm, even more preferably from about 0.075mm to about 0.1 mm. In some embodiments, bond pad 341 has a height (in the z-axis) of about 0.01mm, about 0.02mm, about 0.03mm, about 0.04mm, about 0.05mm, about 0.06mm, about 0.07mm, about 0.08mm, about 0.09mm, about 0.1mm, about 0.11mm, about 0.12mm, about 0.13mm, about 0.14mm, about 0.15mm, about 0.16mm, about 0.17mm, about 0.18 mm. In some embodiments, bond pad 341 has a height (in the z-axis) of about 0.09 mm.

In some embodiments, the bonding pad 341 is centered with respect to the x-axis of the dispensing channel 342. Other embodiments are also included in which the bonding pad 341 is not centered on the x-axis of the dispensing channel 342.

In the embodiment shown in fig. 14, a close-up view of the longitudinal (in the x-axis) dispensing passage 342 is shown connecting the transverse (in the y-axis) eductor 343.

In some embodiments, the dispensing passage 342 has a length (in the x-axis) ranging from about 1mm to about 50mm, preferably from about 1mm to about 25mm, more preferably from about 1mm to about 10mm, even more preferably from about 2.5mm to about 5 mm. In some embodiments, the dispensing passage 342 has a length (on the x-axis) of about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, or more. In some embodiments, the dispensing channel 342 has a length (on the x-axis) of about 4 mm.

In some embodiments, the dispensing passage 342 has a width (in the y-axis) ranging from about 0.01mm to about 1mm, preferably from about 0.025mm to about 0.75mm, more preferably from about 0.05mm to about 0.5mm, even more preferably from about 0.075mm to about 0.25 mm. In some embodiments, the dispensing passage 342 has a width (on the y-axis) of about 0.01mm, about 0.025mm, about 0.05mm, about 0.075mm, about 0.1mm, about 0.25mm, about 0.5mm, about 0.75mm, about 1mm, or more. In some embodiments, the distribution channel 342 has a width (in the y-axis) of about 0.125 mm.

In some embodiments, the dispensing passage 342 has a height (in the z-axis) ranging from about 0.01mm to about 0.175mm, preferably from about 0.025mm to about 0.15mm, more preferably from about 0.05mm to about 0.125mm, even more preferably from about 0.075mm to about 0.1 mm. In some embodiments, the distribution channel 342 has a height (in the z-axis) of about 0.01mm, about 0.02mm, about 0.03mm, about 0.04mm, about 0.05mm, about 0.06mm, about 0.07mm, about 0.08mm, about 0.09mm, about 0.1mm, about 0.11mm, about 0.12mm, about 0.13mm, about 0.14mm, about 0.15mm, about 0.16mm, about 0.17mm, about 0.18 mm. In some embodiments, the distribution channel 342 has a height (in the z-axis) of about 0.09 mm.

In some embodiments, droplet generator 340 includes at least 1, preferably at least 2,3,4, 5,6, 7, 8, 9, 10, or more ejectors 343. In the embodiment shown in fig. 7, 11, and 12, droplet generator 340 includes 6 ejectors 343.

In some embodiments, the ejector 343 has a length (on the y-axis) ranging from about 0.1mm to about 5mm, preferably from about 0.25mm to about 2.5mm, more preferably from about 0.5mm to about 1 mm. In some embodiments, the ejector 343 has a length (on the y-axis) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 3mm, about 4mm, about 5mm, or more. In some embodiments, the ejector 343 has a length (on the y-axis) of about 0.8 mm.

In some embodiments, the injectors 343 have a width (on the x-axis) ranging from about 0.01mm to about 0.5mm, preferably from about 0.02mm to about 0.25mm, more preferably from about 0.03mm to about 0.1mm, and even more preferably from about 0.04mm to about 0.08 mm. In some embodiments, the injector 343 has a width (on the x-axis) of about 0.01mm, about 0.02mm, about 0.03mm, about 0.04mm, about 0.05mm, about 0.06mm, about 0.07mm, about 0.08mm, about 0.09mm, about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, or more. In some embodiments, the ejector 343 has a width (on the x-axis) of about 0.075 mm. In some embodiments, the ejector 343 has a width (on the x-axis) of about 0.045 mm.

In some embodiments, the ejector 343 has a variable width (in the x-axis). As shown in fig. 14, the width (in the x-axis) of the eductor 343 may gradually decrease from the proximal end (on the side of the dispensing passage 342) to the distal end (on the side of the sloped region 344). In some embodiments, the eductor 343 has a diameter at the proximal end position ranging from about 0.03mm to about 0.3mm, preferably from about 0.04mm to about 0.2mm, more preferably from about 0.05mm to about 0.1 mm; at the distal position, has a variable width (in the x-axis) ranging from about 0.01mm to about 0.2mm, preferably from about 0.02mm to about 0.1mm, more preferably from about 0.03mm to about 0.08 mm. In some embodiments, the ejector 343 has a variable width (in the x-axis) of about 0.075mm in the proximal position and about 0.045mm in the distal position.

In some embodiments, the ejector 343 has a height (in the z-axis) ranging from about 0.005mm to about 0.05mm, preferably from about 0.01mm to about 0.03mm, and more preferably from about 0.015mm to about 0.02 mm. In some embodiments, the injectors 343 have a height (in the z-axis) of about 0.005mm, about 0.0075mm, about 0.01mm, about 0.011mm, about 0.012mm, about 0.013mm, about 0.014mm, about 0.015mm, about 0.016mm, about 0.017mm, about 0.018mm, about 0.019mm, about 0.02mm, about 0.025mm, about 0.03mm, about 0.035mm, about 0.04mm, about 0.045mm, about 0.05mm, or more. In some embodiments, the ejector 343 has a height (in the z-axis) of about 0.018 mm.

In the embodiment shown in fig. 15, the operative coupling between the loading aperture 320 and the inlet 330 is shown. Fig. 16 is a close-up view showing an inlet 330 operably coupling a loading aperture 320 on the one hand to a bond pad 341 on the other hand. The bonding pads 341 are further open in the longitudinal (in the x-axis) dispensing channel 342.

In the embodiment shown in fig. 17, the location of the drop generator 340 below the loading aperture 320 and its operative connection with the drop chamber 350 is shown. Fig. 18 is a close-up view showing the longitudinal (in the x-axis) dispensing channel 342 opening on the eductor 343. Fig. 19 is a close-up view showing the connection between ejector 343 and droplet chamber 350 through angled region 344.

In some embodiments, the drop generator 340 is located below a bottom wall portion 3212 of the loading aperture 320 and included within the bottom wall portion 3212 in projection into the base plane (x/y). In some embodiments, the drop generator 340 is located below a bottom wall portion 3212 of the loading aperture 320 and is surrounded by a side wall portion 3211 of the loading aperture 320 in projection in the base plane (x/y). In an embodiment, the drop generator 340 is located below the bottom wall portion 3212 of the loading aperture 320 and does not extend beyond the side wall portion 3211 of the loading aperture 320 in projection in the base plane (x/y).

In some embodiments, the sloped region 344 has a width (on the x-axis) ranging from about 1mm to about 50mm, preferably from about 1mm to about 25mm, more preferably from about 1mm to about 10mm, even more preferably from about 2mm to about 7.5mm, even more preferably from about 4mm to about 6 mm. In some embodiments, the sloped region 344 has a width (on the x-axis) of about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, or more. In some embodiments, the sloped region 344 has a width (in the x-axis) of about 5 mm.

The width (in the x-axis) of the sloped region 344 depends on the number of ejectors 343 in the drop generator 340. In some embodiments, the sloped region 344 occupies at least the width (in the x-axis) required to operatively couple all of the injectors 343 (if more than 1). Thus, the present disclosure includes the case where each injector 343 opens on a single sloped region 344. However, the present disclosure also includes the case where each ejector 343 opens on one sloped region 344, multiple sloped regions ultimately operably coupled to the drop chamber 350.

In some embodiments, the sloped region 344 has a length (on the y-axis) ranging from about 0.1mm to about 3mm, preferably from about 0.1mm to about 2mm, from about 0.1mm to about 1mm, from about 0.2mm to about 0.75mm, more preferably from about 0.3mm to about 0.5 mm. In some embodiments, the sloped region 344 has a length (on the y-axis) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2mm, about 2.1mm, about 2.2mm, about 2.3mm, about 2.4mm, about 2.5mm, about 2.6mm, about 2.7mm, about 2.8mm, about 2.9mm, about 3mm, or more. In some embodiments, the sloped region 344 has a length (on the y-axis) of about 0.4 mm.

In some embodiments, the sloped region 344 has a variable height (in the z-axis), i.e., the upper and lower surfaces of the sloped region diverge with respect to each other in at least one direction, e.g., in the y-axis.

As shown in fig. 19, the height (in the z-axis) of the sloped region 344 may gradually increase from the proximal end (on the side of the ejector 343) to the distal end (on the side of the droplet chamber 350). In some embodiments, the angled region 344 has a width in the proximal position ranging from about 0.005mm to about 0.05mm, preferably from about 0.01mm to about 0.03mm, more preferably from about 0.015mm to about 0.02 mm; at the distal location, a variable height (in the z-axis) of from about 0.02mm to about 0.5mm, preferably from about 0.04mm to about 0.2mm, more preferably from about 0.06mm to about 0.15mm, even more preferably from about 0.08mm to about 0.1 mm. In some embodiments, the angled region 344 has a variable height (in the z-axis) of about 0.018mm in the proximal position and about 0.09mm in the distal position.

In some embodiments, the sloped region 344 has a variable height with a slope over the length (on the y-axis) of the sloped region 344 having a value ranging from about 1% ± 5% to about 30% ± 5%, preferably from about 5% ± 2.5% to about 25% ± 2.5%, more preferably from about 10% ± 2% to about 20% ± 2%, even more preferably from about 14% ± 1% to about 18% ± 1%. In some embodiments, the sloped region 344 has a variable height with a slope over the length (in the y-axis) of the sloped region 344 having a value ranging from about 1% ± 0.25% to about 30% ± 0.25%, preferably from about 5% ± 0.25% to about 25% ± 0.25%, more preferably from about 10% ± 0.25% to about 20% ± 0.25%, even more preferably from about 14% ± 0.25% to about 18% ± 0.25%. In some embodiments, sloped region 344 has a variable height with a slope value over the length (in the y-axis) of sloped region 344 of about 1% ± 0.5%, about 2% ± 0.5%, about 3% ± 0.5%, about 4% ± 0.5%, about 5% ± 0.5%, about 6% ± 0.5%, about 7% ± 0.5%, about 8% ± 0.5%, about 9% ± 0.5%, about 10% ± 0.5%, about 11% ± 0.5%, about 12% ± 0.5%, about 13% ± 0.5%, about 14% ± 0.5%, about 15% ± 0.5%, about 16% ± 0.5%, about 17% ± 0.5%, about 18% ± 0.5%, about 19% ± 0.5%, about 20% ± 0.5%, about 21% ± 0.5%, about 22% ± 0.5%, about 23% ± 0.5%, about 24% ± 0.5%, about 25% ± 0.5%, about 29.5%, about 28% ± 0.5%, about 28.5%. In some embodiments, sloped region 344 has a variable height with a slope value over the length (in the y-axis) of sloped region 344 of about 1% ± 0.25%, about 2% ± 0.25%, about 3% ± 0.25%, about 4% ± 0.25%, about 5% ± 0.25%, about 6% ± 0.25%, about 7% ± 0.25%, about 8% ± 0.25%, about 9% ± 0.25%, about 10% ± 0.25%, about 11% ± 0.25%, about 12% ± 0.25%, about 13% ± 0.25%, about 14% ± 0.25%, about 15% ± 0.25%, about 16% ± 0.25%, about 17% ± 0.25%, about 18% ± 0.25%, about 19% ± 0.25%, about 20% ± 0.25%, about 21% ± 0.25%, about 22% ± 0.25%, about 23% ± 0.25%, about 24% ± 0.25%, about 25% ± 0.25%, about 25%, about 29% ± 0.25%, about 25%. In some embodiments, the sloped region 344 has a variable height with a slope value of about 16% ± 0.5%. In some embodiments, the sloped region 344 has a variable height with a slope value of about 16% ± 0.25%.

In some embodiments, the slope of the sloped region 344 is smooth.

In some embodiments, the slope of the sloped region 344 comprises a step. In some embodiments, the slope of the sloped region 344 includes at least 2, preferably at least 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more steps. In the embodiment shown in fig. 19, the slope of the sloped region 344 includes 16 steps. In some embodiments, the step of the slope of the sloped region 344 has a length (on the y-axis) ranging from about 0.001mm to about 0.1mm, preferably from about 0.005mm to about 0.075mm, more preferably from about 0.01mm to about 0.05mm, even more preferably from 0.02mm to about 0.03 mm. In some embodiments, the step of the slope of the sloped region 344 has a length slope of about 0.001mm, about 0.005mm, about 0.01mm, about 0.02mm, about 0.03mm, about 0.04mm, about 0.05mm, about 0.06mm, about 0.07mm, about 0.08mm, about 0.09mm, about 0.1mm, or greater. In some embodiments, the step of the slope of the sloped region 344 has a length (on the y-axis) of about 0.025 mm.

Similar drop generators are disclosed in patent applications US20130078164 and US20180037934, the entire contents of which are incorporated herein by reference. The dimensions of the embodiments disclosed herein, and in particular the various elements of the droplet generator 340, are not limiting, and a skilled artisan can determine that some of these dimensions, and in particular the dimensions of the ejector 343, can be modified to obtain smaller or larger droplets 314.

As shown in fig. 11 and 19, the droplet generator 340 opens through an inclined region 344 onto a droplet chamber 350, the droplet chamber 350 being configured or adapted to store the droplet 314. In some embodiments, droplet chamber 350 has a length (on the y-axis) ranging from about 1mm to about 100mm, preferably from about 2.5mm to about 75mm, more preferably from about 5mm to about 50mm, even more preferably from about 7.5mm to about 25mm, even more preferably from about 10mm to about 18 mm. In some embodiments, droplet chamber 350 has a length (on the y-axis) of about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, about 11mm, about 12mm, about 13mm, about 14mm, about 15mm, about 16mm, about 17mm, about 18mm, about 19mm, about 20mm, about 21mm, about 22mm, about 23mm, about 24mm, about 25mm, about 30mm, about 35mm, about 40mm, about 45mm, about 50mm, about 60mm, about 70mm, about 80m, about 90mm, about 100mm, or more. In some embodiments, droplet chamber 350 has a length (on the y-axis) of about 14.3 mm.

In some embodiments, droplet chamber 350 has a width (on the x-axis) ranging from about 1mm to about 80mm, preferably from about 2mm to about 65mm, more preferably from about 3mm to about 50mm, even more preferably from about 4mm to about 40mm, even more preferably from about 5mm to about 25mm, even more preferably from about 6mm to about 15mm, even more preferably from about 7mm to about 10 mm. In some embodiments, droplet chamber 350 has a width (on the x-axis) of about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, about 11mm, about 12mm, about 13mm, about 14mm, about 15mm, about 16mm, about 17mm, about 18mm, about 19mm, about 20mm, about 21mm, about 22mm, about 23mm, about 24mm, about 25mm, about 30mm, about 35mm, about 40mm, about 45mm, about 50mm, about 60mm, about 70mm, about 80m, or more. In some embodiments, droplet chamber 350 has a width (on the x-axis) of about 8.5 mm.

In the embodiment shown in fig. 11, the droplet chamber 350 has an L-shape. Such a shape is useful in the configuration shown in fig. 11, accommodating air tanks 360 associated with adjacent droplet chambers at the corners of the droplet chambers. However, the present disclosure includes droplet chambers 350 having any suitable shape, particularly in the x-axis and y-axis, depending on the available surfaces on the microfluidic chip 300 and the number of microfluidic cells on the microfluidic chip 300.

In some embodiments, droplet chamber 350 has a height (in the z-axis) ranging from about 0.01mm to about 0.175mm, preferably from about 0.025mm to about 0.15mm, more preferably from about 0.05mm to about 0.125mm, even more preferably from about 0.075mm to about 0.1 mm. In some embodiments, droplet chamber 350 has a height (in the z-axis) of about 0.01mm, about 0.02mm, about 0.03mm, about 0.04mm, about 0.05mm, about 0.06mm, about 0.07mm, about 0.08mm, about 0.09mm, about 0.1mm, about 0.11mm, about 0.12mm, about 0.13mm, about 0.14mm, about 0.15mm, about 0.16mm, about 0.17mm, about 0.18mm, or more. In some embodiments, droplet chamber 350 has a height (in the z-axis) of about 0.09 mm.

In some embodiments, droplet chamber 350 is configured or adapted to store droplet population 314. For example, a droplet population 314 ranging from about one thousand to about five million droplets or more may be stored in the droplet chamber 350. The number of droplets 314 stored in the droplet chamber 350 depends on the size of the droplet chamber 350 and the diameter of the droplets 314. In some embodiments, a droplet population 314 ranging from about ten thousand to about twenty five thousand droplets 314 may be stored in droplet chamber 350, such as about twenty five thousand or about twenty thousand droplets 314.

In some embodiments, droplet chamber 350 is not a droplet channel, i.e., droplet chamber 350 has at least two dimensions, specifically a length (in the y-axis) and a width (in the x-axis), that are at least two times larger than the diameter of droplet 314, e.g., two times, three times, four times, five times, ten times, fifty times, one hundred times, one thousand times, five thousand times, or even more.

In the embodiment shown in fig. 7 and 11, the droplet chamber 350 can include a chamber post 370.

In some embodiments, the chamber column 370 does not have a cylindrical shape in a cross-section parallel to the base plane (x/y).

In some embodiments, the chamber pillars 370 have a diamond (rhombus) shape in a cross-section parallel to the base plane (x/y). In some embodiments, the chamber pillars 370 have a diamond (Lozenge) or diamond (Diamond) shape in a cross-section parallel to the base plane (x/y).

As shown in fig. 23, a diamond shape or diamond shape may be defined by four sides s of equal or equal length, a long diagonal of ldl length, a short diagonal of sdl length (the latter two forming a right angle at their intersection), two opposite acute angles ζ and two opposite obtuse angles η,

whereinAnd is

In some embodiments, the length of the side s of the chamber column 370 is an integer multiple of the diameter of the droplet 314 in the droplet chamber 350, such as about 2 times the diameter, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, or more the diameter of the droplet 314 in the droplet chamber 350.

In some embodiments, the long diagonal (in the y-axis) of the chamber column 370 has a length ldl ranging from about 0.1mm to about 5mm, preferably from about 0.5mm to about 4mm, more preferably from about 1mm to about 3mm, even more preferably from about 1.5mm to about 2 mm. In some embodiments, the long diagonal (in the y-axis) of the chamber column 370 has a length l of about 0.1mm, 0.2mm, 0.3mm, 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2mm, about 2.1mm, about 2.2mm, about 2.3mm, about 2.4mm, about 2.5mm, about 2.6mm, about 2.7mm, about 2.8mm, about 2.9mm, about 3mm, about 3.5mm, about 4mm, about 4.5mm, about 5mm, or greater. In some embodiments, the long diagonal (in the y-axis) of the chamber column 370 has a length ldl of about 1.8 mm.

In some embodiments, the acute angle ζ of the chamber column 370 ranges from about 10 ° to about 50 °, preferably from about 20 ° to about 40 °, more preferably from about 25 ° to about 35 °. In some embodiments, the acute angle ζ of the chamber column 370 ranges from about 40 ° to about 80 °, preferably from about 50 ° to about 70 °, more preferably from about 55 ° to about 65 °. In some embodiments, the acute angle ζ of the chamber column 370 is about 10 °, about 15 °, about 20 °, about 25 °, about 30 °, about 35 °, about 40 °, about 45 °, about 50 °, about 55 °, about 60 °, about 65 °, about 70 °, about 75 °, about 80 °, about 85 °, or greater. In some embodiments, the acute angle ζ of the chamber column 370 is about 30 °. In some embodiments, the acute angle ζ of the chamber column 370 is about 60 °.

In some embodiments, at least one, and in particular at least two, three, or even four, of the vertices 371 of the chamber column 370 is sharp-edged.

In some embodiments, at least one, specifically at least two, three, or even four, of the vertices 371 of the chamber column 370 is rounded. For example, as in the embodiment shown in FIG. 23, the four vertices 371 of the chamber post 370 may be rounded, each vertex 371 having a radius of curvature ranging from about 0.01mm to about 0.5mm, preferably from about 0.05mm to about 0.4mm, more preferably from about 0.1mm to about 0.3mm, even more preferably from about 0.15mm to about 0.2 mm. In some embodiments, each apex 371 of the cell post 370 has a radius of curvature of about 0.01mm, about 0.05mm, about 0.1mm, about 0.125mm, about 0.15mm, about 0.175mm, about 0.2mm, about 0.225mm, about 0.25mm, about 0.3mm, about 0.35mm, about 0.4mm, about 0.45mm, about 0.5mm, or more. In some embodiments, each apex 371 of the chamber column 370 has a radius of curvature of about 0.175 mm.

In some embodiments, the upper plate 310 of the microfluidic chip 300 forms a base that supports at least one air tank 360. In its mode of operation, the microfluidic chip 300 includes a lower plate 311 (shown in fig. 6) such that the air tank 360 is a closed cavity 364.

In the embodiment shown in fig. 11, the air tank 360 is operatively coupled to the droplet chamber 350 through an output channel 361.

In an alternative embodiment, the air tank 360 is operably coupled to the drop generator 340, specifically to the sloped region 344, without contacting the drop chamber 350.

In the embodiment shown in fig. 20-21, the air tank 360 is bounded by a wall 363, the wall 363 including a bottom wall portion 3632 coupled to a sidewall portion 3631.

In some embodiments, the bottom wall portion 3632 extends integrally according to a tank top plane ttp substantially parallel to the base plane (x/y) (see also fig. 8).

In some embodiments, the sidewall portion 3631 extends in a can transverse direction tld (in the z-axis) that is disposed according to an angle κ relative to a can top plane ttp, as shown in fig. 8 and 20-21. In some embodiments, angle κ has a value ranging from about 75 ° to about 120 °, preferably from about 85 ° to about 110 °, more preferably from about 90 ° to about 105 °. In some embodiments, the angle κ has a value of about 75 °, about 80 °, about 85 °, about 90 °, about 95, about 100 °, about 105 °, or more. In some embodiments, the angle κ has a value of about 98 °.

As shown in the embodiment of fig. 8, the wall 363 of the air tank 360 and the wall 321 of the loading aperture 320 may share a common section. Specifically, the side wall portion 3631 of the air tank 360 and the side wall portion 3211 of the loading hole 320 may share a common section.

In some embodiments, in a cross-sectional view perpendicular to the base plane (x/y), air tank 360 has the general shape of a truncated isosceles triangle having a base 362, a truncation formed by base wall portion 3632, and a pair of equal sides formed by lateral wall portions 3631.

In some embodiments, in a cross-sectional view perpendicular to the base plane (x/y), the air tank 360 has the general shape of a truncated pyramid (e.g., a truncated pyramid having a square base, a rectangular base, a diamond (rhombus) base, a diamond (loggen) base, a diamond (diamond) base, a circular base [ i.e., a truncated cone ], etc.) having a base 362, a truncated apex formed by the bottom wall portion 3632, and four sides formed by the side wall portions 3631.

In some embodiments, air tank 360 has a length (on the y-axis) at the level of bottom 362 ranging from about 0.1mm to about 10mm, preferably from about 0.5mm to about 7.5mm, more preferably from about 1mm to about 5mm, even more preferably from about 3mm to about 4 mm. In some embodiments, the air tank 360 has a length (on the y-axis) at the level of the base 362 of about 0.1mm, about 0.25mm, about 0.5mm, about 0.75mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 2.25mm, about 2.5mm, about 2.75mm, about 3mm, about 3.25mm, about 3.5mm, about 3.75mm, about 4mm, about 4.25mm, about 4.5mm, about 4.75mm, about 5mm, about 5.5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, or more. In some embodiments, the air tank 360 has a length (on the y-axis) of about 3.7mm at the level of the bottom 362.

In some embodiments, the air tank 360 has a length (on the y-axis) at the truncated level ranging from about 0.1mm to about 10mm, preferably from about 0.5mm to about 7.5mm, more preferably from about 1mm to about 5mm, even more preferably from about 1.5mm to about 2.5 mm. In some embodiments, the air tank 360 has a length (on the y-axis) at the truncated level of about 0.1mm, about 0.25mm, about 0.5mm, about 0.75mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 2.25mm, about 2.5mm, about 2.75mm, about 3mm, about 3.25mm, about 3.5mm, about 3.75mm, about 4mm, about 4.25mm, about 4.5mm, about 4.75mm, about 5mm, about 5.5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, or more. In some embodiments, the air tank 360 has a length (on the y-axis) of about 2.1mm at the truncated level.

In some embodiments, air tank 360 has a width (on the x-axis) at the level of bottom 362 ranging from about 0.1mm to about 5mm, preferably from about 0.5mm to about 4mm, more preferably from about 1mm to about 3mm, even more preferably from about 1.5mm to about 2.5 mm. In some embodiments, the air tank 360 has a width (on the x-axis) at the level of the bottom 362 of about 0.1mm, about 0.25mm, about 0.5mm, about 0.75mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 2.25mm, about 2.5mm, about 2.75mm, about 3mm, about 3.25mm, about 3.5mm, about 3.75mm, about 4mm, about 4.25mm, about 4.5mm, about 4.75mm, about 5mm, or more. In some embodiments, the air tank 360 has a width (on the x-axis) of about 2mm at the level of the bottom 362.

In some embodiments, the air tank 360 has a width (on the x-axis) at the truncated apex ranging from about 0.1mm to about 5mm, preferably from about 0.2mm to about 2.5mm, more preferably from about 0.3mm to about 1mm, even more preferably from about 0.4mm to about 0.75 mm. In some embodiments, the air tank 360 has a width (on the x-axis) at the truncated apex of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 2.5mm, about 3mm, about 3.5mm, about 4mm, about 4.5mm, about 5mm, or more. In some embodiments, the air tank 360 has a width (on the x-axis) of about 0.5mm at the truncated apex.

In some embodiments, the air tank 360 has a depth (in the z-axis) from the bottom 362 to the truncated top ranging from about 1mm to about 15mm, preferably from about 2mm to about 12mm, more preferably from about 3mm to about 10mm, even more preferably from about 4mm to about 8mm, even more preferably from about 5mm to about 7 mm. In some embodiments, the air tank 360 has a depth (in the z-axis) from the bottom 362 to the truncated top of about 1mm, about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, about 11mm, about 12mm, about 13mm, about 14mm, about 15mm, or more. In some embodiments, the air tank 360 has a depth (in the z-axis) from the bottom 362 to the truncated top of about 6.4 mm.

In some embodiments, base 362 is crowned with a recess 3621 that extends around base 362. As shown in fig. 20-21, the recess 3621 defines an inner edge 3622 and an outer edge 3623.

In some embodiments, grooves 3621 have a width (from inner edge 3622 to outer edge 3623) ranging from about 0.05mm to about 3mm, preferably from about 0.1mm to about 2mm, more preferably from about 0.25mm to about 1mm, even more preferably from about 0.25mm to about 0.75 mm. In some embodiments, grooves 3621 have a width (from inner edge 3622 to outer edge 3623) of about 0.01mm, about 0.05mm, about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 2.5mm, about 3mm, or more. In some embodiments, groove 3621 has a width (from inner edge 3622 to outer edge 3623) of about 0.5 mm.

In some embodiments, grooves 3621 have a depth (in the z-axis) ranging from about 0.01mm to about 0.5mm, preferably from about 0.05mm to about 0.4mm, more preferably from about 0.1mm to about 0.3mm, even more preferably from about 0.15mm to about 0.25 mm. In some embodiments, grooves 3621 have a depth (in the z-axis) of about 0.01mm, about 0.05mm, about 0.1mm, about 0.125mm, about 0.15mm, about 0.175mm, about 0.2mm, about 0.225mm, about 0.25mm, about 0.3mm, about 0.35mm, about 0.4mm, about 0.45mm, about 0.5mm, or more. In some embodiments, grooves 3621 have a depth (in the z-axis) of about 0.2 mm.

In some embodiments, air tank 360 has a volume (including recess 3621) ranging from about 5 μ L to about 60 μ L, preferably from about 10 μ L to about 50 μ L, more preferably from about 15 μ L to about 45 μ L, even more preferably from about 20 μ L to about 40 μ L, even more preferably from about 25 μ L to about 35 μ L. In some embodiments, air tank 360 has a volume (including recess 3621) of about 5 μ L, about 10 μ L, about 15 μ L, about 20 μ L, about 25 μ L, about 30 μ L, about 35 μ L, about 40 μ L, about 45 μ L, about 50 μ L, about 55 μ L, about 60 μ L, or more. In some embodiments, air tank 360 has a volume of about 30 μ L (including recess 3621).

In some embodiments, air tank 360 has a volume (including grooves 3621) that is greater than the volume of sample droplet 313, e.g., at least about 1% greater, at least about 5% greater, at least about 10% greater, at least about 50% greater, at least about 100% greater, at least about 200% greater, at least about 300% greater, at least about 400% greater, at least about 500% greater, or more.

In some embodiments, air tank 360 has a volume (including recess 3621) that is greater than the volume of the droplet population 31 stored in droplet chamber 350, e.g., at least about 1% greater, at least about 5% greater, at least about 10% greater, at least about 50% greater, at least about 100% greater, at least about 200% greater, at least about 300% greater, at least about 400% greater, at least about 500% greater, at least about 600% greater, or more.

In some embodiments, the output channel 361 can be housed in the sidewall portion 3631 or the bottom wall portion 3632 of the wall 363, preferably in the sidewall portion 3631. In some embodiments, the output channel 361 is received at an end of the sidewall portion 3631, toward the bottom of the upper plate 310. In some embodiments, the output channel 361 is received in an end of the sidewall portion 3631, in the recess 3621.

In some embodiments, the outlet channel 361 has a length (on the x-axis) ranging from about 0.1mm to about 5mm, preferably from about 0.2mm to about 2.5mm, more preferably from about 0.3mm to about 1mm, even more preferably from about 0.4mm to about 0.75 mm. In some embodiments, the outlet channel 361 has a length (on the x-axis) of about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1mm, about 1.25mm, about 1.5mm, about 1.75mm, about 2mm, about 2.5mm, about 3mm, about 3.5mm, about 4mm, about 4.5mm, about 5mm, or greater. In some embodiments, the output channel 361 has a length (on the x-axis) of about 0.5 mm.

In some embodiments, the output channel 361 has a width (on the y-axis) ranging from about 0.01mm to about 1mm, preferably from about 0.025mm to about 0.75mm, more preferably from about 0.05mm to about 0.5mm, even more preferably from about 0.075mm to about 0.25 mm. In some embodiments, the output channel 361 has a width (on the y-axis) of about 0.01mm, about 0.05mm, about 0.075mm, about 0.8mm, about 0.9mm, about 0.1mm, about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.75mm, about 1mm, or more. In some embodiments, the output channel 361 has a width (on the y-axis) of about 0.12 mm.

In some embodiments, the outlet channel 361 has a height (in the z-axis) ranging from about 0.005mm to about 0.2mm, preferably from about 0.01mm to about 0.1mm, more preferably from about 0.01mm to about 0.05mm, even more preferably from about 0.01mm to about 0.03mm, even more preferably from about 0.015mm to about 0.025 mm. In some embodiments, the output channel 361 has a height (in the z-axis) of about 0.005mm, about 0.0075mm, about 0.01mm, about 0.011mm, about 0.012mm, about 0.013mm, about 0.014mm, about 0.015mm, about 0.016mm, about 0.017mm, about 0.018mm, about 0.019mm, about 0.02mm, about 0.025mm, about 0.03mm, about 0.035mm, about 0.04mm, about 0.045mm, about 0.05mm, or more. In some embodiments, the output channel 361 has a height (in the z-axis) of about 0.02 mm.

In some embodiments, the width (in the y-axis) and/or height (in the z-axis) of output channel 361 is less than the diameter of droplet 314. In some embodiments, the output channel 361 has a width (in the y-axis) that is at least about one times larger than the diameter of the droplets 314, preferably at least about two times, about three times, about four times, about five times, about ten times, about fifteen times, about twenty times, or more larger than the diameter of the droplets 314, i.e., in a plane parallel to the direction of flow of the continuous phase 312. In some embodiments, output channel 361 has a height (in the z-axis) that is at least about one times smaller than the diameter of droplet 314, preferably at least about 0.75 times, about 0.5 times, about 0.25 times, about 0.1 times, about 0.01 times or less smaller than the diameter of droplet 314, i.e., in a plane perpendicular to the direction of flow of continuous phase 312.

In some embodiments, the minimum distance between the output channel 361 and the droplet generator 340 is at most about 50%, preferably at most about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or less, of the largest dimension in the base plane (x/y) of the droplet chamber 350. In some embodiments, the minimum distance between the output channel 361 and the droplet generator 340 is at most about 50%, preferably at most about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or less, of the smallest dimension in the base plane (x/y) of the droplet chamber 350. In some embodiments, the minimum distance between the output channel 361 and the drop generator 340 is zero, i.e., the output channel 361 and the drop generator 340 are directly adjacent.

In some embodiments, the smallest surface in a base plane (x/y) between one output channel 361, one droplet generator 340, and one corner of the droplet chamber 350 (preferably the closest corner of the droplet chamber 350 relative to the output channel 361) covers at most about 50% of the surface of the droplet chamber 350, preferably at most about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or less of the surface of the droplet chamber 350.

In some embodiments, a straight line between the output channel 361 and the drop generator 340 divides the drop chamber 350 into two unequal surfaces. In this embodiment, the ratio of the smallest area to the largest area of the two unequalities is at most 1:2, preferably at most 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50, 1:100 or less.

Exemplary operations

This section describes exemplary operations of the components, devices, and systems disclosed above.

Fig. 24-32 schematically illustrate an exemplary operation of the loading aperture 320 seen in fig. 8-10.

The loading well 320 according to the above-described embodiments can reduce the dead volume of a sample droplet to be loaded into the microfluidic chip 300. Typically, in a biphasic microfluidic chip, the continuous phase 312 is first loaded and at least partially fills the microfluidic network (e.g., in the presence of the air tank 360, the microfluidic chip 300 is only partially filled with the continuous phase 312 and the air tank 360 is entirely filled with air), and then a drop of the dispersed phase (typically the sample 313) is placed in the loading well 320 at the continuous phase/air interface. It is desirable to move the sample 313 to and capture it at a defined location within the loading well 320 to reproducibly load the sample into the microfluidic network while reducing the dead volume of the sample when loaded. In some embodiments, the defined position is adjacent to the inlet 330 in at least one axis. In another embodiment, the defined position is adjacent to the inlet 330 in at least two axes. In yet another embodiment, the defined position is adjacent to the inlet 330 in three axes.

By "immediately adjacent" is meant a distance of no more than about 1 times the diameter of the sample droplet, preferably no more than about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 times the diameter of the sample droplet.

In the embodiments shown in fig. 8-10, the loading well 320 is thus intended to capture the sample droplet 313 in a defined position and/or to move the sample droplet 313 to a defined position within said loading well 320, in close proximity to the inlet 330, irrespective of the position of the sample droplet 313 deposited within the loading well 320.

Fig. 24-26 schematically represent different sequential steps of a first operation of a loading well 320, the loading well 320 being configured to move and/or capture a sample droplet 313 to an in-plane (x and/or y) location defined within the loading well 320.

As shown in fig. 24, the sample droplet 313 placed in the loading well 320 filled with the continuous phase 312 contacts the bottom wall portion 3212 while deforming the continuous phase/air interface. This deformation increases the continuous phase/air contact area, forming a meniscus. The system eventually moves towards reducing the continuous phase/air contact area due to surface tension.

This phenomenon moves and captures the sample droplet 313 to a deeper position d of the loading well 320, as shown in fig. 25.

In some embodiments, the height of the continuous phase 312 in the loading well 320 at the deeper position d of the loading well 320 is less than about the diameter of the sample droplet 313, preferably less than about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1 times the diameter of the sample droplet 313.

In some embodiments, the inlet 330 is vertically (in the z-axis) proximate to the deeper position d of the loading aperture 320, as shown in fig. 26.

Fig. 27-32 schematically represent different steps of another exemplary operation of a loading well 320, the loading well 320 being configured to move and/or capture a sample droplet 313 to an in-plane (x and/or y) location defined within the loading well 320.

As shown in fig. 27, the sample droplet 313 placed in the loading well 320 filled with the continuous phase 312 floats on the continuous phase, thereby deforming the continuous phase/air interface. This deformation increases the continuous phase/air contact area, forming a meniscus. The system eventually moves towards reducing the continuous phase/air contact area due to surface tension.

This phenomenon moves the sample droplet 313 to the side wall portion 3211 of the loading hole 320, as shown in fig. 28.

Here, sample droplet 313 migrates along sidewall portion 3211 toward the lower in-plane curvature of sidewall portion 3211 (if the local curvature value of sidewall portion 3211 is greater than 1/2 for the diameter of sample droplet 313, i.e. the radius of sample droplet 313) or toward the higher in-plane curvature of sidewall portion 3211 (if the local curvature value of sidewall portion 3211 is less than 1/2 for the diameter of sample droplet 313, i.e. the radius of sample droplet 313), as shown in fig. 29 (see thick black arrows showing sample droplet migration). In other words, the sample droplet 313 migrates along the sidewall portion 3211 in a direction in which the local curvature value more conforms to the sample droplet 313 (i.e., the local curvature value is closer to the radius). The most advantageous location of the inlet is then below the center of the sample droplet, at a distance from sidewall portion 3211, where sidewall portion 3211 has a radius of curvature closest to half the diameter of the sample droplet. The optimal location of the inlet may be defined by the curvature of the sidewall portion 3211 and the diameter of the sample droplet in general use.

In some embodiments, the distance d in the base plane (x/y)_inlet-wallIs the distance between the inlet and the curved portion of the sidewall portion 3211 and ranges from about 0.5mm to about 2.5 mm. Preferably, the curved portion of the side wall portion 3211 has a radius of curvature ranging from about 0.5mm to about 2.5mm, more preferably about d_inlet-wallThe radius of curvature of (a).

The "local curvature" (cp (X)) at position X refers to the average curvature value of sidewall portion 3211 over a portion of sidewall portion 3211 centered at position X (a) having a length equal to the diameter of sample droplet 313. The change in curvature Cp along sidewall portion 3211 is derived therefromIt is given.

The sample droplet 313 stops and remains captured at the position: in this position, the sidewall portion 3211 local radius of curvature is equal to half the diameter (i.e. radius) of the sample droplet 313 or closest to it relative to the local curvature on each side; or the curvature of the side wall portion 3211 has an extreme value as shown in fig. 30.

In other words, the meniscus surface between sample droplet 313 and sidewall portion 3211 when in contact with sidewall portion 3211 depends on the curvature of sample droplet 313And the average local curvature Cp of the sidewall portion 3211 at the position X of the sample droplet 313. To minimize the total surface energy of the system, the sample droplet 313 is moved along the sidewall portion 3211 toward a position where the difference in curvature δ — Cp is small. The sample droplet 313 ends up inAnd then stop. In particular, ifThe droplet is stably captured at that position of the sidewall portion 3211, i.e., the droplet spontaneously returns to that position after a perturbation that causes the droplet to move away from that position.

In some embodiments, the inlet 330 is laterally (in the x-axis and/or y-axis) proximate to the optimal curvature location of the sidewall portion 3211, as shown in fig. 31.

In some embodiments, the inlet 330 is vertically (in the z-axis) proximate to the optimal curvature location of the sidewall portion 3211, as shown in fig. 32.

Fig. 33-36 schematically represent various exemplary operations of the chamber column 370 according to different embodiments of the present disclosure.

The chamber pillars 370 can maintain a constant height of the droplet chamber 350 (in the z-axis, i.e., between opposing surfaces defined by the upper plate 310 and the lower plate 311), and/or increase the concentration of droplets 314 on each surface of the droplet chamber 350 filled with the droplet population 314. Typically, large droplet chambers will collapse or expand during operation of the microfluidic chip, for example because of pressure differences across their walls and/or temperature changes, etc. Therefore, a plurality of small cell columns are regularly placed so that the cell height (in the z-axis) is kept constant. This limits the number of droplets 314 per surface area (i.e., the concentration of droplets 314 per surface of the droplet chamber 350) and thus the overall throughput of the operation.

In addition, the cell pillars can interfere with the droplet 314 lattice within the droplet cell. In fact, the cell columns currently used are cylindrical (i.e. have a circular or elliptical cross section parallel to the base plane (x/y)). However, the droplets 314 spontaneously coalesce into a hexagonal close-packed lattice, as shown in fig. 33. However, near the cell pillars, default values in the lattice can form and propagate, leaving empty space in the lattice, which limits the number of droplets 314 per surface area, and thus limits the overall throughput of the operation. This default in the droplet lattice is well observed in fig. 34A-C, where a light gray droplet 314 arranged around the cell column (in the center of the light) can be seen. The black areas between the droplets 314 represent the lattice default values. 34A-B illustrate a lattice of droplets 314 in a droplet chamber 350 that includes a circular cross-section chamber column. Fig. 34C shows a lattice of droplets 314 in a droplet chamber 350 that includes an elliptical cross-section chamber column.

The number of droplets 314 in a droplet chamber 350 for a given surface can be defined as follows:

wherein:

the surface is in mm²Is the surface of the droplet chamber 350, and

d is the average diameter of the droplets 314 in mm.

Per surface unit (mm) in the droplet chamber 350²) The concentration of droplets 314 may be defined as follows:

wherein:

d is the average diameter of the droplets 314 in mm.

The surface concentration of droplets 314, i.e., the number of droplets 314 per surface unit, may be defined as follows:

wherein:

d is the average diameter of the droplets 314 in mm.

The formulae given aboveThe "best achievable concentration" of the droplets in the droplet chamber is defined, i.e. there is no default formation at all in the droplet lattice. However, the use of chamber columns may be required in the droplet chamber. These cell columns are described as destructive means of the droplet lattice, having a negative impact on droplet concentration.

In some embodiments, the droplet population 314 stored in the droplet chamber 350 is monodisperse. In other words, each droplet 314 in the population of droplets 314 stored in the droplet chamber 350 has the same diameter and/or volume with a margin of error of less than about 20%, preferably less than about 15%, about 10%, about 5%, or less. In some embodiments, the error magnitude is about 10%.

In some embodiments, the droplet population 314 stored in the droplet chamber 350 is arranged in a two-dimensional (2D) droplet lattice, particularly in the base plane (x/y) (in other words, arranged in a 2D droplet layer). In some embodiments, the droplet population 314 stored in the droplet chamber 350 is arranged in a 3-dimensional (3D) droplet lattice.

In some embodiments, a population of droplets 314 (including droplets having an average diameter of about 75 μm and/or an average volume of about 220 pL) stored in droplet chamber 350 and arranged in a two-dimensional (2D) droplet lattice in a base plane (x/y) has about 148 droplets/mm as defined above²The best achievable concentration.

In the embodiment shown in fig. 7 and 11, the purpose of the chamber column 370 is to keep the height (in the z-axis) of the droplet chamber 350 constant (i.e., between the opposing surfaces defined by the upper plate 310 and the lower plate 311); while increasing the concentration of droplets 314 on each surface of droplet chamber 350. This may be accomplished by streamlining, reducing, or otherwise eliminating default formation in the droplet lattice.

As shown in fig. 35-36, the diamond shape of cell pillars 370 (in cross-section along the base plane (x/y)) fits into the natural pattern of the lattice of droplets 314. Specifically, fig. 36 is a photograph showing the arrangement of droplets 314 in the droplet chamber 350 including the diamond-shaped cross-section chamber pillars 370. Thus, the cell pillars 370 allow for avoiding default formation in the lattice, particularly near the cell pillars 370 and increasing the concentration of droplets 314 on each surface of the droplet chamber 350.

In some embodiments, the droplet concentration on each surface of the droplet chamber using chamber pillars 370 is increased by at least about 0.5%, preferably by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25% or more, as compared to a chamber pillar having a circular shape in cross-section along the base plane (x/y).

Fig. 37-40 schematically illustrate exemplary operation of a drop generator 340 positioned below a bottom wall portion 3212 of the loading aperture 320 and included within the bottom wall portion 3212 according to the present disclosure.

The microfluidic chip may include a precision microfluidic channel and a loading well. The microfluidic channels and the loading wells are well separated in projection in the base plane (x/y). This constraint helps to avoid any distortion or warping of the precision microfluidic channel due to the difference in aspect ratio. This results in microfluidic chips with narrow and tall loading wells that are separated from the precision microfluidic channels, thus leaving only limited space for the droplet chambers. For example, the injection molded part of the microfluidic chip with the high sidewall portion of the loading well exhibits an indentation in the bottom of the sidewall portion as shown in fig. 37 (indicated by thick black arrows) because the parts have different shrinkage rates during molding. Thus, if a precision microfluidic channel (e.g., a drop generator) is placed in close proximity to a sidewall portion, or more generally, a loading well, the precision microfluidic channel will deform or otherwise warp during molding and its function will be affected.

Therefore, placing the loading well containing the inlet 330 close to the sensitive microfluidic area is challenging and limits the design of microfluidic chips comprising large droplet chambers, as shown in fig. 38 (the indented area under the sidewall portion of the loading well 320 is indicated by a double-headed arrow; the useful area of the microfluidic channel comprising the sensitive microfluidic channel is indicated as "microfluidic area").

The present disclosure provides a solution to this problem that includes the use of a wide loading aperture 320. The precision microfluidic channel (e.g., droplet generator 340) may be placed below the loading well 320 in projection in the base plane (x/y) and the sidewall portion 3211 of the loading well 320 is placed farther away, e.g., on top of the microfluidic network that is unaffected by potential shape changes during fabrication (e.g., droplet chamber 350).

Fig. 39 shows an exemplary embodiment of the solution provided herein (the indented area under the side wall portion of the loading well is indicated by a double-headed arrow; the useful area of the microfluidic channel including the sensitive microfluidic channel is indicated as "microfluidic area"). As shown in this figure, a precision microfluidic channel (e.g., a droplet generator 340) may be placed immediately below an inlet 330 below a bottom wall portion 3212 of a loading well 320 in projection in the base plane (x/y). Thus, the space occupation of the microfluidic area and the droplet chamber 350 is increased compared to current microfluidic chip designs.

Fig. 40 further shows this exemplary embodiment of a microfluidic chip 300 and shows in a transparent manner the spatial organization of elements placed in the top part (black) of the upper plate 310 (including the loading wells 320) and elements placed in the bottom part (grey) of the upper plate 310 (including the droplet generator 340 and the droplet chamber 350).

Fig. 41-52 schematically represent an exemplary operation of an air tank 360 according to the present disclosure.

Typically, the microfluidic chip comprises a microfluidic network directly connected to an upstream inlet microchannel for loading the sample and a downstream output channel for releasing the continuous phase overflowing in the microfluidic chip during droplet generation and storage in the microfluidic network.

However, there are several problems with this microfluidic design, particularly with regard to keeping the droplets and the continuous phase around the droplets in place, regardless of any further flow in the microfluidic network. This is the case, for example, in microfluidic chips that use pressure and air springs to drive droplet generation (such as the microfluidic chip disclosed in international patent application WO 2016170126). In such systems, pressure release (e.g., after droplet generation) can produce continuous phase backflow, which can disrupt the droplet population.

The solution provided herein allows a continuous phase to flow from at least one inlet microchannel to at least one outlet channel while leaving the population of droplets in the continuous phase at rest, i.e., without destroying the integrity of the population of droplets.

By "not disrupting the integrity of the droplet population" is meant that the droplets remain in their relative positions in the microfluidic network, particularly that the droplet population retains its spatial organization in the microfluidic network (e.g., as a 2-dimensional layer of droplets organized in a droplet lattice).

Another problem with existing solutions, where droplet loading or generation means pushing the sample in a locally static continuous phase, is to stabilize the droplet population, resulting in a gradual depletion or depletion of surfactants and/or other components contained in the continuous phase near the input channel or droplet generator.

By "locally static continuous phase" is meant that the continuous phase does not flow in the direction of droplet flow in close proximity to the input channel or droplet generator in the microfluidic channel of the microfluidic chip. Alternatively, it is meant that the continuous phase is not updated in close proximity to the input channel or droplet generator.

The solution provided herein allows homogenizing the local static continuous phase and its concentration in surfactants and/or other ingredients throughout the droplet loading or generation process in the local static continuous phase.

Fig. 41-44 illustrate a first exemplary architecture and operation of a microfluidic chip that includes an inlet microchannel 345 operatively coupled to a droplet chamber 350. Inlet microchannel 345 is further operably coupled to an output channel 361, which is comprised of a capillary trap 3611 and an outlet 3612 from a proximal end to a distal end. In some embodiments, outlet 3612 is a dead end, such as air tank 360.

By "capillary trap" is meant that at least a portion or all of the output channel 361, characterized by width (in the y-axis) and/or height (in the z-axis), is smaller than the diameter of the droplet 314.

As shown in fig. 42, during loading of the droplet chamber with a population of droplets (in this case, loading a population of droplets or an emulsion generated outside and loaded in the microfluidic chip to be stored in the droplet chamber and optionally further processed), the droplets flow from the inlet microchannel 345 to the droplet chamber 350 (stream 3141). When passing through the output channel 361, the droplets do not flow out towards the outlet, since the height of the capillary trap 3611 is smaller than the diameter of the droplet 314. Next, as droplet chamber 350 fills with droplets 314, continuous phase 312 flows toward output channel 361 (stream 3121).

As shown in fig. 43, droplet chamber 350 advantageously has a height (in the z-axis) greater than the average diameter of droplets 314 (but less than twice the average diameter of droplets 314, provided that a 2D droplet layer is intended) to facilitate circulation of stream 3121 through droplets 314.

As shown in fig. 44, after the droplet chamber 350 is loaded with the droplet population 314, the continuous phase 312 can flow from the output channel 361 back to the inlet microchannel 345 or reverse flow (stream 3122) without contact, thus not disrupting the droplet population 314 and the continuous phase 312 in the droplet chamber 350.

Fig. 45A-B illustrate an alternative exemplary architecture and operation of a microfluidic chip that includes an inlet microchannel 345 operatively coupled to a droplet chamber 350. In this alternative architecture, an output channel 361 consisting of a capillary trap 3611 and an outlet 3612 from the proximal end to the distal end is operably coupled to the droplet chamber 350, preferably near the inlet microchannel 345 that joins with the droplet chamber 350. In some embodiments, outlet 3612 is a dead end, such as air tank 360. As previously described. As described, since the height of capillary trap 3611 is less than the diameter of droplet 314, stream 3141 of droplet 314 does not enter output channel 361. Next, when the droplet chamber 350 is filled with droplets 314, the continuous phase 312 flows (stream 3121) toward the output channel 361 (fig. 45A). After the droplet chamber 350 is loaded with the droplet population 314, the continuous phase 312 can flow from the output channel 361 back to the inlet microchannel 345, or reverse flow (stream 3122) is made without contact (or at least with minimal contact), and thus without disrupting (or at least with minimal disruption) the droplet population 314 and the continuous phase 312 in the droplet chamber 350 (fig. 45B).

Fig. 46A-B illustrate alternative exemplary architectures and operations of microfluidic chips. In this alternative architecture, the inlet microchannel 345 includes or corresponds to or is comprised of a droplet generator 340 that opens onto a droplet chamber 350. In this alternative architecture, an output channel 361 consisting of a capillary trap 3611 and an outlet 3612 from the proximal end to the distal end is operatively coupled to the droplet chamber 350, preferably near the droplet generator 340 engaged with the droplet chamber 350. In some embodiments, outlet 3612 is a dead end, such as air tank 360. In this alternative embodiment, the sample is ejected (stream 3142) through inlet microchannel 345. As the sample passes through the droplet generator 340, a population of droplets 314 is generated, the population of droplets 314 eventually being stored in a droplet chamber 350 (stream 3141). As previously described, because the height of capillary trap 3611 is less than the diameter of droplet 314, the resulting stream 3141 of droplet 314 does not enter output channel 361. Next, when the droplet chamber 350 is filled with the droplet group 314, the continuous phase 312 flows toward the output channel 361 (stream 3121) (fig. 46A). After the droplet chamber 350 is loaded with the droplet population 314, the continuous phase 312 can flow from the output channel 361 back to the inlet microchannel 345, or reverse flow (stream 3122) is made without contact (or at least with minimal contact), and thus without disrupting (or at least with minimal disruption) the droplet population 314 and the continuous phase 312 in the droplet chamber 350 (fig. 46B).

Fig. 47 illustrates an alternative exemplary architecture and operation of a microfluidic chip. In this alternative architecture, several inlet microchannels 345 include or correspond to or are made up of drop generators 340 that open together on a single drop chamber 350. In this alternative architecture, an output channel 361 consisting of a capillary trap 3611 and an outlet 3612 from the proximal end to the distal end is operatively coupled to the droplet chamber 350, preferably near the droplet generator 340 engaged with the droplet chamber 350. In some embodiments, outlet 3612 is a dead end, such as air tank 360. In this alternative embodiment, the sample is ejected (stream 3142) through inlet microchannel 345. As the sample passes through the droplet generator 340, a population of droplets 314 is generated, the population of droplets 314 eventually being stored in a droplet chamber 350 (stream 3141). As previously described, because the height of capillary trap 3611 is less than the diameter of droplet 314, the resulting stream 3141 of droplet 314 does not enter output channel 361. Next, when the droplet chamber 350 is filled with the droplet group 314, the continuous phase 312 flows toward the output channel 361 (stream 3121) (fig. 47A). After the droplet chamber 350 is loaded with the droplet population 314, the continuous phase 312 can flow from the output channel 361 back to the nearest inlet microchannel 345, or reverse flow (stream 3122) is made without contact (or at least with minimal contact), and thus without disrupting (or at least with minimal disruption) the droplet population 314 and the continuous phase 312 in the droplet chamber 350 (fig. 47B).

Fig. 48 illustrates an alternative exemplary architecture and operation of a microfluidic chip. In this alternative architecture, several inlet microchannels 345 include or correspond to or are made up of drop generators 340 that open together on a single drop chamber 350. In this alternative architecture, two output channels 361 consisting of a capillary trap 3611 and an outlet 3612, respectively, from the proximal end to the distal end are operatively coupled to the droplet chamber 350, preferably near the droplet generator 340 engaged with the droplet chamber 350. In some embodiments, one or both of outlets 3612 are dead-ends, such as air tank 360. In this alternative embodiment, the sample is ejected (stream 3142) through inlet microchannel 345. As the sample passes through the droplet generator 340, a population of droplets 314 is generated, the population of droplets 314 eventually being stored in a droplet chamber 350 (stream 3141). As previously described, because the height of capillary trap 3611 is less than the diameter of droplet 314, the resulting stream 3141 of droplet 314 does not enter either output channel 361. Next, when the droplet chamber 350 is filled with the droplet population 314, the continuous phase 312 flows toward both output channels 361 (stream 3121) (fig. 48A). After the droplet chamber 350 is loaded with the droplet population 314, the continuous phase 312 can flow from both output channels 361 back to the nearest inlet microchannel 345, or reverse flow (stream 3122) is made without contact (or at least with minimal contact), and thus without disrupting (or at least with minimal disruption) the droplet population 314 and the continuous phase 312 in the droplet chamber 350 (fig. 48B).

Fig. 49-52 show an exemplary design of a microfluidic cell 301 adapted or configured to allow (1) a continuous phase to flow from an inlet microchannel to an output channel while keeping a population of droplets in the continuous phase at rest, and (2) to homogenize a localized static continuous phase and its concentration in surfactants and/or other components throughout droplet loading or generation in the localized static continuous phase.

Fig. 49 is a perspective view of a design of such a microfluidic cell 301 comprising an inlet 330 operatively coupled to a drop generator 340 that opens onto a drop chamber 350. The exemplary chip includes two air tanks 360 operatively coupled to a drop generator 340 by output channels 361.

Fig. 50 is a plan view of the microfluidic cell 301 design shown in fig. 19. As can be seen in FIG. 50, the drop generator 340 includes nine ejectors 343 and a single sloped region 344, the sloped region 344 occupying the width required to operatively couple all of the ejectors 343. Note also that the output channel 361 operably couples the air tank 360 and the sloped region 344.

Fig. 51A-B illustrate an exemplary operation of the microfluidic cell 301 of fig. 49-50. Fig. 52A-B illustrate an alternative exemplary operation of the microfluidic cell 301 of fig. 11.

The sample flowing from inlet 330 is ejected (stream 3142) through inlet microchannel 345. As the sample passes through the droplet generator 340, a population of droplets 314 is generated, the population of droplets 314 eventually being stored in the droplet chamber 350. As previously described, the resulting stream 3141 of droplets 314 does not enter any of the output channels 361 because the height of the capillary trap is less than the diameter of the droplets 314. Next, when the droplet chamber 350 is filled with the droplet group 314, the continuous phase 312 flows toward both the output channels 361 (stream 3121), as shown in fig. 51A and 52A.

After the droplet chamber 350 is loaded with the droplet population 314, the continuous phase 312 can flow from both output channels 361 back to the nearest inlet microchannel 345 (stream 3122) without contact (or at least with minimal contact), and thus without disrupting (or at least with minimal disruption) the droplet population 314 and the continuous phase 312 in the droplet chamber 350, as shown in fig. 51B and 52B.

Current microfluidic technology utilizes pores that tend to contract when cooled after fabrication. This shrinkage can produce sink marks at the bottom of the wells, which can affect the underlying microfluidic network. For this reason, the diameter of the wells currently used in microfluidic chips is small, and the microfluidic network is usually far from these wells, minimizing their surface on the microfluidic chip.

The present disclosure may be described in an alternative manner by one or more numbered paragraphs:

[1] a microfluidic chip (300) comprising an upper plate (310), said upper plate (310) being in contact with at least one loading well (320) and at least one microfluidic channel, extending according to a base plane (x/y) and forming a base for the at least one loading well (320) and the at least one microfluidic channel,

wherein the loading aperture (320) comprises a wall (321) comprising a bottom wall portion (3212) extending integrally according to a bottom plane (wbp) parallel to the base plane (x/y) and a side wall portion (3211) extending along an aperture transverse direction (wld) arranged according to an angle 0 ≦ α ≦ 180 ° relative to the bottom plane (wbp),

wherein the at least one microfluidic channel extends parallel to and below the bottom wall portion (3212) of the loading well (320), and

wherein the bottom wall portion (3212) and the at least one microfluidic channel, which are located in projection in the base plane (x/y), are such that the microfluidic channel is comprised within the bottom wall portion (3212) of the loading well (320).

[2] The microfluidic chip (300) according to numbered paragraph [1], wherein the at least one microfluidic channel has a level of accuracy of ± 5% in its smallest dimension ranging from about 10 μ ι η to about 200 μ ι η.

[3] The microfluidic chip (300) according to numbered paragraphs [1] to [2], wherein the at least one microfluidic channel is a droplet generator (340).

[4] The microfluidic chip (300) according to any of the numbered paragraphs [1] to [3], further comprising a continuous phase (312), preferably wherein the continuous phase (312) partially or completely fills the microfluidic network of the microfluidic chip (300), more preferably wherein the microfluidic network of the microfluidic chip (300) comprises at least a droplet generator (340) and a droplet chamber (350).

[5] The microfluidic chip (300) according to any of the numbered paragraphs [1] to [4], wherein the microfluidic chip (300) is made of molded plastic, injected plastic, or injection compression molded plastic.

[6] A system for preventing warping of a microfluidic channel immediately adjacent to a loading well (320), the system comprising at least one microfluidic chip (300) according to any one of the numbered paragraphs [1] to [5], wherein the system is configured to avoid warping of the microfluidic channel.

[7] A method of preventing warping of a microfluidic channel immediately adjacent to a loading well (320), the method comprising:

-manufacturing a microfluidic chip (300), wherein:

the microfluidic chip (300) comprising an upper plate (310), the upper plate (310) extending according to the base plane (x/y) and forming at least one loading well (320) and a base of at least one microfluidic channel,

the loading aperture (320) comprises a wall (321) comprising a bottom wall portion (3212) extending integrally according to a bottom plane (wbp) parallel to the base plane (x/y) and a side wall portion (3211) extending along an aperture transverse direction (wld) arranged according to an angle 0 ≦ α ≦ 180 ° relative to the bottom plane (wbp),

said at least one microfluidic channel extends parallel to and below said bottom wall portion (3212) of said loading well (320), and

the bottom wall portion (3212) and the at least one microfluidic channel being located in projection in the base plane (x/y) such that the microfluidic channel is comprised in the bottom wall portion (3212) of the loading well (320),

thereby avoiding warping of the sensitive microfluidic channel.

[8] A system for optimizing the space occupation of microfluidic elements in a microfluidic chip, the system comprising at least one microfluidic chip (300) according to any one of the numbered paragraphs [1] to [5], wherein the system is configured to avoid sink marks on the microfluidic channels.

[9] A method for optimizing the space occupation of a microfluidic element in a microfluidic chip, the method comprising:

-manufacturing a microfluidic chip (300), wherein:

the microfluidic chip (300) comprising an upper plate (310), the upper plate (310) extending according to the base plane (x/y) and forming at least one loading well (320) and a base of at least one microfluidic channel,

the loading aperture (320) comprises a wall (321) comprising a bottom wall portion (3212) extending integrally according to a bottom plane (wbp) parallel to the base plane (x/y) and a side wall portion (3211) extending along an aperture transverse direction (wld) arranged according to an angle 0 ≦ α ≦ 180 ° relative to the bottom plane (wbp),

said at least one microfluidic channel extends parallel to and below said bottom wall portion (3212) of said loading well (320), and

the bottom wall portion (3212) and the at least one microfluidic channel being located in projection in the base plane (x/y) such that the microfluidic channel is comprised in the bottom wall portion (3212) of the loading well (320),

thereby optimizing the space occupation of the microfluidic elements in the microfluidic chip.

In the numbered paragraphs, the upper features of the upper plate (310) correspond to normal use of the microfluidic chip (300). When in use, the microfluidic chip (300) is held nearly horizontal with the loading well (320) above the upper plate (310): the plate is the base where the holes are located. Furthermore, the microfluidic channel, the droplet generator (340) and the droplet chamber (350) are located below the upper plate (310): the plate is a support for the microfluidic channel below the support. Finally, the upper features are associated with the microfluidic channel, the droplet generator (340) and the droplet chamber (350).

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