SYSTEM AND METHOD FOR LOADING REAGENTS-CONTAINING MICROFLUIDIC CHIPS - Patent application

Abstract

The microfluidic device can include a microfluidic circuit including an inlet port, a reagent-containing chamber configured to receive fluid from the inlet port, a non-aqueous liquid-containing reservoir configured to receive liquid from the chamber, and a droplet generation region configured to receive the liquid from the reservoir and generate droplets of the liquid. The circuit can also include first and second valves or frangible members. The first valve or frangible member can have a closed position that prevents fluid from entering or leaving the chamber therethrough and an open position that allows fluid to enter or leave the chamber therethrough. The second valve or frangible member can have a closed position that prevents fluid from flowing therethrough between the chamber and the reservoir and an open position that allows fluid to flow therethrough between the chamber and the reservoir. TIFF2024528068000002.tif119149

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 227,303, filed July 29, 2021, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates generally to microfluidic chip loading, and in particular to microfluidic chip loading for reagent testing. [Background technology]

[0003] background Microfluidic chips are increasingly being used in a wide variety of fields, including cosmetics, pharmaceuticals, pathology, chemistry, biology, and energy. Microfluidic chips typically have one or more grooves arranged to transport, mix, and / or separate one or more samples for analysis thereof. At least one of the groove(s) can have dimensions on the order of one micrometer or tens of micrometers, allowing for analysis of relatively small volumes of sample (e.g., nanoliters and picoliters). The small sample volumes used in microfluidic chips offer several advantages over traditional benchtop methods. For example, due to the scale of the chip's components, more precise biological measurements, including manipulation and analysis of single cells and / or molecules, may be achievable with microfluidic chips. Microfluidic chips can also provide improved control of the cellular environment therein to facilitate experiments related to cell growth, aging, antibiotic resistance, and the like. Microfluidic chips are also well suited for diagnostic applications, including pathogen identification and point-of-care diagnostics, due to their small sample volumes, low cost, and disposability.

[0004] In some applications, microfluidic chips are configured to generate droplets to facilitate analysis of samples. Traditionally, such chips are loaded by increasing the pressure at an inlet port of the chip to cause liquid to flow toward a test volume of the chip and form droplets that enter the test volume. During this process, the pressure in the test volume increases above ambient pressure. If subsequent processing of the droplets in the microfluidic chip outside the instrument is required, the pressure in the test volume must be returned to ambient pressure or sealed to prevent the flow of droplets outside the test volume. This depressurization process can be time consuming to mitigate droplet fusion, and sealing the test volume adds significant complexity.

[0005] It can be beneficial to test the effect of multiple reagents on a sample. For example, in the case of antibiotic susceptibility testing, testing multiple antibiotics can allow for the selection of the antibiotic most effective at inhibiting bacterial growth to treat an infection. Traditionally, such testing is performed by placing different reagents in individual wells of a test device and manually introducing a portion of the sample into each of the wells using a pipette or robot. However, such a process can be susceptible to error, expensive and complicated.

[0006] In droplet microfluidics, droplets can encapsulate the cells or molecules under investigation, in effect amplifying their concentration and increasing the number of reactions. Thus, droplet-based microfluidic chips, even with the chip loading inefficiencies mentioned above, have excellent potential for high-throughput reagent testing, such as antibiotic susceptibility testing. To test for the interaction of the reagent with the sample (e.g., the ability of an antibiotic to inhibit bacterial growth), the reagent is introduced into the sample. This has been done in part by introducing the reagent (or reagents) into the device during the test, e.g., by generating a set of droplets from a test reagent-containing liquid and fusing those droplets with droplets generated from the sample liquid. However, adding reagents during the test can reduce test throughput and further complicate the testing process. Summary of the Invention

[0007] overview Thus, there is a need in the art for an apparatus and method for efficiently loading a sample into a microfluidic device and introducing one or more reagents to the sample. To address this need, some of the microfluidic devices can be preloaded with one or more reagents and configured to allow the sample to flow to each of the reagent(s). The microfluidic device can include at least one inlet port and a chamber for each of the reagent(s) that contains the reagent and is configured to receive fluid from at least one of the inlet port(s). Additionally, for each chamber, the microfluidic device can include a reservoir that contains a non-aqueous liquid and first and second isolation members (e.g., valves or frangible members) each having a closed position and an open position.

[0008] For each chamber, reagents can be introduced to the sample by increasing the pressure at the inlet port(s) fluidly communicating with the chamber such that the sample in the inlet port(s) flows into the chamber. Prior to this pressure increase, it is preferable to decrease the pressure at the inlet port(s) such that gas flows out of the chamber and out of the inlet port(s). During this loading, the first and second isolation members can be closed such that no fluid can enter or leave the chamber through the first isolation member, and no fluid can flow between the chamber and the reservoir through the second isolation member. Thus, the closed isolation members can prevent fluid flow between the chambers other than that occurring along the flow path(s) that fluidly connects the chambers to the inlet port(s), which facilitates the formation of a pressure gradient that induces the above-mentioned flows during loading.

[0009] After the sample is received in the chamber, droplets can be generated from the sample. To do so, the first and second isolation members can be opened (e.g., by drilling holes in them if they comprise frangible members) so that fluid can enter and exit the chamber through the first isolation member and fluid can flow between the chamber and the reservoir through the second isolation member. With the isolation members open, the sample liquid with the introduced reagent can enter the reservoir, and loading for droplet generation can be performed by increasing the pressure in the first isolation member so that at least a portion of the sample and at least a portion of the non-aqueous liquid flow from the reservoir through the droplet generation region of the microfluidic device to form droplets for analysis. It is preferable to reduce the pressure in the opened first isolation member before increasing the pressure so that gas flows from the droplet generation region through the reservoir and through the chamber.

[0010] Since the reagent(s) do not need to be introduced into the microfluidic device with the sample, reagent testing can be made easier and more efficient. It can also be more reliable than traditional pipetting methods. The two-step loading process of first introducing the reagent into the sample and then generating the droplets also facilitates consistency in the amount of sample introduced into the microfluidic device. For example, when multiple reagents are tested in the microfluidic device, such consistency can allow each chamber to receive substantially the same amount of sample to facilitate accurate analysis when comparing the effects of the reagents therein. Furthermore, if the gas is evacuated before the liquid is introduced into the microfluidic device, the pressure within the microfluidic device can return to ambient pressure as the sample flows through it. Thus, the movement of the droplets after loading can be mitigated without the time-consuming return to ambient pressure that occurs in traditional loading microfluidic devices.

[0011] A portion of the microfluidic device includes a microfluidic circuit including an inlet port, a chamber containing a reagent configured to receive fluid from the inlet port, and a first valve or frangible member. The first valve or frangible member, in some embodiments, has a closed position in which fluid is prevented from passing through the first valve or frangible member to or from the chamber, and an open position in which fluid is allowed to pass through the first valve or frangible member to or from the chamber. In some embodiments, the first valve or frangible member comprises a first fluid-impermeable membrane.

[0012] In some embodiments, the microfluidic circuit includes a reservoir configured to receive liquid from the chamber. The reservoir, in some embodiments, contains a non-aqueous liquid. In some embodiments, the microfluidic circuit includes a second valve or frangible member having a closed position in which fluid is prevented from flowing between the chamber and the reservoir through the second valve or frangible member and an open position in which fluid is allowed to flow between the chamber and the reservoir through the second valve or frangible member. The second valve or frangible member, in some embodiments, comprises a second fluid-impermeable membrane. In some embodiments, the first and second fluid-impermeable membranes are aligned such that an axis extends through each.

[0013] The microfluidic circuit, in some embodiments, includes a droplet generation region configured to receive a liquid from a reservoir and generate droplets of the liquid. The droplet generation region, in some embodiments, includes a flow path having a minimum cross-sectional area that increases along the flow path in a direction away from the reservoir.

[0014] In some embodiments, the microfluidic circuit comprises a third valve or frangible member separating the chamber into a first portion and a second portion. The third valve or frangible member, in some embodiments, has a closed position that allows gas, but not liquid, to flow through the third valve or frangible member between the first portion and the second portion, and an open position that allows fluid to flow through the third valve or frangible member between the first portion and the second portion. In some embodiments, the third valve or frangible member comprises an air-permeable membrane. In some embodiments, the first fluid-impermeable membrane, the second fluid-impermeable membrane, and the air-permeable membrane are aligned such that an axis extends through each. The air-permeable membrane, in some embodiments, contains a reagent.

[0015] In some embodiments, the microfluidic device comprises a penetrator that in some embodiments is movable relative to the membrane along an axis, the penetrator configured to pierce the membrane such that the membrane is in an open position.

[0016] Some of the present methods of loading a microfluidic device include placing an aqueous liquid in an inlet port of the microfluidic device and introducing a reagent into the aqueous liquid. In some aspects, the step of introducing the reagent is performed by at least decreasing the pressure at the inlet port such that gas flows out of the inlet port from a chamber of the microfluidic device containing the reagent, and increasing the pressure at the inlet port such that at least a portion of the aqueous liquid flows from the inlet port into the chamber.

[0017] Some methods include generating droplets of an aqueous liquid by opening at least a first and a second port, each in fluid communication with a chamber. In some methods, generating the droplets includes reducing the pressure at the first port such that gas flows from a droplet generation region of the microfluidic device, through a reservoir of the microfluidic device containing the non-aqueous liquid, and through the chamber via the first and second ports. In some methods, generating the droplets includes increasing the pressure at the first port such that at least a portion of the aqueous liquid and at least a portion of the non-aqueous liquid flow from the reservoir through the droplet generation region. The droplet generation region, in some methods, includes a flow path having a minimum cross-sectional area that increases along the flow path in a direction away from the reservoir.

[0018] In some methods, opening the first and second ports includes opening a first valve or frangible member that, if not opened, prevents fluid from flowing through the first port into the chamber and from exiting the chamber through the first port, and opening a second valve or frangible member that, if not opened, prevents fluid from flowing through the second port into the chamber and from exiting the chamber through the second port.

[0019] In some methods, for each valve or frangible member, the valve or frangible member comprises a membrane, and opening the valve or frangible member comprises piercing the membrane.

[0020] In some methods, the device includes a valve or membrane in fluid communication with the chamber. Some such methods involve increasing the pressure at the inlet port so that gas, but not liquid, flows through the valve or membrane.

[0021] In some methods, the device includes a third valve or frangible member separating the chamber into a first portion and a second portion. In some such methods, increasing the pressure at the inlet port is performed such that gas, but not liquid, flows between the first portion and the second portion through the third valve or frangible member. The step of generating droplets of the aqueous liquid in some methods includes opening the third valve or frangible member to allow liquid to flow between the first portion and the second portion through the third valve or frangible member.

[0022] Some devices for introducing liquid to a reagent for receipt by a microfluidic chip include a body having an internal volume and an end including a first opening in fluid communication with the internal volume. Some devices include a reagent disposed within the internal volume. In some devices, the body is configured to couple to a port of the microfluidic chip such that the end receives or is received by the port and the body includes a passage configured to allow liquid to flow into the internal volume and contact the reagent without flowing out of the port.

[0023] In some devices, the body includes a second opening in fluid communication with the internal volume. Some devices include a first valve or frangible member having a closed position in which fluid is prevented from entering or leaving the internal volume through the first valve or frangible member and an open position in which fluid is allowed to enter or leave the internal volume through the first valve or frangible member. Some devices include a second valve or frangible member separating the internal volume into a first portion and a second portion, the second valve or frangible member having a closed position in which gas, but not liquid, is allowed to flow between the first portion and the second portion through the second valve or frangible member and an open position in which fluid is allowed to flow between the first portion and the second portion through the second valve or frangible member. The passageway is configured in some devices to allow liquid to flow into the first portion to contact the reagent without flowing out of the port.

[0024] The term "coupled" is defined as connected, although not necessarily directly and not necessarily mechanically, and two things that are "coupled" may be integral to one another. The terms "a" and "an" are defined as one or more, unless the disclosure expressly requires otherwise. The term "substantially" is defined as and includes most, but not necessarily all, of what is specified, as understood by one of ordinary skill in the art, e.g., substantially 90 degrees includes 90 degrees, and substantially parallel includes parallel. In any disclosed embodiment, the term "substantially" may be replaced with "within [a percentage]" of what is specified, with percentages including 0.1, 1, 5, and 10 percent.

[0025] The term "comprise" and any of its forms, e.g., "comprises" and "comprising", "have" and any of its forms, e.g., "has" and "having", "include" and any of its forms, e.g., "includes" and "including", as well as "contain" and any of its forms, e.g., "contains" and "containing", are open-ended linking verbs. As a result, an apparatus that "comprises", "has", "includes", or "contains" one or more elements may hold or contain those one or more elements, but is not limited to holding or containing only those elements. Similarly, a method that "comprises", "has", or "includes" one or more steps may have those one or more steps, but is not limited to having only those one or more steps.

[0026] Any aspect of any of the apparatus, systems, and methods can consist of or consist essentially of any of the described steps, elements, and / or features, rather than comprising / including / having any of the described steps, elements, and / or features. Thus, in any of the claims, the terms "consisting of" or "consisting essentially of" can be used in place of the above open-ended linking verbs in order to modify the scope of a given claim from that which would otherwise use an open-ended linking verb.

[0027] Furthermore, a device or system configured in a particular way is configured in at least that way, but may be configured in other ways other than as specifically described.

[0028] One or more features of one embodiment may be applied to other embodiments even if not described or illustrated, unless expressly prohibited by the nature of this disclosure or the embodiment.

[0029] Certain details associated with the above and other aspects are described below. [Brief description of the drawings]

[0030] The following drawings are presented by way of example and not limitation. For the sake of brevity and clarity, every feature of a given structure is not necessarily labeled in every figure in which the structure appears. Identical reference numbers do not necessarily refer to identical structures. Rather, the same reference numbers may be used to refer to similar features or features with similar functionality, as well as non-identical reference numbers. The figures in the drawings are drawn to scale unless otherwise noted, which means that the sizes of the illustrated elements are accurate relative to each other, at least with respect to the aspects within the figures.

[0031] [Figure 1A]FIG. 1 is a perspective view of one of the present microfluidic devices that can be pre-loaded with one or more reagents and vacuum loaded. [Figure 1B] 1B-1E are side, front, rear, and bottom views of the microfluidic device of FIG. 1A. [Figure 1C] See legend to Figure 1B. [Figure 1D] See legend to Figure 1B. [Figure 1E] See legend to Figure 1B. [Diagram 2] FIG. 1B is a perspective view of the microfluidic device of FIG. 1A with the lid removed. [Diagram 3] FIG. 1B is a perspective view of the microfluidic device of FIG. 1A with the shell removed, showing the relative positioning of the penetrator assembly and the microfluidic chip of the microfluidic device. [Figure 4A] FIG. 1B is a perspective view of one of the microfluidic chips of the microfluidic device of FIG. 1A. [Figure 4B] 4B is a bottom view of the chip of FIG. 4A showing the test volume of the chip of FIG. 4A. [Figure 4C] FIG. 4C is a cross-sectional view of the chip of FIG. 4A taken along line 4C-4C. [Figure 4D] FIG. 4D is a cross-sectional view of the chip of FIG. 4A taken along line 4D-4D. [Diagram 5] Figure 5A is a bottom view of a plug device of the microfluidic device of Figure 1A configured to be attached to a microfluidic chip to define a chamber containing a reagent, and Figure 5B is a top view of the plug device of Figure 5A without an overflow cap. [Figure 6] Figure 6A is a bottom view of a portion of the chip of Figure 4A, showing the droplet generation region and test volume, and Figure 6B is a cross-sectional view of the chip of Figure 4A along line 6B-6B in Figure 6A, showing the droplet generation region. [Figure 7A] FIG. 4B is a cross-sectional view of the microfluidic chip of FIG. 4A with an aqueous sample liquid disposed within the receptacle. [Figure 7B]FIG. 4B is a cross-sectional view of the microfluidic chip of FIG. 4A showing gas evacuation through the sample during vacuum loading. [Figure 7C] FIG. 4B is a cross-sectional view of the microfluidic chip of FIG. 4A showing the entry of a sample into a chamber defined by one of the plug devices coupled to the chip. [Figure 7D] 4B is a cross-sectional view of the microfluidic chip of FIG. 4A showing the penetrator assembly piercing the first and second frangible members to open the first and second ports thereof. [Figure 7E] FIG. 4B is a cross-sectional view of the microfluidic chip of FIG. 4A showing sample entry into the reservoir. [Figure 7F] FIG. 4B is a cross-sectional view of the microfluidic chip of FIG. 4A showing gas evacuation through the sample and non-aqueous liquid disposed in the reservoir during vacuum loading. [Figure 7G] FIG. 4B is a cross-sectional view of the microfluidic chip of FIG. 4A showing droplet formation in a droplet generation region of the chip. [Figure 8] FIG. 1B is a schematic diagram showing the microfluidic device of FIG. 1A positioned within a loading chamber that can be used to load a sample into the microfluidic chip of the device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description 1A-1E, an embodiment 10 of the present microfluidic device for introducing one or more reagents to a sample is shown. The device 10 can include a shell 14 and one or more microfluidic circuits 22, such as any one or two of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more microfluidic circuits, with the device including two microfluidic circuits as shown. With further reference to FIG. 2, each of the microfluidic circuits 22 can include at least one inlet port 26 capable of receiving a liquid sample for analysis. The device 10 can include a lid 18 movable (e.g., pivotable or removable) between a closed position that engages the inlet port(s) 26 and an open position that allows liquid to be introduced into the inlet port(s). As described in further detail below, each microfluidic circuit 22 can contain one or more reagents and can be configured so that a sample introduced to the inlet port(s) 26 can flow to each of the reagent(s) and into a test volume 98 where interactions between the sample and the reagents can be analyzed.

[0033] With reference to FIG. 3, which shows the device 10 with a portion of the shell 14 removed, the microfluidic circuit(s) 22 can be at least partially defined by one or more, optionally two or more, microfluidic chips 30 within the shell. As shown, the device 10 includes two microfluidic chips 30, each defining a respective portion of the circuit 22. With further reference to FIGS. 4A-4D, each microfluidic circuit 22 can include one or more chambers 34, at least one of the chamber(s) containing a reagent 38 (FIG. 4D). To allow for analysis of multiple reagents 38, the microfluidic circuit(s) 22 of the device 10, whether part of a single circuit or part of multiple circuits, can have multiple chambers 34, such as any one of or between any two of 2 or more, 3 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 20 or more, 24 or more, 28 or more, or 32 or more. For example, in the embodiment shown, each of the two microfluidic circuits 22 has 16 chambers 34 such that the device 10 includes a total of 32 chambers. At least one of the chambers 34 can be devoid of a reagent (e.g., so that a control analysis can be performed) and the others of the chambers can have a different reagent 38.

[0034] To allow each reagent 38 to be introduced to the sample, each chamber 34 can be in fluid communication with at least one inlet port 26 of its microfluidic circuit 22. Such fluid communication can be achieved via a flow path 42 extending between the inlet port 26 and the chamber 34. As illustrated, for example, the flow path 42 can include a receptacle 46 of the chip 30 coupled to the inlet port 26 such that the receptacle can receive the sample therefrom (FIGS. 3 and 4A). The flow path 42 can further comprise one or more grooves 50 extending between the receptacle 46 and a passage 66 through which fluid can enter the chamber 34 (FIGS. 4B-4D). By way of illustration, for each microfluidic circuit 22, a portion of the liquid sample from the inlet port 26 can flow into the receptacle 46, through the groove(s) 50, and, for each chamber 34, through the passage 66 into the chamber.

[0035] Each chamber 34 can be defined by a plug device 54 coupled to the microfluidic chip 30. As further illustrated in FIGS. 5A and 5B, the plug device 54 can include a body 58 having an internal volume 62 that contains the chamber 34. An end of the body 58 can define an opening that communicates with the internal volume 62 and can receive or be received by an inlet port 70 of the chip 30. When coupled to the chip inlet port 70, the body 58 of the plug device 54 can also define a passageway 66 that allows sample liquid from one of the grooves 50 (one or more) of the chip 30 to enter the chamber 34 (e.g., without flowing out of the chip inlet port) and contact the reagent 38 in the chamber, if present, as described above. The chip inlet port 70 can be configured to receive sample liquid from the chamber 34, as described in more detail below, and can define a reservoir 74 that can contain a non-aqueous liquid (e.g., 194) for droplet generation.

[0036] Each microfluidic circuit 22 may include at least a first and a second isolation member 78a, 78b, and an optional third isolation member 78c, for each chamber 34, which facilitates loading of the chambers with a sample and prevents the sample from entering the reservoir 74 during reagent introduction. The first, second, and third isolation members 78a-78c may each have a closed position and an open position. When the first isolation member 78a is in the closed position, fluid is prevented from passing through the first isolation member to or from the chamber 34. Thus, when the microfluidic device 10 is exposed to a change in pressure, the change in pressure may be transmitted to the inlet port(s) 26 without passing through the closed isolation member 78a, which may result in a pressure gradient that causes fluid to flow between the inlet port(s) 26 and the chamber 34. When the second isolation member 78b is in the closed position, fluid is prevented from flowing between the chamber 34 and the reservoir 74 through the second isolation member such that the chamber can fill before the sample enters the reservoir. This can prevent liquid from entering portions of the microfluidic circuit 22 downstream of the reservoir 74, described in more detail below, when a reagent is introduced to the sample, which can further facilitate metering of a constant amount of sample into multiple chambers 34 such that each chamber contains substantially the same amount of sample for subsequent droplet generation. Additionally, the closed second isolation member 78b can prevent non-aqueous liquid contained within the reservoir 74 from entering the chamber 34 (e.g., to prevent unintended contact with the reagent 38 contained in the chamber).

[0037] The third isolating member 78c can further facilitate metering of a fixed amount of sample. The third isolating member 78c can separate the chamber 34 into first and second portions 82a, 82b and, when in the closed position, can allow gas, but not liquid, to flow between the first and second portions. Thereby, the liquid sample flowing into the chamber 34 can be confined within the first portion 82a of the chamber. Meanwhile, any gas within the microfluidic circuit 22 that may flow into the chamber 34 as the sample flows in can pass through the third isolating member 78c into the second portion 82b, which can be bounded by the first isolating member 78a, the third isolating member, and the overflow cap 86 of the body 58 of the plug device 54. By allowing the gas to flow into the second portion 82b, the sample can easily occupy the entire volume of the first portion 82a of the chamber 34, facilitating delivery of substantially the same amount of sample to multiple chambers, even if some are filled before others. The third isolating member 78c can also include a reagent 38 such that the reagent can be introduced to the sample when the sample fills the first portion 82a and contacts the third isolating member. For example, the reagent 38 can be added to the third isolating member 78c by introducing a reagent-containing liquid into the microfluidic device prior to assembling the microfluidic device 10 and drying the isolating member (e.g., by lyophilization) such that the reagent remains in the isolating member. However, in other embodiments, the chamber 34 may not include a third isolating member 78c such that it is not divided into the first and second portions 82a, 82b, and in some such embodiments, the first isolating member 78a can allow gas, but not liquid, to pass therethrough and out of the chamber when closed, and can optionally include the reagent 38 (e.g., if it includes an air-permeable membrane).

[0038] The first, second, and third isolation members 78a-78c can be opened so that a sample can enter the reservoir 74 once loaded with reagents and be directed into one of the test volume(s) 98 of the device 10 for analysis. Each of the isolation members 78a-78c can comprise any suitable structure that can be opened, such as a valve or frangible member, and as shown, each comprises a frangible member, the first and second isolation members each comprise a fluid impermeable membrane, and the third isolation member comprises an air permeable (and liquid impermeable) membrane. To open the frangible members 78a-78c, the microfluidic device 10 can include, for each chamber 34, a penetrator assembly 90 that comprises a penetrator 94 (FIG. 3). The penetrator assembly 90 can be movable from a first position in which the frangible members 78a-78c of each chamber 34 are closed, to a second position in which each of the penetrator(s) 94 of the assembly pierces the respective frangible member of the chamber(s) to open them. For example, the shell 14 of the microfluidic device 10 can include one or more openings 178 through which a plunger can be engaged, thereby moving the penetrator assembly 90 to the second position. For each chamber 34, the frangible members 78a-78c can be aligned such that an axis extends through each, thereby allowing the penetrator 94 aligned with the axis to penetrate the frangible member.

[0039] 6A and 6B, each microfluidic circuit 22 can have, for each of its chamber(s) 34, a test portion including a reservoir 74 (e.g., defined by chip inlet ports 70), a test volume 98, and one or more flow paths 102 extending between the reservoir and the test volume. Each flow path 102 can include a droplet generation region 106 along which fluid can flow from the reservoir 74, through the droplet generation region, to the test volume 98 such that droplets are formed and introduced into the test volume for analysis. Each flow path 102 may be defined by one or more grooves and / or other passages through which fluid may flow, and may have any suitable maximum transverse dimension to facilitate microfluidic flow, such as a maximum transverse dimension taken perpendicular to a centerline of the flow path, such as any one of or between any two of 2,000 μm or less, 1,500 μm or less, 1,000 μm or less, 500 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, 50 μm or less, or 25 μm or less. Each test portion of each microfluidic network 22 optionally includes an outlet port 146 through which at least some (e.g., excess) droplets may enter from the test volume 98, and the outlet port may be sealed to prevent fluid from entering or exiting the outlet port, except via the flow path(s) between the outlet port and the test volume.

[0040] Droplet generation can be accomplished in any suitable manner. For example, as shown in FIG. 6B, at droplet generation region 106, the minimum cross-sectional area of ​​channel 102 can increase along the channel in a direction away from reservoir 74. By way of example, channel 102 can include a constriction section 110 and an expansion region 114, with the minimum cross-sectional area of ​​the channel being greater in the expansion region than in the constriction section. Thus, a liquid including an aqueous sample in the presence of a non-aqueous liquid can expand to form droplets as it flows along channel 102 from constriction section 110 to expansion region 114.

[0041] Such a change in cross-sectional area of ​​the flow channel 102 may result from a variation in the depth of the flow channel. For example, in the expansion region 114, the flow channel 102 may include constant sections (e.g., along which the flow channel depth is substantially the same) and / or expansion sections (e.g., along which the flow channel depth increases along the flow channel), each having a maximum depth 126b greater than the maximum depth 122 of the constriction section 110, e.g., at least 10%, 50%, 100%, 150%, 200%, 250%, or 400% greater. By way of example, the maximum depth 122 of the narrowed section 110 can be any one of 20 μm or less, 15 μm or less, 10 μm or less, or 5 μm or less, or between any two of them (e.g., 10 to 20 μm), and the maximum depth 126b of the expansion region 114 can be any one of 15 μm or more, 30 μm or more, 45 μm or more, 60 μm or more, 75 μm or more, 90 μm or more, 105 μm or more, or 120 μm or more, or between any two of them (e.g., 65 to 85 μm).

[0042] As shown, the expansion region 114 comprises an expansion section including a ramp 118 having a sloped portion 134 angled relative to the constricted section 110 by an angle 138 such that the depth of the expansion section increases away from the constricted section (e.g., from a minimum depth 126a to a maximum depth 126b). The angle 138 can be any one of 5° or more, 10° or more, 20° or more, 30° or more, 40° or more, 50° or more, 60° or more, 70° or more, or 80° or more, or between any two of them (e.g., 20°-40°), as measured relative to a direction parallel to the centerline of the constricted section 110. As shown, the ramp 118 is defined by a plurality of steps 142 having a rise and depth such that the ramp has any of the sloped portions 134 described above, however, in other embodiments, the ramp can be defined by a single flat surface.

[0043] The droplet generation region 106 can have other configurations for forming droplets. For example, in other embodiments, liquid expansion can be accomplished using only a fixed section, a fixed section upstream of an expanding section, or an expanding section upstream of a fixed section. Also, in other embodiments, the droplet generation region 106 can be configured to form droplets via a T-junction (e.g., where two channels, i.e., an aqueous liquid flowing through one and a non-aqueous liquid flowing through the other, connect such that the non-aqueous liquid shears the aqueous liquid to form droplets), flow focusing, co-flow, etc. In some such alternative embodiments, each of the microfluidic network(s) 22 can include multiple chip inlet ports 70, and the aqueous and non-aqueous liquids can be received at different inlet ports (e.g., so that they can meet at a junction for droplet generation).

[0044] Due at least in part to the geometry of droplet generation region 106, droplets generated within droplet generation region 106 can have a relatively small volume, such as, for example, a volume of 10,000 picoliters (pL) or less, 5,000 pL or less, 1,000 pL or less, 500 pL or less, 400 pL or less, 300 pL or less, 200 pL or less, 100 pL or less, 75 pL or less, or 25 pL or less, or between any two of them (e.g., 25-500 pL). Each droplet can have a diameter of, for example, 100 μm or less, 95 μm or less, 90 μm or less, 85 μm or less, 80 μm or less, 75 μm or less, 70 μm or less, 65 μm or less, or 60 μm or less, or between any two of them (e.g., 60-85 μm). The relatively small volume of droplets can facilitate, for example, the analysis of microorganisms contained by the aqueous sample liquid. During droplet generation, one or more of the microorganisms can each be encapsulated by one of the droplets (e.g., such that each encapsulating droplet contains a single microorganism and, optionally, its progeny). The concentration of the encapsulated microorganism(s) in the droplet can be relatively high due to the small droplet volume, which can allow for the detection of the microorganism(s) without requiring prolonged incubation to grow them.

[0045] Droplets from droplet generation region 106 can flow into test volume 98, which can have a droplet capacity to accommodate droplets sufficient for analysis. For example, test volume 98 can be sized to accommodate any one of 1,000 or more, 5,000 or more, 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, or 100,000 or more droplets, or any two therebetween (e.g., 13,000-25,000 droplets). To do so, the test volume 98 can have a length 130 and width 132, each of which is greater than its maximum depth, such as a length and width, each of which is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 times greater than the maximum depth of the test volume. By way of example, the length 130 and width 132 can each be any one of, or between any two of, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, 15 mm or more, 16 mm or more, or 17 mm or more, with the length being greater than the width, as shown (e.g., the length is 11-15 mm and the width is 5-9 mm). The depth of the test volume 98 can accommodate the droplets while mitigating droplet stacking (e.g., without compressing the droplets). The depth can be, for example, any one of 15 μm or more, 30 μm or more, 45 μm or more, 60 μm or more, 75 μm or more, 90 μm or more, 105 μm or more, or 120 μm or more, or between any two of them (e.g., 15-90 μm, 65-85 μm, etc.) (e.g., substantially the same as maximum depth 126b of extension region 114), and optionally can be substantially the same throughout test volume 98.

[0046] 7A-7G, to load a microfluidic device (e.g., 10) (e.g., any of those described herein), some methods include placing an aqueous liquid (e.g., 186) (e.g., a liquid containing a sample for analysis, such as urine, saliva, blood, soft tissue, mucus, etc. from a patient) into one or more inlet ports (e.g., 26) thereof. As described above, the aqueous liquid in the inlet port can flow into a receptacle (e.g., 46) of a chip (e.g., 30) that is in fluid communication with the inlet port (FIG. 7A).

[0047] Some methods include introducing one or more, optionally two or more, reagents (e.g., 38) into the aqueous liquid, each of the reagent(s) contained in a respective one or more chambers (e.g., 34) of the microfluidic device. For example, the aqueous liquid can contain one or more microorganisms, and each of the reagent(s) can include a drug, such as an antibiotic (e.g., an antibacterial agent or an antifungal agent), such that the microfluidic device can be used to evaluate the ability of the antibiotic(s) to kill or inhibit the growth of the microorganism(s). Vacuum loading can be used to introduce the reagent(s) into the aqueous liquid. As shown, in vacuum loading, some methods include, for each of the chamber(s) in fluid communication with the inlet port, reducing the pressure at each of the inlet port(s) such that gas (e.g., 190) flows out of the chamber (e.g., through the aqueous liquid disposed therein) and out of the inlet port (FIG. 7B). The pressure at the inlet port(s) can be reduced below ambient pressure. For example, the pressure can be reduced such that the pressure at the inlet port(s) is any one or between any two of 0.5 atm or less, 0.4 atm or less, 0.3 atm or less, 0.2 atm or less, 0.1 atm or less, or 0 atm or less. A larger pressure drop can increase the amount of gas exhausted from each of the chamber(s).

[0048] Then, for each of the chamber(s) in fluid communication with the inlet port, the pressure at each of the inlet port(s) can be increased (e.g., to ambient pressure) such that at least a portion of the aqueous liquid flows from the inlet port into the chamber (FIG. 7C). For example, as described above, for each of the chamber(s), a portion of the aqueous liquid can flow from the receptacle of the chip, along one or more grooves (e.g., 50), through a passage (e.g., 66) into the chamber. By evacuating gas before introducing liquid into the chamber, the pressure in the chamber can return to ambient pressure when liquid is introduced therein, allowing subsequent steps to be performed without the need for reduced pressure. However, in other embodiments, positive pressure loading can be used without evacuating gas (e.g., without reducing the pressure at the inlet port(s) of the device before increasing the pressure at the inlet port(s).

[0049] The portion of the aqueous liquid received in the reagent-containing chamber can contact the reagent in the chamber such that the aqueous liquid contains the reagent. If the device includes multiple chambers, at least one of the chamber(s) can be devoid of reagent so that a control experiment can be performed.

[0050] As described above, each of the chamber(s) can include a first and second isolation member, and optionally a third isolation member (e.g., 78a-78c). The first and second isolation members can control flow through a first and second port (e.g., 148a and 148b), respectively. As shown, the first and second ports can each be in fluid communication with the chamber, and when open, the first port allows fluid flow into and out of the chamber (without flowing through a passageway), and the second port allows fluid flow between the chamber and a reservoir (e.g., 74) containing a non-aqueous liquid (e.g., 194). For each of the chamber(s), the first and second ports can each be closed when the reagent(s) are introduced into the aqueous liquid. Thus, a pressure drop and / or a pressure increase transmitted to the inlet port(s) of the microfluidic device is not transmitted to the chamber through the first port, which can facilitate the inflow of fluid into the chamber. Additionally, with the second port closed, the aqueous liquid received in the chamber is prevented from flowing into the reservoir before the chamber is fully loaded. Also, the third isolation member, if used, can separate the chamber into first and second portions (e.g., 82a and 82b) and allow gas, but not liquid, to pass through such that when pressure at the inlet port(s) increases, the portion of the aqueous liquid received in the chamber fills the first portion of the chamber and can be confined to the first portion of the chamber while the gas flows between the first and second portions of the chamber. However, as noted above, in other embodiments, the chamber may not include a third isolation member such that it is not divided into first and second portions, and in some such embodiments, the first isolation member can allow gas, but not liquid, to flow therethrough such that when pressure at the inlet port(s) increases, gas flows through the first isolation member.

[0051] Some methods include generating droplets of aqueous liquid for each of the chamber(s). Droplet generation can include opening a first and second port for each of the chamber(s) so that fluid can communicate through each (e.g., by opening the first and second isolating members) (FIG. 7D). As shown, the ports are opened by piercing the first and second isolating members (e.g., first and second frangible members, each of which can be fluid-impermeable membranes) with a piercer (e.g., 94). A third isolating member, if present, can also be opened (e.g., by piercing the isolating member, which can be an air-permeable membrane) to allow fluid flow across it so that pressure changes at the first port can be more easily transmitted through the chamber to the second port.

[0052] For each of the chambers (one or more), with the first and second ports open, the aqueous liquid in the chamber can enter the reservoir through the second port (FIG. 7E), changing the pressure at the first port to provide fluid flow for droplet generation. Similar to reagent introduction, droplet generation can be achieved by vacuum loading. To do so, the pressure at the first port can be reduced so that gas flows from the droplet generation region (e.g., 106) of the microfluidic device, through the reservoir, through the first and second ports, and through the chamber (FIG. 7F). The pressure at the first port can be reduced to below ambient pressure, for example, such that the pressure at the first port is any one of or between any two of 0.5 atm or less, 0.4 atm or less, 0.3 atm or less, 0.2 atm or less, 0.1 atm or less, or 0 atm or less. As shown, gas can flow as bubbles through the aqueous and non-aqueous liquids in the reservoirs, which can advantageously agitate and thereby mix the aqueous liquids to facilitate their loading and / or analysis.

[0053] The pressure at the first port can then be increased (e.g., to ambient pressure) such that at least a portion of the aqueous liquid and at least a portion of the non-aqueous liquid flow from the reservoir through the droplet generation region (FIG. 7G). The aqueous liquid can form droplets (e.g., 198) as it passes through the droplet generation region and then enter the test volume (e.g., 98) for analysis. As described above, in the droplet generation region, the minimum cross-sectional area of ​​the flow path can increase along the flow path in a direction away from the reservoir, which allows the aqueous liquid to form droplets in the presence of the non-aqueous liquid. To facilitate droplet generation, the non-aqueous liquid can be relatively dense compared to water, e.g., the specific gravity of the non-aqueous liquid can be any one of 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, or 1.7 or more, or between any two of them (e.g., 1.5 or more). With vacuum loading, as the aqueous and non-aqueous liquids enter the test volume, the pressure in the test volume can increase until it substantially reaches ambient pressure.

[0054] Vacuum loading offers several benefits. In conventional loading techniques using a positive pressure gradient, the test volume may be pressurized above ambient pressure when the droplets are loaded, and thus droplets loaded in that manner may tend to migrate and be ejected from the test volume when the environment surrounding the microfluidic device returns to ambient pressure. To mitigate that ejection, conventionally loaded devices may require a seal or other retention mechanism to hold the droplet within the test volume, and the pressure in the external environment may need to be slowly returned to ambient pressure. By using a negative pressure gradient to achieve pressure equalization between the test volume and the environment external to the microfluidic device (e.g., to ambient pressure), the position of the droplet within the test volume can be maintained for analysis without the need for additional seals or other retention mechanisms, and pressure equalization can occur faster. Additionally, the negative pressure gradient used to load the microfluidic device can reinforce seals (e.g., between different parts thereof) to prevent delamination, and can contain unintentional leaks by drawing gas into the leak in the event of a failure. Leak containment can promote safety, for example, when the aqueous liquid contains pathogens. Nonetheless, in some embodiments, droplet generation can be achieved using a positive pressure gradient.

[0055] After the droplets are generated and placed in the test volume(s) of the microfluidic device, some methods include, for each of the test volume(s), capturing an image of the liquid (e.g., the droplets) in the test volume. The aqueous liquid may contain a fluorescent compound, such as a viability indicator (e.g., resazurin), that may have a specific fluorescence that changes over time in the presence of a microorganism. For example, in droplets that encapsulate a microorganism, the microorganism may interact with the viability indicator to exhibit a fluorescent signature. The droplets may be illuminated with one or more light sources such that the droplets (if present) may exhibit such fluorescence, which may be measured using image capture to evaluate the impact of a reagent introduced into the aqueous liquid. For example, an antibiotic may inhibit the growth of the microorganism(s) encapsulated in the droplet, and fewer droplets exhibiting a fluorescent signature relative to droplets in a control test volume may demonstrate the effectiveness of the antibiotic.

[0056] As shown, multiple chambers and test volumes can be loaded simultaneously so that multiple reagents (e.g., multiple antibiotics) can be evaluated along with controls. To do so, a pressure change at the inlet port(s) of the microfluidic device and the first port of the chamber can be effected by placing the device in the chamber and changing the pressure therein. By way of example, referring to FIG. 8, a system 150 is shown that can be used to perform the above-mentioned loading of a microfluidic device. The system 150 can include a chamber 152 configured to receive and house the microfluidic device. A pressure source 154 (e.g., a vacuum source) and one or more control valves 158a-158d can be configured to adjust the pressure in the chamber 152. For example, in the case of a vacuum source, the pressure source 154 can be configured to remove gas from the chamber 152, thereby reducing the pressure therein, and thus the pressure at the inlet port(s) 26 (and, after the isolation members 78a-78c are opened, at the first port(s) 148a) (e.g., to below ambient pressure). The pressure reduction can facilitate outgassing of the microfluidic circuit(s) 22. Each of the control valve(s) 158a-158d can be moved between a closed position and an open position in which the control valves block and allow, respectively, movement of fluid between the chamber 152, the pressure source 154, and / or the external environment 162. For example, after a vacuum is created in the chamber 152, opening at least one of the control valve(s) 158a-158d can allow gas (e.g., from the external environment 162) to flow into the vacuum chamber, increasing the pressure therein, and thus at the inlet port(s) 26 (and, after the isolation members 78a-78c are opened, at the reservoir(s) 74) (e.g., to ambient pressure). The pressure increase can facilitate the flow of the aqueous sample into the chamber(s) 34 and the test volume(s) 98.The pressure source 154 can be a vacuum source to enable vacuum loading of the microfluidic device 10, although in other aspects the pressure source can be a positive pressure source configured to increase the pressure within the chamber 152 (e.g., by introducing a gas therein) to load the device using positive pressure.

[0057] The system 150 can include a controller 166 configured to control the pressure source 154 and / or the control valve(s) 158a-158d to regulate the pressure in the chamber 152. The controller 166 can be configured to receive chamber pressure measurements from a pressure sensor 170. Based at least in part on those pressure measurements, the controller 166 can be configured to actuate the pressure source 154 and / or at least one of the control valve(s) 158a-158d, for example, to achieve a target pressure in the chamber 152 (e.g., using a proportional-integral-derivative controller). For example, the control valve(s) 158a-158d of the system 150 can include a slow valve 158a and a fast valve 158b that, when in an open position, allow fluid flow between the chamber 158a and at least one of the pressure source 154 and the external environment 162. The system 150 can be configured such that the maximum rate at which gas can flow through the slow valve 158a is lower than the maximum rate at which gas can flow through the fast valve 158b. As shown, for example, the system 150 includes a restriction 146 in fluid communication with the slow valve 158a. The controller 166 can control the rate at which gas enters or leaves the chamber 152, and thus the rate of change of pressure within the chamber, by selecting at least one of the slow valve 158a (e.g., for low flow rates) and / or the fast valve 158b (e.g., for high flow rates) to open and closing any unselected valve(s). Thus, while suitable control can be achieved without the need for a variable power pressure source or proportional valves, in some embodiments the pressure source 154 can provide different levels of vacuum power and / or at least one of the control valves 158a-158d can include a proportional valve.

[0058] The control valve(s) 158a-158d of the system 150 may comprise a source valve 158c and a vent valve 158d. When the pressure source 154 exhausts gas (in the case of a vacuum source) or introduces gas (in the case of a positive pressure source), the source valve 158c may be opened and the vent valve 158d may be closed so that the pressure source draws gas out of or drives gas into the chamber 152 and the chamber is isolated from the external environment 162. To return the pressure in the chamber 152 to ambient pressure, the source valve 158c may be closed and the vent valve 158d may be opened so that gas (e.g., air) may flow from the external environment 162 into the chamber 152 (if a vacuum load is used) or from the chamber into the external environment (if a positive pressure load is used). The low speed valve 158a and the high speed valve 158b can be in fluid communication with both the source valve 158c and the vent valve 158d such that the controller 166 can regulate the flow rate into and out of the chamber 152 using the low speed and high speed valves in both stages.

[0059] System 150 may also include one or more plungers 174 configured to engage penetrator assembly 90 of microfluidic device 10 through opening(s) 178 of the device such that penetrator(s) 94 open isolation member(s) 78a-78c as described above. System 150 may also include an optical sensor 182 (e.g., a camera) for analyzing droplets in test volume(s) 98 as described above. For example, shell 14 of microfluidic device 10 may include one or more transparent portions through which optical sensor 182 may capture images of droplets in test volume(s) 98 (FIG. 1E).

[0060] The above specification and examples provide a complete description of the structure and use of the exemplary embodiments. Although certain embodiments have been described above with a certain degree of particularity or with reference to one or more individual embodiments, those skilled in the art may make many modifications to the disclosed embodiments without departing from the scope of the present invention. Thus, the various exemplary embodiments of the method and system are not intended to be limited to the particular forms disclosed. Rather, they include all modifications and alternatives that fall within the scope of the claims, and embodiments other than those illustrated may include some or all of the features of the illustrated embodiments. For example, elements may be omitted or combined in an integral structure and / or connections may be substituted. Furthermore, where appropriate, any aspect of the above-described examples may be combined with any aspect of the other examples described to form further examples having equivalent or different characteristics and / or functions and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or to several embodiments.

[0061] The claims are not intended to, and should not be construed as including, means-plus-function or step-plus-function limitations, unless such limitations are expressly recited in a given claim using the phrase(s) "means for" or "step for," respectively.

Claims

1. An inlet port; A chamber containing a reagent, configured to receive fluid from the inlet port; A closed position in which fluid is prevented from entering or leaving the chamber through a first valve or frangible member, and An open position in which fluid is allowed to enter or leave the chamber through the first valve or frangible member The first valve or frangible member having; A reservoir containing a non-aqueous liquid, configured to receive liquid from the chamber; A closed position in which fluid is prevented from flowing between the chamber and the reservoir through a second valve or frangible member, and An open position in which fluid is allowed to flow between the chamber and the reservoir through the second valve or frangible member The second valve or frangible member having; and A droplet generation region configured to receive liquid from the reservoir and generate droplets of the liquid A microfluidic device comprising a microfluidic circuit comprising.

2. The microfluidic circuit comprises a third valve or frangible member that separates the chamber into a first portion and a second portion, The third valve or frangible member, A closed position in which gas, rather than liquid, is allowed to flow between the first portion and the second portion through the third valve or frangible member, and An open position in which fluid is allowed to flow between the first portion and the second portion through the third valve or frangible member Having, The microfluidic device according to claim 1.

3. The microfluidic device according to claim 2, wherein the third valve or frangible member comprises an air-permeable membrane.

4. The microfluidic device according to claim 3, wherein the air-permeable membrane contains a reagent.

5. The microfluidic device according to any one of claims 1 to 4, wherein the first valve or frangible member comprises a first fluid-impermeable membrane.

6. The microfluidic device according to any one of claims 1 to 4, wherein the second valve or frangible member comprises a second fluid-impermeable membrane.

7. The first valve or frangible member comprises a first fluid-impermeable membrane, The second valve or frangible member comprises a second fluid-impermeable membrane, The first fluid-impermeable membrane, the second fluid-impermeable membrane, and the air-permeable membrane are aligned such that one axis extends through each of them, The microfluidic device according to claim 3 or 4.

8. The microfluidic device according to claim 6, wherein the first fluid-impermeable membrane and the second fluid-impermeable membrane are aligned such that one axis extends through each of them.

9. The microfluidic device according to claim 7, comprising a penetrator movable relative to the membrane along the axis, the penetrator being configured to pierce the membrane so that the membrane is in the open position.

10. The microfluidic device according to claim 8, comprising a penetrator movable relative to the membrane along the axis, the penetrator being configured to pierce the membrane so that the membrane is in the open position.

11. The microfluidic device according to claim 1, wherein the droplet generation region includes the flow path having a minimum cross-sectional area that increases along the flow path in a direction away from the reservoir.

12. A method of loading a microfluidic device, comprising the following steps: Placing an aqueous liquid in an inlet port of the microfluidic device; At least, Reducing the pressure at the inlet port such that gas flows out of the chamber of the microfluidic device containing the reagent and out of the inlet port, and Increasing the pressure at the inlet port such that at least a portion of the aqueous liquid flows from the inlet port into the chamber To introduce a reagent into the aqueous liquid; and At least, Opening first and second ports each in fluid communication with the chamber, Gas is, From the droplet generation region of the microfluidic device, Through the reservoir of the microfluidic device containing the non-aqueous liquid, Through the chamber through the first and second ports Flowing, reducing the pressure at the first port, Increasing the pressure at the first port such that at least a portion of the aqueous liquid and at least a portion of the non-aqueous liquid flow from the reservoir through the droplet generation region To generate droplets of the aqueous liquid.

13. The device comprises a valve or membrane in fluid communication with the chamber, The method according to claim 12, wherein the pressure at the inlet port is increased such that gas, rather than liquid, flows through the valve or membrane. The method according to claim 12.

14. The device comprises a third valve or frangible member that separates the chamber into a first portion and a second portion. The pressure at the inlet port is increased such that gas, rather than liquid, flows between the first portion and the second portion through the third valve or the frangible member. The method according to claim 12.

15. The method according to claim 14, wherein the step of generating the droplets of the aqueous liquid includes opening the third valve or the frangible member such that liquid can flow between the first portion and the second portion through the third valve or the frangible member.

16. Opening the first and second ports comprises opening a first valve or frangible member that, if not opened, prevents fluid from flowing through the first port into the chamber and from flowing out of the chamber through the first port, and opening a second valve or frangible member that, if not opened, prevents fluid from flowing through the second port into the chamber and from flowing out of the chamber through the second port The method according to any one of claims 12 to 15.

17. For each of the valves or frangible members, the valve or frangible member comprises a membrane, opening the valve or frangible member includes creating a hole in the membrane, The method according to claim 15.

18. For each of the valves or frangible members, the valve or frangible member comprises a membrane, opening the valve or frangible member includes creating a hole in the membrane, The method according to claim 16.

19. The method according to claim 12, wherein the droplet generation region includes a flow path having a minimum cross-sectional area that increases along the flow path in a direction away from the reservoir.

20. A body having an internal volume and an end portion including a first opening in fluid communication with the internal volume; and a reagent disposed within the internal volume comprising At a port of the microfluidic chip, the end portion receives or is received by the port, and the body includes a passage configured to allow liquid to flow into the internal volume and contact the reagent without flowing out of the port. The body is configured to be coupled A device for introducing liquid received by a microfluidic chip to a reagent.

21. The body includes a second opening in fluid communication with the internal volume, The device has a closed position in which fluid is prevented from entering and leaving the internal volume through a first valve or frangible member, an open position in which fluid can enter and exit the internal volume through the first valve or frangible member comprising the first valve or frangible member having The device according to claim 20.

22. a second valve or frangible member that separates the internal volume into a first portion and a second portion, a closed position in which gas rather than liquid can flow between the first portion and the second portion through the second valve or frangible member, an open position in which fluid can flow between the first portion and the second portion through the second valve or frangible member the second valve or frangible member having comprising the passageway is configured to allow liquid to flow into the first portion without flowing out of the port and contact the reagent The device according to claim 20 or 21.

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