Microfluidic chip including grooves to facilitate loading thereof and related methods

Abstract

A microfluidic chip can include a body and a microfluidic network defined by the body. The network can include one or more inlet ports, a test space, and one or more flow paths extending between the inlet port and the test space. Along each flow path, a fluid is allowed to flow from one of the inlet ports through at least one droplet generation region and flow to the test space, and the minimum cross-sectional area of ​​the flow path in the at least one droplet generation region increases along the flow path. The network can include grooves arranged along at least a portion of the periphery of the test space, so that fluid from the flow path is not allowed to flow into the groove without flowing through the test space, wherein the depth of the groove along the groove is at least 10% greater than the depth of the test space at the periphery.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 889,420, filed on August 20, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates generally to microfluidic chips, and more particularly, but not limited to, to droplet generation microfluidic chips defining one or more networks, each having a test space and channels capable of receiving droplets from the test space. Background Art

[0004] Microfluidic chips have been increasingly used in many fields, including cosmetics, pharmaceuticals, pathology, chemistry, biology, and energy. Microfluidic chips typically have one or more channels that are configured to transmit, mix, and / or separate one or more samples for analysis. At least one of the channels may have a size of about one micron or tens of microns, allowing relatively small (e.g., nanoliters or picoliters) sample volumes to be analyzed. The smaller sample volumes used in microfluidic chips provide many advantages over conventional desktop techniques. For example, due to the scale of chip components, more accurate biometric results can be achieved using microfluidic chips, including the manipulation and analysis of single cells and / or molecules. Microfluidic chips can also provide improved control of the cell environment therein, so as to facilitate experiments related to cell growth, aging, antibiotic resistance, and the like. Moreover, microfluidic chips are very suitable for diagnostic applications including identification of pathogens and instant diagnosis due to their small sample volume, low cost, and disposability.

[0005] In some applications, microfluidic chips are configured to generate droplets to facilitate analysis of samples. Droplets can encapsulate the cells or molecules being studied, effectively amplifying their concentration and increasing the number of reactions. Therefore, droplet-based microfluidic chips can be well suited for high-throughput applications such as chemical screening and PCR.

[0006] The test space of the microfluidic network of the chip traditionally utilizes the pressure at the network inlet port to be increased to above the ambient pressure to load the sample so that the sample flows into the test space. These microfluidic chips must usually balance the pressure between the test space and the surrounding environment after the droplets are formed, for example by allowing at least a portion of the liquid to leave through the second port. To prevent the loss of droplets during pressure balance, these chips may require additional mechanisms to keep the droplets in the test space. In many chips, the droplets in the test space preferably form a two-dimensional array, in which the droplets overlap, stack and / or are compressed to a minimum, so as to facilitate analysis thereof. For example, when the droplets overlap, stack and / or are compressed, they may be more difficult to distinguish from each other.

[0007] The test space can have a droplet capacity that, if exceeded, would undesirably result in overlapping, stacking, and / or compression of droplets in the test space, particularly when the chip has a droplet retention mechanism. Attempts to mitigate this adverse effect have been largely unsatisfactory and are expensive and / or complex. For example, it may be difficult and impractical to control the volume of liquid introduced into the inlet port (e.g., such that the volume is capable of producing sufficient droplets for analysis without overloading the test space). In addition, flow control mechanisms that stop the flow when the droplet capacity of the test space is reached are typically both expensive and complex.

[0008] These challenges associated with volume control and flow control may be increased when multiple microfluidic networks are loaded simultaneously. In these cases, one of the test spaces of a network may reach its capacity before the other networks because a larger volume of liquid may have been introduced into the inlet port of that network and / or the test spaces may have different droplet capacities. If not independently controlled, the full-capacity test space may continue to receive droplets when loading of the partially loaded test space is complete, which can produce undesirable droplet overlap, stacking, and / or compression. Preventing this volume mismatch can become particularly difficult as the number of microfluidic networks increases. And the expense and complexity of flow control may also increase with the number of microfluidic networks, as such a system may require independent flow control for each of the networks. Summary of the invention

[0009] Therefore, there is a need in the art for a microfluidic chip that can effectively (and in a simple and cost-effective manner) mitigate the overlap, stacking and / or compression of droplets that may be caused when the test space continues to load droplets after reaching its droplet capacity. The chip of the present invention can address this need by using a groove, which is arranged along at least a portion (e.g., at least a large portion) of the periphery of the test space, and the depth of the groove along the groove is at least 10% greater than the depth of the test space at the periphery. Unlike conventional chips, in this way, the groove can provide a relatively large area through which droplets can leave the test space, so that when the droplet capacity of the test space is reached, the rate at which droplets are removed can be similar to or greater than the rate at which additional droplets enter the test space. Therefore, even when additional droplets are introduced into the full capacity test space, droplet overlap, stacking and / or compression can be mitigated. In this way (whether loading a single microfluidic network or loading multiple microfluidic networks simultaneously), the groove can facilitate the formation of a two-dimensional array of droplets, and the formation of the two-dimensional array promotes accurate analysis of the droplets without the need for precise, expensive and / or complex volume control and flow control.

[0010] Some microfluidic chips of the present invention include a body and a microfluidic network defined by the body, the network includes one or more inlet ports, and some methods of the present invention include setting a liquid in a first inlet port of the one or more inlet ports of the microfluidic network. In some embodiments, the network includes one or more inlet ports, a test space, and one or more flow paths extending between the inlet port and the test space. In some embodiments, along each of the flow paths, the fluid is allowed to flow from one of the inlet ports through at least one droplet generation area and flow to the test space, and the minimum cross-sectional area of ​​the flow path in the at least one droplet generation area increases along the flow path. Some methods include guiding at least a portion of the liquid along a first flow path in the flow path so that a portion of the liquid flows from the first inlet port through at least one droplet generation area and flows to the test space, and the minimum cross-sectional area of ​​the first flow path in the at least one droplet generation area increases along the first flow path.

[0011] In some embodiments, the network includes grooves arranged along at least a portion, optionally at least a majority, of the periphery of the test space, so that fluid from the flow path is not allowed to flow into the grooves without flowing through the test space. In some embodiments, along the grooves, the depth of the grooves is at least 10%, optionally at least 90%, greater than the depth of the test space at the periphery. In some embodiments, the depth of the test space is 15 to 90 microns (μm) and / or is substantially the same over the entire test space. In some embodiments, the depth of the grooves is at least 100 μm. In some embodiments, the maximum lateral dimension of the grooves taken perpendicular to the centerline of the grooves is less than or equal to 10% of each of the width and length of the test space. In some embodiments, the network includes one or more outlet ports in fluid communication with the grooves, so that the fluid is allowed to flow from the grooves to the outlet ports without flowing through the test space.

[0012] In some methods, at least a portion of the liquid is directed along a first flow path so that droplets are formed from the portion of the liquid and directed to the testing volume, at least one of the droplets flows from the testing volume to the gutter and optionally to one of the outlet ports. In some methods, each droplet has a volume of 25 to 500 picoliters.

[0013] In some embodiments, each of the flow paths includes a contraction section, a constant section, and an expansion section in at least one droplet generation region, so that the fluid is allowed to leave the contraction section, enter the constant section, and flow to the expansion section. In some embodiments, the depth of the constant section is at least 10% greater than the depth of the contraction section, and the depth is optionally substantially the same along at least 90% of the length of the constant section. In some embodiments, the depth of the expansion section increases as it moves away from the constant section. In some methods, at least a portion of the liquid is guided along the first flow path so that the portion of the liquid leaves the contraction section, enters the constant section, and flows to the expansion section.

[0014] In some embodiments, the microfluidic network is a first microfluidic network, and optionally, the body defines a second microfluidic network. In some embodiments, the second network includes one or more inlet ports, a test space, and one or more flow paths extending between the inlet port and the test space. In some embodiments, along each of the flow paths of the second network, the fluid is allowed to flow from one of the inlet ports through at least one droplet generation region and flow to the test space, and the minimum cross-sectional area of ​​the flow path in the at least one droplet generation region increases along the flow path. In some embodiments, the second network includes a groove disposed along at least a portion of the periphery of the test space, so that the fluid from the flow path is not allowed to flow into the groove without flowing through the test space. In some embodiments, along the grooves of the second network, the depth of the groove is at least 10% greater than the depth of the test space at the periphery.

[0015] In some methods, the liquid is a first liquid and the methods include disposing a second liquid in a first inlet port of the inlet ports of the second network. Some such methods include directing at least a portion of the first liquid along a first flow path of the flow paths of the second network while directing at least a portion of the second liquid along a first flow path of the flow paths of the second network such that a portion of the second liquid flows from the first inlet port through at least one droplet generation region and flows to a test space, wherein a minimum cross-sectional area of ​​the first flow path in the at least one droplet generation region increases along the first flow path. In some methods, for at least one of the first network and the second network, at least a portion of the liquid is directed along the first flow path by at least (1) reducing the pressure at the first port such that gas flows out of the first port from the test space along at least one of the flow paths; and increasing the pressure at the first port such that a portion of the liquid flows from the first port through at least one of the droplet generation regions and flows to the test space.

[0016] The term "coupled" is defined as connected, but not necessarily directly and not necessarily mechanically; two items that are "coupled" can be integral with each other. Unless otherwise expressly required by the present disclosure, the terms "a" and "an" are defined as one or more. The term "substantially" is defined as to a large extent, but not necessarily entirely, the specified content, and includes the specified content; for example, substantially 90 degrees includes 90 degrees, and substantially parallel includes parallel, as understood by one of ordinary skill in the art. In any disclosed embodiment, the term "substantially" can be replaced with "within [percentage]" as specified, where percentages include 0.1%, 1%, 5%, and 10%.

[0017] The terms "include" and any form thereof (such as "includes" and "containing"), "have" and any form thereof (such as "have" and "with"), and "include" and any form thereof (such as "containing" and "including") are open-ended linking verbs. As a result, a device that "includes," "has," or "includes" one or more elements possesses the one or more elements, but is not limited to possessing only these elements. Similarly, a method that "includes," "has," or "includes" one or more steps possesses the one or more steps, but is not limited to possessing only the one or more steps.

[0018] Any embodiments of any apparatus, system, and method may consist of or consist essentially of (rather than include / have / contain) any described steps, elements, and / or features.

[0019] Furthermore, a device or system that is configured in a certain way is configured in at least that way, but it may also be configured in other ways other than the specifically described way.

[0020] Unless expressly prohibited by the disclosure or the nature of the embodiments, one or more features of one embodiment may be applied to other embodiments even if not described or illustrated.

[0021] Some details associated with the above-described embodiments and other embodiments are described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following figures are shown by way of example and not limitation. For the sake of brevity and clarity, every feature of a given structure is not always labeled in every figure in which the structure appears. The same reference numerals do not necessarily indicate the same structure. On the contrary, the same reference numerals may be used to indicate similar features or features having similar functions, as may different reference numerals. Unless otherwise indicated, the views in the drawings are drawn to scale, meaning that, at least for the embodiments in the views, the sizes of the depicted elements are accurate relative to each other.

[0023] Figure 1A is an exploded perspective view of one of the microfluidic chips of the present invention having a body defining a plurality of microfluidic networks. Each of the microfluidic networks is configured to generate droplets that can be collected in a test volume of the network.

[0024] Figure 1B yes Figure 1A A top view of the chip showing the inlet and outlet ports of the chip.

[0025] Figure 1C to Figure 1F They are Figure 1A Left view, right view, front view and back view of the chip.

[0026] Figure 1G yes Figure 1A A bottom view of a first workpiece of a chip with a second workpiece of the chip removed. Figure 1G Shown is the microfluidic network defined by the chip.

[0027] Figure 1H yes Figure 1A A magnified view of one of the microfluidic networks of the chip.

[0028] Figure 2 is along Figure 1B The line 2-2 is intercepted Figure 1A Cross-sectional view of a chip. Figure 2 An inlet port of one of the microfluidic networks of the chip and a portion of a flow path connected to the inlet port are shown.

[0029] Figure 3A yes Figure 1AA magnified view of one of the droplet generation regions of one of the microfluidic networks of a chip. In the droplet generation region, the flow path includes a contraction segment, a constant segment, and an expansion segment, so that the minimum cross-sectional area of ​​the flow path increases along the flow path.

[0030] Figure 3B is along Figure 3A The line 3B-3B is intercepted Figure 1A A partial cross-sectional view of a chip. Figure 3B The relative size of the constriction and the upstream passage connected to the constriction are shown.

[0031] Figure 3C is along Figure 3A The line 3C-3C is intercepted Figure 1A Partial cross-sectional view of the microfluidic chip. Figure 3C The geometry of the constant section and the expanding section relative to the contracting section is shown, with the expanding section having a ramp defined by a single planar surface.

[0032] Figure 4 FIG. 1 is a partial cross-sectional view of a droplet generation region of another embodiment of a microfluidic chip of the present invention, which is similar to FIG. Figure 1A The chips are basically similar, with the main exceptions being Figure 4 The ramp of the expansion section in the chip is defined by a plurality of steps.

[0033] FIG. 5A to FIG. 5D It shows that when the liquid enters the constant section from the contraction section and flows toward the expansion section Figure 1A Generation of droplets in a chip.

[0034] Figure 6 is along Figure 1H The line 6-6 is intercepted Figure 1A A partial cross-sectional view of a chip is shown, showing the grooves arranged around the test space.

[0035] Fig. 7A and Figure 7B shows when a droplet enters the groove from the test space Figure 1A The functionality of the trenches in the chip.

[0036] Figure 8 Schematic diagram of a system including a vacuum chamber that can be used to change the pressure at the inlet port of some microfluidic chips of the present invention to exhaust gas from the test space of the chip and load liquid into the test space of the chip. The system can include a vacuum source, one or more control valves and a controller to adjust the rate of generating or exhausting a vacuum.

[0037] 9A to 9Dis a schematic diagram showing some of the inventive methods of loading a microfluidic chip, wherein liquid is loaded into a port, gas is expelled from a test space through the liquid, and the liquid flows through at least one droplet generation region to form droplets. DETAILED DESCRIPTION

[0038] from Figures 1A to 1H Initially, a first embodiment of a microfluidic chip 10 of the present invention is shown. The chip 10 may include a body 14 defining one or more (optionally two or more) microfluidic networks 18 ( Figure 1G ); as shown, the chip defines multiple networks. The body 14 can be made of any suitable material and can include a single workpiece or multiple workpieces (e.g., 22a and 22b), wherein at least one of the workpieces defines at least a portion of the microfluidic network 18. For example, as shown, the body 14 of the chip 10 includes two workpieces 22a and 22b, wherein at least one of the workpieces can include a (e.g., rigid) polymer, and optionally, one of the workpieces can include a polymer film.

[0039] Especially refer to Figure 1H , which shows one of the microfluidic networks 18 of the chip 10, each of which may include a test space 30 configured to receive a liquid (e.g., a droplet) for analysis. For example, the chip 10 can be configured to allow identification of pathogens encapsulated in a microfluidic droplet disposed in the test space 30. However, in other embodiments, the chip 10 can be used for any other suitable microfluidic application (e.g., DNA analysis, drug screening, cell experiments, electrophoresis, and / or similar applications).

[0040] To allow for loading of the test space 30, each of the microfluidic networks 18 can include one or more inlet ports 26, a test space 30, and one or more flow paths 34 extending between the inlet ports and the test space. Along each of the flow paths 34, a fluid can flow from one of the inlet ports 26 through at least one droplet generation region 38 (described in further detail below) and into the test space 30, so that droplets can be formed and introduced into the test space for analysis. The flow paths 34 can be defined by one or more channels and / or other passages through which a fluid can flow. Each of the flow paths 34 can have any suitable maximum lateral dimension (e.g., a maximum lateral dimension taken perpendicular to a centerline of the flow path that is less than or equal to any one of 2000, 1500, 1000, 500, 300, 200, 100, 50, or 25 μm, or between any two thereof) to facilitate microfluidic flow.

[0041] Each of the microfluidic networks 18 can be configured to allow vacuum loading of the test space 30, such as by allowing gas from the test space to be vented before introducing liquid into the test space. For example, when liquid is disposed in at least one of the inlet ports 26, gas venting can be achieved by reducing the pressure at the inlet port so that gas in the test space 30 flows through at least one of the flow paths 34, through the liquid, and out of the inlet port. Liquid can be introduced into the test space 30 (e.g., for analysis) by increasing the pressure at the inlet port 26 so that the liquid flows from the inlet port through at least one of the flow paths 34 and into the test space.

[0042] See also Figure 2 The relative sizes of each of the inlet ports 26 and the portion 42 of the flow path 34 connected thereto can promote the formation of bubbles as the gas passes through the liquid and can minimize or prevent liquid loss (e.g., liquid loss that may result if slug flow is generated). For example, the portion 42 of the flow path 34 can have a minimum cross-sectional area 46 (taken perpendicular to the centerline 58 of the inlet port) that is smaller than the minimum cross-sectional area 54 of the inlet port 26 (taken perpendicular to the centerline 58 of the portion), for example, the minimum cross-sectional area is less than or equal to any one of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the minimum cross-sectional area of ​​the inlet port, or between any two thereof (e.g., less than or equal to 90% or 10%). The smaller cross-sectional area of ​​the portion 42 can promote the formation of bubbles having a diameter less than the diameter of the inlet port 26, so that slug flow is reduced during gas discharge and thereby liquid loss is reduced. The bubbles can agitate and thereby mix the liquid in the inlet port 26 to facilitate loading and / or analysis of the liquid in the test space 30.

[0043] The droplet generation region 38 can be configured to form droplets in any suitable manner. FIG. 3A to FIG. 3C , for each of the flow paths 34, the minimum cross-sectional area of ​​the flow path in at least one of the droplet generation regions 38 can increase along the flow path. To illustrate, in the droplet generation region 38, the flow path 34 can include a contraction section 62, a constant section 66, and / or an expansion section 70.

[0044] The contraction section 62 can be configured to promote droplet generation. As shown, for example, the contraction section 62 can extend between an inlet 74a and an outlet 74b, wherein the inlet is connected to a channel 78 so that liquid can enter the contraction section from the channel ( Figure 3A and 3B). The channel 78 can have a maximum transverse dimension 82 (taken perpendicular to the centerline of the portion of the channel) and / or a maximum depth 86 (taken perpendicular to the centerline and transverse dimension of the portion of the channel) that are respectively greater than the maximum transverse dimension 90 and the maximum depth 94 of the constricted section 62. For example, at least one of the maximum transverse dimension 82 and the maximum depth 86 of the channel 78 can be greater than or equal to any one of 10, 25, 50, 75, 100, 125, 150, 175, or 200 μm, or between any two thereof (e.g., 75 to 170 μm), while the maximum transverse dimension 90 of the constricted section 62 can be less than or equal to any one of 200, 175, 150, 125, 100, 75, or 50 μm, or between any two thereof, and the maximum depth 94 can be less than or equal to any one of 20, 15, 10, or 5 μm, or between any two thereof (e.g., 10 to 20 μm). Furthermore, the contraction section 62 can define a contraction between the inlet 74a and the outlet 74b, and the minimum cross-sectional area 98 (taken perpendicular to the centerline of the contraction) of the contraction section of the flow path 34 at the contraction section can be smaller (e.g., at least 10% smaller) than at the inlet and / or the outlet. The minimum transverse dimension 102 of the contraction section 62 (e.g., at the contraction section) can be less than or equal to any one of 40, 35, 30, 25, 20, or 15 μm, or between any two thereof, and the length 106 of the contraction section between the inlet 74a and the outlet 74b can be greater than or equal to any one of 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 μm, or between any two thereof, (e.g., 450 to 750 μm), which ensures that the contraction section 62 remains ready during droplet pinch-off.

[0045] The formation of droplets can be achieved by allowing the liquid to expand after it contracts. Along the flow path 34, the liquid from the contraction section 62 can enter the expansion region 110, and the minimum cross-sectional area 114 of the flow path in the expansion region is greater than the minimum cross-sectional area 98 ( Figure 3C). For example, the cross-sectional area 114 can be at least 10%, 50%, 100%, 200%, 300%, 400%, 500%, or 1000% greater than the cross-sectional area 98. Such expansion can include a change in the depth of the flow path 34. The depth of the flow path 34 in the expansion region 110 (e.g., 118, 126a, and / or 126b) can be at least 10%, 50%, 100%, 150%, 200%, 250%, or 400% greater than the maximum depth 94 of the contraction section 62, such as greater than or equal to any one of 5, 15, 30, 45, 60, 75, 90, 105, or 120 μm, or between any two thereof (e.g., 35 to 45 μm or 65 to 85 μm). Liquid flowing from the contraction section 62 to the expansion region 110 along the flow path 34 can thereby expand and form droplets.

[0046] These depth changes can occur in the constant section 66 and / or the expansion section 70 of the flow path 34, wherein liquid flowing from one of the inlet ports 26 to the test volume 30 is allowed to exit the contraction section 62 into the constant and / or expansion section. Figure 3C In the illustrated embodiment, expansion of the liquid can be achieved using both the constant section 66 and the expansion section 70, whose geometry can promote the formation of droplets of substantially the same size and facilitate suitable droplet placement in the test space 30. The constant section 66 and the expansion section 70 can be arranged so as to allow a fluid flowing from one of the inlet ports 26 to the test space 30 to flow from the contraction section 62 through the constant section to the expansion section. The constant section 66 can have a depth 118 that can be equal to the minimum depth of the expansion region 110 and greater than (e.g., at least 10% or at least 50%) the maximum depth 94 of the contraction section 62, such as greater than or equal to any one of 5, 20, 35, 50, 65, or 80 μm, or between any two thereof (e.g., 35 to 45 μm). The depth 118 of the constant section 66 can be substantially the same along at least 90% of its length 122 between the contraction section 62 and the expansion section 70. The constant segment 66 can have any suitable length 122 to allow complete droplet formation (including droplet pinch-off), for example, a length greater than or equal to any one of 15, 25, 50, 100, 200, 300, 400 or 500 μm, or between any two thereof (e.g., 150 to 200 μm).

[0047] The expansion section 70 can be expanded to move toward the test space 30 along the flow path 34, and the depth of the expansion section increases from the first depth 126a to the second depth 126b. The first depth 126a and the second depth 126b can be, for example, the minimum depth and the maximum depth of the expansion region 110, respectively. For illustration, the expansion section 70 can define a ramp 130 with a slope 134, which is arranged at an angle 138 relative to the contraction section 62, so that the depth of the expansion section increases as it moves away from the constant section. The angle 138 measured as measured relative to the direction parallel to the centerline of the contraction section 62 can be greater than or equal to any one of 5°, 10°, 20°, 30°, 40°, 50°, 60°, 70° or 80°, or between any two thereof (e.g., 20° to 40°). The ramp 130 can extend from the constant section 66 (e.g., such that the depth 126a is substantially the same as the depth 118) to a point where the expansion region 110 reaches its maximum depth 126b, which can be greater than or equal to any one of 15, 30, 45, 60, 75, 90, 105, or 120 μm, or between any two thereof (e.g., 65 to 85 μm). As shown, the ramp 130 is defined by a (e.g., single) planar surface. However, referring to Figure 4 In other embodiments, the ramp 130 can be defined by a plurality of steps 142 (e.g., which can be cost-effective if the chip 10 is manufactured using a mold produced using lithography), each of which has an appropriate rise 146 and run 150 so that the ramp has any of the above-described slopes 134.

[0048] See also FIG. 5A to FIG. 5D (which shows the use of relative Figure 3C As the liquid flows from the contraction section to the constant section, the droplet 154 can be formed from the aqueous liquid 158 in the presence of the non-aqueous liquid 162. The constant section 66 can compress the droplet 154 to prevent it from fully expanding ( Figure 5A and Figure 5B The constant section 66 can thereby prevent the droplets 154 from stacking on each other, so that the droplets can be arranged in a two-dimensional array in the test space 30. Such an array can facilitate accurate analysis of the droplets 154. The compressed droplets 154 flowing from the constant section 66 to the expansion section 70 can travel along the ramp 130 and decompress ( Figure 5C and Figure 5D). The reduced pressure can reduce the surface energy of the droplet 154, so that the droplet is propelled along the ramp 130 and away from the expansion section 70 (e.g., toward the test space 30). At least by pushing the droplet 154 out of the expansion section 70, the ramp 130 can alleviate the accumulation of droplets at the interface between the outlet 74b of the contraction section 62 and the constant section 66, so that the droplet 154 does not hinder the formation of subsequent droplets. Because such obstruction can cause inconsistent droplet sizes, by alleviating blockage, the expansion section 70 can promote the formation of droplets of uniform size, for example, the diameter of each of these droplets is within 3-6% of the diameter of each other.

[0049] The droplet generation region 38 can have other configurations to form droplets. For example, the expansion of the liquid can be achieved using the constant section 66 alone, the expansion section 70 alone, or the expansion section upstream of the constant section. And in other embodiments, at least one of the droplet generation regions 38 can be configured to form droplets via a T-joint (e.g., two channels (aqueous liquid 158 flows through one channel, and non-aqueous liquid 162 flows through another channel) connected at the T-joint so that the non-aqueous liquid shears the aqueous liquid to form droplets), flow focusing, co-flowing, and / or the like. In some such alternative embodiments, each of the microfluidic networks 18 can include a plurality of inlet ports 26, and the aqueous liquid 158 and the non-aqueous liquid 162 can be arranged in different inlet ports (e.g., so that they can meet at the joint for droplet generation).

[0050] Due at least in part to the geometry of the droplet generation region 38, the droplets 154 can have a relatively low volume, for example, less than or equal to any one of 10,000, 5,000, 1,000, 500, 400, 300, 200, 100, 75, or 25 picoliters (pL), or between any two thereof (e.g., 25 to 500 pL). The relatively low volume of the droplets 154 can facilitate analysis of microorganisms contained, for example, by an aqueous liquid. During droplet generation, each of the one or more microorganisms can be encapsulated by one of the droplets 154 (e.g., such that each of the encapsulated droplets includes a single microorganism and optionally its progeny). Due to the small droplet volume, the concentration of the encapsulated microorganisms in the droplets can be relatively high, which can allow for their detection without requiring prolonged culturing to propagate the microorganisms.

[0051] Droplets from the droplet generation region 38 can flow to the test space 30, which can have a droplet capacity to accommodate enough droplets for analysis. For example, the size of the test space 30 can be set to accommodate any one of greater than or equal to 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 or 100000, or a number of droplets between any two thereof (e.g., 13000 to 25000 droplets). To this end, the test space 30 can have a length 166 and a width 170, the length being greater than or equal to any one of 9, 10, 11, 12, 13, 14, 15, 16, or 17 mm, or between any two thereof (e.g., 11 to 15 mm), and the width being greater than or equal to any one of 3, 4, 5, 6, 7, 8, 9, 10, or 11 mm, or between any two thereof (e.g., 5 to 9 mm). The test space 30 can also have a depth 186 that can accommodate droplets while mitigating droplet stacking (e.g., without compressing the droplets). The depth 186 can, for example, be greater than or equal to any one of 15, 30, 45, 60, 75, 90, 105, or 120 μm, or between any two thereof (e.g., 15 to 90 μm, such as 65 to 85 μm) (e.g., substantially the same as the maximum depth 126 b of the expansion region 110), and optionally, can be substantially the same across the test space 30.

[0052] In conventional chips, when the droplet capacity of the test space is reached, droplets may overlap, stack, and / or compress, which can adversely affect their analysis. For example, when droplets are analyzed using an imaging system, the overlapping, stacked, and / or compressed droplets may be difficult to distinguish, which can reduce the quality of the information captured during analysis. Figure 6, each of the microfluidic networks 18 can include grooves 174 that can mitigate these undesirable effects when the test space 30 reaches its droplet capacity. The grooves 174 can be arranged along at least a portion (e.g., at least a majority) of the perimeter 178 of the test space 30 so that fluid from the flow path 34 is not allowed to flow into the groove without flowing through the test space; this does not exclude the possibility that one or more other flow paths of the network can allow fluid to flow into the groove without flowing through the test space. Along the groove 174, the depth 182 of the groove can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 110% (e.g., at least 90%) greater than the depth 186 of the test space 30 at the periphery 178, for example, greater than or equal to any one of 100, 115, 130, 145, 160, 175, 190, 205, 220, 235 or 250 μm, or between any two thereof (e.g., 140 to 160 μm). And the maximum lateral dimension 190 of the groove 174 (taken perpendicularly to the centerline of the groove) can be less than or equal to any one of 12%, 10%, 8%, 6%, 4% or 2% of each of the length 166 and the width 170 of the test space 30, or between any two thereof, such as less than or equal to any one of 210, 200, 190, 180, 170 or 160 μm, or between any two thereof.

[0053] See also Fig. 7A and Figure 7B (which shows the use of groove 174), when the test space reaches a certain capacity ( Figure 7B ), (e.g., due at least in part to the buoyancy difference between the aqueous liquid 158 and the non-aqueous liquid 162), the droplet 154 ( Fig. 7A ) can rise or fall in the groove. The depth 182 of the groove 174 can (but does not have to) increase away from the perimeter 178 of the test volume 30 (e.g., until the depth reaches a maximum value) to facilitate this movement. By being positioned along at least a portion (e.g., at least a majority) of the perimeter 178, the groove 174 can provide a relatively large area through which droplets can exit the test volume 30. In this way, when the test volume reaches full capacity, the rate at which droplets are removed from the test volume 30 can be similar to or faster than the rate at which droplets enter the test volume from the droplet generation region 38, thereby reducing accumulation and thereby reducing droplet stacking, overlapping, and / or compression in the test volume.

[0054] The grooves 174 can be particularly advantageous when liquid is loaded into multiple microfluidic networks 18 in parallel (e.g., when the chip 10 has multiple networks and / or when multiple chips are loaded). If different amounts of liquid are introduced into each microfluidic network 18 and / or if the test spaces 30 of the network have different droplet capacities, at least one of the test spaces can reach capacity before the other test spaces are fully loaded. In conventional chips, continued loading of partially loaded test spaces may cause droplets in the full-capacity test spaces to be undesirably stacked, overlapped, and / or compressed. The microfluidic network 18 can at least address this problem because each of the microfluidic networks 18 includes a groove 174, and droplets in the full-capacity test space 30 can leave at a rate sufficient to mitigate the stacking, overlap, and / or compression of these droplets, while the partially loaded test spaces continue to be loaded in parallel. Therefore, even if the test spaces reach capacity at different times, a suitable array of droplets can be loaded into each of the test spaces 30. And this parallel loading can be achieved without expensive and complex independent flow control for each of the microfluidic networks 18.

[0055] One or more outlet ports 194 can be in fluid communication with the groove 174 via one or more outlet channels 198, so that the fluid can flow from the groove to the outlet port without flowing through the test space 30. Each of the outlet ports 194 can be substantially similar to the inlet port 26 (e.g., the size of each of these outlet ports relative to the portion of the outlet channel 198 connected thereto can be the same as the size of each of the inlet ports relative to the portion 42). In this way, droplets entering the groove 174 from the test space 30 can continue to flow to the outlet port 194, which can accommodate droplets so as to allow a large number of droplets to be removed from the test space 30 to alleviate the stacking, overlapping and / or compression of these droplets. In other embodiments, as an alternative or in addition to the outlet port 194, the chip 10 can include one or more containers that are sealed separately (e.g., so that liquid cannot be introduced into the chip via the container), which can also receive droplets from the groove 174 via the outlet channel 198. For embodiments in which chip 10 does not include egress port 194 , the chip can be a single port chip (eg, where ingress port 26 consists of a single ingress port).

[0056] Reference Figure 8, shows a system 202, which can be used to load the test space 30 of each of one or more microfluidic networks 18 of at least one chip (e.g., 10) of the present invention. System 202 can include a vacuum chamber 206, which is configured to receive and accommodate a microfluidic chip. A vacuum source 210 and one or more control valves (e.g., 214a-214d) can be configured to regulate the pressure in the vacuum chamber 206. For example, the vacuum source 210 can be configured to remove gas from the vacuum chamber 206, and thus reduce the pressure in the vacuum chamber (e.g., reduced to below ambient pressure), thereby reducing the pressure at the inlet port (e.g., 26) of each in the microfluidic chip. The reduced pressure can promote the gas discharge of the microfluidic chip. Each of the control valves can move between a closed position and an open position, wherein the control valves prevent and allow the fluid transfer between vacuum chamber 206, vacuum source 210, and / or external environment 218, respectively. For example, after the vacuum is generated in the vacuum chamber 206, opening at least one control valve can allow gas (e.g., from the external environment 218) to enter the vacuum chamber to increase the pressure in the vacuum chamber (e.g., to ambient pressure), thereby increasing the pressure at the inlet port of each of the microfluidic chips. The increased pressure can facilitate the generation of droplets and the liquid loading of the test space 30.

[0057] The system 202 can include a controller 222 configured to control the vacuum source 210 and / or the control valve to adjust the pressure in the vacuum chamber 206. The controller 222 can be configured to receive vacuum chamber pressure measurements from a pressure sensor 226. Based at least in part on these pressure measurements, the controller 222 can be configured to activate at least one of the vacuum source 210 and / or the control valve, for example, to achieve a target pressure within the vacuum chamber 206 (e.g., using a proportional integral derivative controller). For example, the control valves of the system 202 can include a slow valve 214a and a fast valve 214b, each of which (when in an open position) allows fluid to flow between the vacuum chamber 206 and at least one of the vacuum source 210 and the external environment 218. The system 202 can be configured so that the maximum rate at which gas can flow through the slow valve 214a is lower than the maximum rate at which gas can flow through the fast valve 214b. As shown, for example, the system 202 includes a restriction device 230 in fluid communication with the slow valve 214a. The controller 222 can control the rate at which gas enters or escapes the vacuum chamber 206, thereby controlling the rate of change of pressure in the vacuum chamber, by at least selecting and opening at least one of the slow valve 214a (e.g., for low flow rates) and the fast valve 214b (e.g., for high flow rates) and closing the unselected valves (if any). Thus, suitable control can be achieved without requiring a variable power vacuum source or a proportional valve, although in some embodiments, the vacuum source 210 can provide different levels of vacuum power and / or at least one of the control valves 214a-214d can include a proportional valve.

[0058] The control valves of the system 202 can include a vacuum valve 214c and an exhaust valve 214d. During gas exhaust, the vacuum valve 214c can be opened and the exhaust valve 214d can be closed, so that the vacuum source 210 can extract gas from the vacuum chamber 206 and isolate the vacuum chamber from the external environment 218. During liquid introduction, the vacuum valve 214c can be closed and the exhaust valve 214d can be opened, so that gas (e.g., air) can flow from the external environment 218 into the vacuum chamber 206. The slow valve 214a and the fast valve 214b can be in fluid communication with both the vacuum valve 214c and the exhaust valve 214d, so that the controller 222 can adjust the flow rate in and out of the vacuum chamber 206 using the slow valve and the fast valve during the two stages.

[0059] Reference 9A to 9D, shows a schematic diagram of some of the methods of the present invention for loading a microfluidic chip (e.g., 10), which can be any of those microfluidic chips described above, and the chip can have a body (e.g., 14) defining one or more microfluidic networks (e.g., 18), each of which has any of the above-mentioned features (e.g., inlet ports, flow paths, test spaces, droplet generation areas, grooves, outlet channels, and / or outlet ports). For each of the networks, some methods include the following steps: placing a liquid (e.g., 156) in a first inlet port (e.g., 26) of the inlet ports; Fig.9A ). The liquid can include an aqueous liquid (e.g., 158) (e.g., a liquid containing a sample for analysis (such as a pathogen and / or a drug)) and a non-aqueous liquid (e.g., 162) (e.g., an oil that can include a surfactant (such as a fluorinated oil)). In order to promote the generation of droplets, the non-aqueous liquid can be relatively dense compared to water, for example, the specific gravity of the non-aqueous liquid can be greater than or equal to any one of 1.3, 1.4, 1.5, 1.6 or 1.7, or between any two thereof (e.g., greater than or equal to 1.5).

[0060] Some methods include the steps of: for each of the microfluidic networks, directing at least a portion of the liquid along a first flow path in the flow paths (e.g., 34) such that the portion of the liquid flows from a first inlet port through at least one droplet generation region (e.g., 38) (e.g., a minimum cross-sectional area of ​​the first flow path in the at least one droplet generation region increases along the first flow path) and flows to a test space (e.g., 30) ( Fig. 9B and Fig. 9C As described above, this can be accomplished via vacuum loading. Some methods, for example, include the steps of reducing the pressure at the first port so that the gas (e.g., 164) flows from the test space through at least one flow path and out of the first port ( Fig. 9B ). Gas flowing out of the first port can pass through the liquid. As described above, the relative sizes of the first port and the portion of the flow path connected thereto (e.g., 42) can promote the formation of bubbles when the gas passes through the liquid. Advantageously, the bubbles can stir and mix the aqueous liquid to facilitate loading and / or analysis of the aqueous liquid in the test space.

[0061] Prior to reducing the pressure, the pressure at the first port (and optionally in the test space) can be substantially ambient pressure; in order to exhaust gas from the test space, the pressure at the first port can be reduced to below ambient pressure. For example, the pressure can be reduced so that the pressure at the first port is less than or equal to any one of 0.5, 0.4, 0.3, 0.2, 0.1 or 0 atm, or between any two thereof. A greater reduction in pressure can increase the amount of gas exhausted from the test space. During gas exhaust, each of the outlet ports (e.g., 194) of the microfluidic network can be sealed (e.g., using a plug 234, a valve, and / or the like) to prevent gas from flowing in through the outlet port; however, in other embodiments, the chip may not have an outlet port.

[0062] To load the liquid into the test space, the pressure at the first port can be increased, optionally such that the pressure at the first port is substantially ambient pressure after the loading is complete. As a result, the portion of the liquid can flow along the first flow path to the test space as described above, and a plurality of droplets (e.g., 154) can be formed in any of the above-described manners ( Fig. 9C ). For example, the first flow path may include a contraction section (e.g., 62), a constant section (e.g., 66), and an expansion section (e.g., 70) in at least one droplet generation region as described above, so that part of the liquid flows from the contraction section to the constant section and then to the expansion section, thereby forming droplets. These droplets can enter the test space; when the liquid is introduced into the test space, the pressure in the test space can be increased until the pressure also substantially reaches the ambient pressure. By achieving a pressure balance between the test space and the chip's external environment (e.g., reaching the ambient pressure), the position of the droplets in the test space can be maintained for analysis without the need for additional sealing or other retaining mechanisms. In addition, a negative pressure gradient can be generated because the pressure in the test space can be below the pressure outside the chip after the gas is exhausted, and this negative pressure gradient can enhance the seal (e.g., between different workpieces of the chip) to prevent chip delamination, and can contain accidental leakage by pumping gas into the leaking liquid in the event of a failure. For example, when the aqueous liquid includes pathogens, leakage containment can improve safety. However, in other embodiments, the chip can be loaded without exhausting the gas (e.g., by increasing the pressure at the first port without reducing the pressure in advance).

[0063] Any suitable system (e.g. Figure 8The system 202 of each microfluidic network is loaded with a test space. For illustration, for vacuum loading, the chip can be set in a vacuum chamber (e.g., 202) substantially at atmospheric pressure. (For example, at least by starting a vacuum source (e.g., 222) and / or opening at least one of one or more control valves (e.g., 214a-214d) to allow gas to be extracted from the vacuum chamber), the pressure in the vacuum chamber can be reduced, and the pressure at the first port can be reduced thereby. A fast valve (e.g., 214b) and a vacuum valve (e.g., 214c) can be opened so that the vacuum source can extract gas from the vacuum chamber at a relatively high flow rate. In order to increase the pressure at the first port, the vacuum chamber can be vented, for example, by controlling one or more of the control valves to allow gas (e.g., air) to enter the vacuum chamber so that the gas flows in the vacuum chamber. For example, an exhaust valve (e.g., 214d) and at least one of a slow valve and a fast valve can be opened so that gas flows into the vacuum chamber from an external environment (e.g., 218). A control valve can be used to control the rate at which gas flows into the vacuum chamber, thereby controlling the rate at which liquid flows to the test space. To illustrate, the fast valve may be first opened so that the gas flows into the vacuum chamber at a relatively high rate. When the fast valve is open, a portion of the liquid may reach the droplet generation region relatively quickly. Thereafter, the fast valve may be closed and the slow valve may be opened so that the gas flows into the vacuum chamber at a relatively low rate. Doing so may reduce the flow rate of the portion of the liquid and may promote the formation of droplets.

[0064] Multiple (e.g., two or more) microfluidic networks (whether defined by the same chip or by different chips) can be loaded simultaneously. For example, one or more microfluidic networks of a chip can include at least a first microfluidic network and a second microfluidic network. A first liquid and a second liquid (e.g., each including an aqueous liquid and a non-aqueous liquid) can be respectively arranged in a first inlet port of the first microfluidic network and in a first inlet port of the second microfluidic network. At least a portion of the second liquid can be guided along the first flow path of the second microfluidic network, while at least a portion of the first liquid (e.g., as described above, for each network in the network) can be guided along the first flow path of the first microfluidic network. For illustration, during loading, the chip can be arranged in a chamber (e.g., a vacuum chamber) so that the inlet ports of each microfluidic network are substantially exposed to pressure changes therein at the same time. As a result, when the pressure in the chamber increases, both the first liquid and the second liquid can be guided to the test space of their respective microfluidic networks.

[0065] The apparatus may be loaded such that, for at least one of the microfluidic networks, at least one of the droplets flows from the test space to a channel (e.g., 174) and, optionally, to one of the outlet ports and / or to a sealed container (e.g., 174) as described above. Fig.9D). In this way, even if the test space reaches capacity, a portion of the droplets can form a suitable two-dimensional array in the test space for analysis. This can facilitate the loading of multiple microfluidic networks in a single chamber, where multiple inlet ports are simultaneously exposed to pressure changes in the chamber, and even if the test space in one of the networks reaches capacity before the others, the droplets in that test space can leave through the grooves at a sufficient rate to mitigate overlap, stacking and / or compression that might otherwise be caused by introducing additional droplets into that test space when loading of other test spaces is complete. Therefore, when loading multiple microfluidic networks, it is not necessary to use independent flow control.

[0066] One or more sensors (e.g., 238) can be used to analyze the droplets in each test space, such as using an imaging sensor. As an illustration, when the aqueous liquid includes a sample containing one or more microorganisms (e.g., bacteria), each of the one or more microorganisms in the sample can be encapsulated in one of the droplets. Substantially all encapsulated droplets (e.g., 242) can include a single microorganism (and optionally its offspring). The liquid (and the droplets obtained therefrom) can include a survival indicator (e.g., resazurin), which can have a specific fluorescence that varies over time based on the interaction of the survival indicator with the encapsulated microorganism. The imaging sensor can capture the data to, for example, identify the type of encapsulated microorganism. However, in other embodiments, any suitable sensor can be used to perform any suitable analysis. Reducing the overlap, stacking, and / or compression of droplets in the test space (features facilitated by the grooves) can improve the accuracy of such analysis.

[0067] The above description and examples provide a complete description of the structure and use of the illustrative embodiments. Although certain embodiments are described above with a certain degree of particularity or with reference to one or more separate embodiments, those skilled in the art may make various changes to the disclosed embodiments without departing from the scope of the present invention. Therefore, the various illustrative embodiments of the method and system are not intended to be limited to the specific forms disclosed. For example, elements may be omitted or combined into an integral structure, and / or connections may be replaced. In addition, where appropriate, aspects of any of the above examples may be combined with aspects of any other examples described to form other examples having comparable or different characteristics and / or functions and solving the same or different problems. Similarly, it should be understood that the above benefits and advantages may relate to one embodiment or may relate to several embodiments.

Claims

1. A microfluidic chip, comprising: main body; as well as a microfluidic network defined by the body, the network comprising: one or more inlet ports; Test space; one or more flow paths extending between the inlet ports and the testing volume, wherein along each flow path, fluid is permitted to flow from one of the inlet ports, through at least one droplet generation region, and to the testing volume, wherein a minimum cross-sectional area of ​​the flow path increases along the flow path in the at least one droplet generation region; and A groove is arranged along at least a portion of the periphery of the test space so that fluid from the flow path is not allowed to flow into the groove without flowing through the test space and droplets generated by the droplet generation area are allowed to flow from the test space into the groove, wherein along the groove, the depth of the groove is at least 10% greater than the depth of the test space at the periphery.

2. The chip according to claim 1, wherein: The groove is disposed along at least a majority of the perimeter of the test space.

3. The chip according to claim 1 or 2, wherein: Along the groove, the depth of the groove is at least 90% greater than the depth of the test space at the periphery.

4. The chip according to claim 1, wherein: The network includes one or more outlet ports in fluid communication with the channels such that fluid is permitted to flow from the channels to the outlet ports without flowing through the test volume.

5. The chip according to claim 1, wherein: The depth of the test space is 15 to 90 µm; and The depth of the groove is at least 100 μm.

6. The chip according to claim 1, wherein: The depth of the test space is substantially the same throughout the test space.

7. The chip according to claim 1, wherein: Each of the flow paths includes a contraction section, a constant section and an expansion section in at least one droplet generation region, such that fluid is allowed to exit the contraction section into the constant section and flow to the expansion section, wherein: The depth of the constant section is at least 10% greater than the depth of the constricted section, and the depth is substantially the same along at least 90% of the length of the constant section; and The depth of the expansion section increases as it moves away from the constant section.

8. The chip according to claim 1, wherein: A maximum lateral dimension of the groove taken perpendicularly to a centerline of the groove is less than or equal to 10% of each of a width and a length of the test space.

9. The chip according to claim 1, wherein: The microfluidic network is a first microfluidic network; and The body defines a second microfluidic network, the second microfluidic network comprising: one or more inlet ports; Test space; one or more flow paths extending between the inlet ports and the testing volume, wherein along each flow path, fluid is permitted to flow from one of the inlet ports, through at least one droplet generation region, and to the testing volume, wherein a minimum cross-sectional area of ​​the flow path increases along the flow path in the at least one droplet generation region; and A groove is arranged along at least a portion of the periphery of the test space so that fluid from the flow path is not allowed to flow into the groove without flowing through the test space and droplets generated by the droplet generation area are allowed to flow from the test space into the groove, wherein along the groove, the depth of the groove is at least 10% greater than the depth of the test space at the periphery.

10. A method for loading a microfluidic chip, the method comprising: Disposing a liquid in a first inlet port of one or more inlet ports of a microfluidic network, the microfluidic network comprising: Test space; one or more flow paths extending between the inlet port and the test volume; and a groove disposed along at least a portion of the perimeter of the test space such that fluid from the flow path is not permitted to flow into the groove without flowing through the test space, wherein along the groove, the depth of the groove is at least 10% greater than the depth of the test space at the perimeter; and At least a portion of the liquid is guided along a first flow path in the flow paths so that the portion of the liquid flows from the first inlet port through at least one droplet generation area to generate droplets formed by the portion of the liquid and the droplets flow into the test space, wherein in the at least one droplet generation area, the minimum cross-sectional area of ​​the first flow path increases along the first flow path, and at least one of the droplets flows from the test space into the groove.

11. The method according to claim 10, wherein: The groove is disposed along at least a majority of the perimeter of the test space.

12. The method according to claim 10 or 11, wherein: Along the groove, the depth of the groove is at least 90% greater than the depth of the test space at the periphery.

13. The method of claim 10, wherein: The network includes one or more outlet ports in fluid communication with the channels; and directing at least a portion of the liquid along the first flow path such that: At least one of the droplets flows from the testing space to the channel and to one of the outlet ports.

14. The method according to claim 13, wherein: The volume of each droplet ranges from 25 to 500 picoliters; The depth of the test space is 15 to 90 µm; and The depth of the groove is at least 100 μm.

15. The method according to claim 10, wherein: The depth of the test space is substantially the same throughout the test space.

16. The method of claim 10, wherein: In the at least one droplet generation region, the first flow path comprises: Contraction section; a constant section having a depth at least 10% greater than the depth of the constricted section, the depth being substantially the same along at least 90% of the length of the constant section; and an expansion section whose depth increases as it moves away from the constant section; and At least a portion of the liquid is directed along the first flow path such that the portion of the liquid leaves the contracting section, enters the constant section, and flows to the expanding section.

17. The method according to claim 10, wherein: Directing at least a portion of the liquid along the first flow path by at least: (1) reducing the pressure at the first port so that gas flows out of the first port from the test space along at least one of the flow paths; as well as (2) Increasing the pressure at the first port so that the portion of the liquid flows from the first port through at least one of the droplet generation regions and flows to the test space.

18. The method according to claim 10, wherein: A maximum lateral dimension of the groove taken perpendicularly to a centerline of the groove is less than or equal to 10% of each of a width and a length of the test space.

19. The method of claim 10, wherein: The microfluidic network is a first microfluidic network; The liquid is a first liquid; and The method comprises: Disposing a second liquid in a first inlet port of one or more inlet ports of a second microfluidic network, the second microfluidic network comprising: Test space; one or more flow paths extending between the inlet port and the test volume; and a groove disposed along at least a portion of the perimeter of the test space such that fluid from the flow path is not permitted to flow into the groove without flowing through the test space, wherein along the groove, the depth of the groove is at least 10% greater than the depth of the test space at the perimeter; and While guiding at least a portion of the first liquid along the first flow path of the first network, at least a portion of the second liquid is guided along a first flow path in the flow paths of the second network, so that the portion of the second liquid flows from the first inlet port through at least one droplet generation area to generate droplets formed by the portion of the second liquid and the droplets flow into the test space, wherein, in the at least one droplet generation area, the minimum cross-sectional area of ​​the first flow path increases along the first flow path, and at least one of the droplets flows from the test space to the groove.

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