SYSTEM AND METHOD FOR LOADING REAGENTS-CONTAINING MICROFLUIDIC CHIPS HAVING SINGLE-USE VALVES - Patent application

Abstract

The microfluidic chip can include a microfluidic network including a port, one or more test volumes, and one or more channels through which fluid must pass from the port to the test volume. A crosslinkable material can also be disposed in the microfluidic network such that the crosslinkable material can flow through the channel. The crosslinkable material of the microfluidic chip can be exposed to light and / or heat to crosslink the material therein and thereby block the channel. A method of loading a microfluidic chip can include disposing a liquid in a port of a microfluidic network including one or more test volumes and one or more channels; flowing each of one or more portions of the liquid from the port through at least one of the channels into a respective one of the test volumes; and directing the crosslinkable material into at least one of the channels and crosslinking the crosslinkable material such that none of the test volumes are in fluid communication with the port when the portion of the liquid is in the test volume. TIFF2024546013000002.tif114148

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 277,945, filed November 10, 2021. The contents of the referenced application are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present invention relates generally to loading of microfluidic chips having single-use valves, and more particularly to loading of microfluidic chips 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 channels arranged to transport, mix, and / or separate one or more samples for analysis thereof. At least one of the channels 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 as integrated fluidic networks for multiplexed applications such as in vitro diagnostic products (IVDs) for infectious diseases, IVDs for cancer, organ chips, multiplexed PCR, multiplexed ELISA, capillary ELISA, gene sequencing (e.g., single cell gene sequencing), gene synthesis for pharmaceuticals (e.g., personalized medicine), multiplexed flow cytometry, primary cell sorting, T cell sorting for therapeutics, algae sorting for green energy, and cell secretion assays (e.g., single cell secretion assays). For example, it may be beneficial to test the effect of multiple reagents on a sample. For example, in the case of antibiotic susceptibility testing, multiplexing and individually testing multiple antibiotics may enable 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 or robotically introducing a portion of the sample into each of the wells using a pipette. However, such a process can be error prone, expensive, and complicated.

[0005] Microfluidic chips configured for multiplexing applications can streamline this process, offering the advantages of reduced biological sample and reagent volumes, faster and simpler diagnostic processing, and reduced false diagnoses resulting from sample contamination. Traditionally, in multiplexing applications where the number of reaction chambers on a chip is maximized, microvalves are utilized to separate individual fluidic reactions or operations. In particular, microvalves are operated between an open position where fluidic samples dispensed at or pumped into the chip's inlet flow into the chip's reaction chambers, and a closed position where the reaction chambers are sealed together, to enable multiplexing of separate and independent reactions, diagnoses, and / or product manufacturing in individual chambers.

[0006] Several types of microvalves with different mechanisms of action for isolating fluid networks or reaction chambers have been previously described, including electrostatic valves, electrochemical valves, piezoelectric valves, magnetic valves, solenoid valves, and gas-actuated valves. Electrostatic microvalves are applied using electrostatic forces of electrodes to move a flexible membrane to close the valve. Electrochemical microvalves utilize hydrogen bubbles generated by electrolyzing a solution to actuate a membrane to close the valve. Piezoelectric microvalves convert an electrical signal into a bending force to displace a membrane. Magnetic microvalves are constructed using soft magnetic materials, such as magnetic cantilever beams or magnetic beads, attached to or embedded in a flexible elastic membrane, and the deflection of the membrane is caused by a magnetic field generated by a nearby magnet. Similarly, electromagnetic microvalves utilize electrical energy to generate an electromagnetic field, which displaces or deflects a membrane. Gas-actuated valves include pneumatic microvalves, which require an external system to pump air in and out of the system to deflect the membrane and open or close the valve, while thermopneumatic microvalves use a heating element, and air expansion caused by an increase in temperature, to actuate the valve movement.

[0007] In particular, the body size of a microfluidic chip for multiplexing applications requiring a large number of reaction chambers is subject to physical space constraints, and it may be difficult to build relatively bulky mechanical, optical, or magnetic microvalves for each reaction chamber on the chip. Furthermore, microfluidic chips are typically fabricated from a select number of materials that allow for optical transparency and biocompatibility. These materials may differ from the materials used to fabricate conventional microvalves and may be difficult to interact with the microvalves on the body of the chip. Summary of the Invention

[0008] overview Thus, there is a need in the art for devices and methods for efficiently loading and distributing fluid samples to microfluidic chips that define integrated microfluidic networks for multiplexing of separate and independent reactions, diagnostics, and / or product manufacturing within the microfluidic network. To address this need, some of the present microfluidic chips utilize valve mechanisms that include a crosslinkable material that upon activation hardens to seal one or more first portions of the microfluidic network from one or more second portions of the network.

[0009] A microfluidic chip can include at least one port that is in fluid communication with one or more test volumes via one or more channels through which fluid must pass from the port to the test volume. In addition to fluid, a microfluidic chip can include crosslinkable (e.g., photocrosslinkable and / or thermally crosslinkable) materials disposed within the microfluidic network of the chip that can also be flowable through the channels from the port to the test volume.

[0010] The microfluidic chip can be loaded by flowing each of one or more portions of the liquid from the port through at least one of the channels into a respective one of the test volumes. Loading the microfluidic chip can further include directing a crosslinkable material into the channel. When the crosslinkable material is directed into the channel of the fluidic network, the crosslinkable material can be crosslinked within the channel such that the test volume is no longer in fluid communication with the port when the portion of the liquid is within the test volume. The crosslinked material can thus occlude the channel and prevent fluid flow into and out of the test volume other than that occurring along one or more flow paths in the microfluidic network following occlusion by the crosslinked material, thereby isolating the test volume from at least a portion of the microfluidic network. This can facilitate the restriction of reagents introduced into the microfluidic network to individual portions of the liquid and can allow simultaneous and separate introduction of multiple reagents to multiple individual portions of the liquid.

[0011] Because the crosslinkable material can be seamlessly integrated with the microfluidic network body in a manner compatible with biological and chemical reactions, multiplexing applications can be simpler and more efficient. Neither extra space on the microfluidic network body nor an interface between the microfluidic chip and the valve mechanism is required to implement the valve mechanism within the fluidic network, and the equipment required to activate the crosslinkable material (e.g., UV / visible light irradiation and heat) is less expensive than physical valves and easier to manage and operate. Furthermore, the valves within the network are not constrained to the individual locations of the physical valves; instead, actuation of the valve mechanism can occur at any location within the microfluidic network that is in fluid communication with the network channels.

[0012] Some of the microfluidic chips of the present invention include a microfluidic network including a port, one or more test volumes, and one or more channels through which fluid must pass from the port to the test volume. In some embodiments, the one or more test volumes include two or more test volumes, and the microfluidic network includes, for each of the test volumes, a chamber through which fluid must pass before entering the test volume. In some embodiments, the chamber has a maximum transverse dimension of 2 millimeters or less. Furthermore, in some embodiments, each of the chambers contains a reagent.

[0013] In some aspects, the microfluidic network includes, for each of the test volumes, a droplet generation region configured to generate droplets from liquid received from the port by the droplet generation region such that droplets flow into the test volumes.

[0014] In some embodiments, the crosslinkable material is disposed within the microfluidic network such that the crosslinkable material can flow through the channel. In some embodiments, the crosslinkable material comprises a photocrosslinkable material and / or a thermally crosslinkable material. In some embodiments, the crosslinkable material comprises a monomer, a crosslinking agent, and an initiator. In some embodiments, the monomer comprises monomethacrylate-terminated poly(dimethylsiloxane), 3-[tris(trimethylsiloxy)silyl]propyl methacrylate, and / or 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate. In some embodiments, the crosslinker comprises polydimethylsiloxane-diacrylamide, poly(propylene glycol) diacrylate, poly(propylene glycol) dimethacrylate, ethylene glycol dimethacrylate, 2,2,3,3,4,4,5,5-octafluoro-1,6-hexyl diacrylate. In some embodiments, the initiator comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 1-hydroxycyclohexyl phenyl ketone, and / or eosin Y.

[0015] Some of the methods include exposing the crosslinkable material of the microfluidic chips disclosed herein to UV / visible light and / or heat to crosslink the material therein. In some methods, crosslinking the crosslinkable material thereby occludes the channel.

[0016] Some of the methods of loading a microfluidic chip of the present invention include placing a liquid into a port of a microfluidic network. In some methods, the microfluidic network includes one or more test volumes and one or more channels, and one or more portions of the liquid flow from the port through at least one of the channels into a respective one of the test volumes. The one or more test volumes, in some methods, include two or more test volumes. The one or more portions of the liquid, in some methods, include two or more portions of the liquid. In some methods, a reagent is introduced into each of the portions of the liquid. The liquid, in some methods, includes an aqueous liquid.

[0017] In some methods, the microfluidic network further comprises, for each of the test volumes, a chamber through which fluid must pass before entering the test volume, the chamber in some methods having a maximum transverse dimension of 2 millimeters or less.

[0018] In some methods, the microfluidic network further includes a droplet generation region for each of the test volumes. For each portion of liquid, in some methods, the flowing step is performed such that the portion flows through a respective one of the droplet generation regions to generate droplets. In some methods, the droplets flow into the test volumes.

[0019] In some methods, a cross-linkable material is introduced into at least one of the channels. In some methods, the cross-linkable material is cross-linked such that none of the test volumes are in fluid communication with the port when the portions of the liquid are in the test volumes. In some methods, the step of introducing and cross-linking the cross-linkable material is performed such that the test volumes are not in fluid communication with any other test volumes when each portion of the liquid is in the test volume. In some methods, the step of introducing the cross-linkable material includes introducing the cross-linkable material from the port into at least one of the channels. In some methods, the cross-linkable material is disposed on the liquid in the port prior to flowing one or more portions of the liquid. In some methods, the step of cross-linking is performed prior to flow of a portion of the liquid into one of the test volumes.

[0020] The crosslinkable material, in some methods, includes a photocrosslinkable material and / or a thermally crosslinkable material. In some methods, the density of the crosslinkable material is less than the density of the liquid. The crosslinkable material, in some methods, includes a monomer, a crosslinking agent, and an initiator. In some methods, the monomer includes monomethacrylate-terminated poly(dimethylsiloxane), 3-[tris(trimethylsiloxy)silyl]propyl methacrylate, and / or 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate. In some methods, the crosslinking agent includes polydimethylsiloxane-diacrylamide, poly(propylene glycol) diacrylate, poly(propylene glycol) dimethacrylate, ethylene glycol dimethacrylate, 2,2,3,3,4,4,5,5-octafluoro-1,6-hexyl diacrylate. In some methods, the initiator includes 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, and / or 1-hydroxycyclohexyl phenyl ketone.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Certain details relating to the above and other aspects are described below. [Brief description of the drawings]

[0027] 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.

[0028] [Figure 1A]Figure 1A is a cross-sectional view of the ports of the microfluidic network of one of the microfluidic chips of the invention taken along line 1A-1A in Figure 1B, which may be loaded with one or more liquids, reagents, and / or crosslinking materials, for example, via the ports, and may be used for multiplexing applications. [Figure 1B] FIG. 1B is a bottom view of a portion of the microfluidic network of FIG. 1A, showing its droplet generation region and test volume. [Figure 1C] FIG. 1C is a cross-sectional view of the microfluidic network of FIG. 1A taken along line 1C-1C in FIG. 1B, showing the droplet generation region thereof. [Figure 2A] FIG. 2A is a cross-sectional view of the microfluidic network of FIG. 1A with an aqueous sample liquid and a cross-linkable material disposed in its ports. [Figure 2B] FIG. 2B is a bottom view of a portion of the microfluidic network of FIG. 1A, showing the flow of sample from a port, through at least one channel of the microfluidic network, and into each test volume of the microfluidic network. [Figure 2C] FIG. 2C is a cross-sectional view of the microfluidic network of FIG. 1A showing droplet formation in a droplet generation region of the network. [Figure 2D] FIG. 2D is a bottom view of a portion of the microfluidic network of FIG. 1A showing aqueous liquid and cross-linkable material disposed in the channels of the network, and also showing the appropriate location of the cross-linkable material after cross-linking. [Diagram 3] Figures 3A-3B are overlapping fluorescent and bright field images of droplets within the test volume of a microfluidic network, generated with (Figure 3A) and without (Figure 3B) a fluorescent dye reagent flowing into the network. [Figure 4]Figure 4 shows the average fluorescence intensity of droplets in the test volume of the microfluidic network that were injected with fluorescent dye reagent (droplet containers 1, 3, 5, 7, 10, 12, 14, and 16) compared to the average fluorescence intensity of droplets in the test volume of the microfluidic network that were not injected with fluorescent dye reagent (droplet containers 2, 4, 6, 8, 9, 11, 13, and 15). [Figure 5A] FIG. 5A shows a schematic of biocompatibility testing of crosslinkable materials. (a) (left panel) shows a comparison of setups for testing the biocompatibility of crosslinkable materials in a microfluidic network (left image) and in a 96-well plate (right image), where the crosslinkable material is placed on top of the aqueous liquid. (b) (right panel) shows an experimental 96-well plate layout for testing the effect of placing a solution containing Tris buffer and varying concentrations of the crosslinkable material on top of an aqueous bacterial solution on subsequent bacterial culture. Test bacterial strains organized by row include: Row A and Row E: control (no bacterial solution); Row B and Row F: Klebsiella pneumoniae 87; Row C and Row G: Pseudomonas aeruginosa 262; Row D and Row H: Staphylococcus aureus 29213. Test crosslinkable material solutions organized by rows and columns include: Rows A-D, columns 1-3: Tris buffer; Rows A-D, columns 4-6: Tris buffer + 10 mg / mL crosslinkable material; Rows A-D, columns 7-9: Tris buffer + 20 mg / mL crosslinkable material; Rows A-D, columns 10-12: Tris buffer + 40 mg / mL crosslinkable material; Rows E-H, columns 1-3: Control (no Tris buffer or crosslinkable material). [Figure 5B] FIG. 5B shows the colorimetric results corresponding to bacterial cell viability after 24 hours of incubation in the 96-well plate described in part (b) of FIG. 5A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description 1A-1C, there is shown a microfluidic network embodiment 10 of one of the microfluidic chips 100 of the present invention for loading one or more liquids, reagents, and / or crosslinkable materials for use in multiplexing applications. Network 10 can include a port 14 capable of receiving a liquid sample for analysis. The network 10 can also include one or more test volumes 22, such as any one 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, or between any two of 2, 3, 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32, and one or more channels 18, such as any one of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or between any two of 1, 2, 3, 4, 5, 6, 7, or 8. As shown in FIG. 1B, the network includes four test volumes 22 and two channels branching off laterally from each side of the port 14. As described in further detail below, each channel 18 can be configured so that a sample introduced at port 14 flows along a flow path 26 into a test volume 22 where interactions between the sample and reagents can be analyzed.

[0030] With further reference to FIG. 1B, which shows a bottom view of a portion of the network 10, each microfluidic network can further include one or more chambers 30 through which the sample passes before entering the test volume 22. At least one of the chambers contains a reagent. To allow for the analysis of multiple reagents, the network 10, whether part of a single network or part of multiple networks, can have multiple chambers 30, such as any one 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, or between any two of 2, 3, 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32 chambers. For example, in the illustrated embodiment, the network 10 has four chambers 30. At least one of the chambers 30 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. The chamber 30 may have any suitable maximum transverse dimension for facilitating microfluidic flow, such as, for example, any one 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, or between any two of 2,000, 1,500, 1,000, 500, 300, 200, 100, 50, or 25 μm.

[0031] The microfluidic network 10 can also include a crosslinkable material 80 (e.g., photocrosslinkable and / or thermally crosslinkable material) disposed within the network 10, as shown in Figures 2A-2B. The crosslinkable material 80 can be flowable through the channels 18 along the flow path 26 and can be crosslinked to form permanent blockages in the channels 18, thereby facilitating separate and independent analysis of multiple reagents within multiple chambers 30, such as any one 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 reagents and / or chambers between any two of 2, 3, 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, or 32.

[0032] The crosslinkable materials used for in situ crosslinking within the network 10 can include a monomer, a crosslinking agent, and an initiator. The monomer can be, for example, monomethacrylate-terminated poly(dimethylsiloxane), 3-[tristo(trimethylsiloxy)silyl]propyl methacrylate, and / or 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate. The crosslinking agent can be, for example, polydimethylsiloxane-diacrylamide, poly(propylene glycol) diacrylate, poly(propylene glycol) dimethacrylate, ethylene glycol dimethacrylate, 2,2,3,3,4,4,5,5-octafluoro-1,6-hexyl diacrylate. The initiator can be, for example, a photoinitiator and / or a thermal initiator configured to cure or harden upon activation by ultraviolet and / or visible light and / or heat, respectively. The photoinitiator can be, for example, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, and / or 1-hydroxycyclohexyl phenyl ketone. Any combination of these monomers, crosslinkers, and initiators can be used to produce the crosslinkable material 80.

[0033] Similar to the liquid sample 76, the crosslinkable material 80 can be received by a port 14 of the network 10. In some cases, the crosslinkable material 80 has a density lower than the density of the liquid sample 76, for example, the specific gravity of the crosslinkable material is less than 0.3 g / cm 3 Below, 0.4g / cm 3 Below 0.5g / cm 3 Below, 0.6g / cm 3 Below, 0.7g / cm 3 Below, 0.8g / cm 3 or less than 0.9g / cm 3 Any one of the following, or 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 g / cm 3 Between any two of these (for example, 0.5 g / cm 3 2A), and is not mixed with the liquid sample 76. In such a case, the crosslinkable material 80 can be disposed above the liquid sample in the port 14, as shown in FIG. 2A, and can be allowed to flow along the flow path 26 through the channel 18 after the sample 76, as shown in FIG. 2D.

[0034] To allow each reagent to be introduced to the sample, each chamber 30 can be in fluid communication with at least one port 14 of the network 10, as shown in FIGS. 1A-1B. Such fluid communication can be achieved via a flow path 26 extending between the port 14 and the chamber 30. As shown, for example, the flow path 26 can include a port 14 for receiving a sample (FIG. 1A). The flow path 26 can further include one or more channels 18 extending between the port 14 and a passage 96 through which fluid can enter the chamber 30 (FIG. 1B). By way of illustration, for each microfluidic network 10, a portion of the liquid sample from the port 14 can flow through the channel 18 and, for each chamber 30, through the passage 96 into the chamber (FIGS. 2A-2B).

[0035] Each chamber 30 can be defined by a body coupled to the microfluidic chip 100. The body can include an internal volume that contains the chamber 30. An end of the body can define an opening that communicates with the internal volume and can receive or be received by an inlet port of the chip 100. When coupled to the chip inlet port, the body can also define a passageway 96 that allows sample liquid from one of the channels 18 of the network 10 to enter the chamber 30 (e.g., without flowing out of the chip inlet port) and contact reagents in the chamber, if present, as described above. Components of a representative chamber 30 and its configuration are described, for example, in U.S. Provisional Patent Application No. 63 / 227,303, which is incorporated herein by reference in its entirety.

[0036] The chip inlet port can be configured to receive sample liquid from chamber 30 and can define a reservoir that can contain a non-aqueous liquid (e.g., 88) for droplet generation. The sample enters the reservoir once loaded with reagents and can be directed to one of the test volumes 22 of network 10 for analysis.

[0037] 1B and 1C, each microfluidic network 10 can have, for each of its chambers 30, a test portion including a reservoir (e.g., defined by a chip inlet port), a test volume 22, and one or more flow paths 34 extending between the reservoir and the test volume. Each flow path 34 can include a droplet generation region 38 along which fluid can flow from the reservoir, through the droplet generation region 38, to the test volume 22 such that droplets are formed and introduced into the test volume for analysis. Each flow path 34 may be defined by one or more channels and / or other passages through which fluid may flow, and may have any suitable maximum transverse dimension to facilitate microfluidic flow, such as any one 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, or between any two of 2,000, 1,500, 1,000, 500, 300, 200, 100, 50, or 25 μm, such as a maximum transverse dimension taken perpendicular to a centerline of the flow path 34. Each test portion of each microfluidic network 10 optionally includes an outlet port 42 through which at least some (e.g., excess) droplets may enter from the test volume 22, and the outlet port 42 may be sealed to prevent fluid from entering or exiting the outlet port, except via a flow path between the outlet port and the test volume.

[0038] Droplet generation can be accomplished in any suitable manner. For example, as shown in FIG. 1C, at droplet generation region 38, the minimum cross-sectional area of ​​flow channel 34 can increase along the flow channel in a direction away from the reservoir. By way of example, flow channel 34 can include a constriction section 46 and an expansion region 50, with the minimum cross-sectional area of ​​the flow 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 flow channel 34 from constriction section 46 to expansion region 50.

[0039] Such a change in cross-sectional area of ​​the flow channel 34 may result from a variation in the depth of the flow channel. For example, in the expansion region 50, the flow channel 34 may include constant sections (e.g., the flow channel depth along which is substantially the same) and / or expansion sections (e.g., the flow channel depth along which increases along the flow channel), each having a maximum depth 62b greater than the maximum depth 58 of the constriction section 46, e.g., at least 10%, 50%, 100%, 150%, 200%, 250%, or 400% greater. By way of example, the maximum depth 58 of the narrowed section 46 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 20, 15, 10, or 5 μm (e.g., 10 to 20 μm), and the maximum depth 62b of the expansion region 50 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 15, 30, 45, 60, 75, 90, 105, or 120 μm (e.g., 65 to 85 μm).

[0040] As shown, the expansion region 50 comprises an expansion section including a ramp 54 having a sloped portion 66 angled relative to the constricted section 46 by an angle 70 such that the depth of the expansion section increases away from the constricted section (e.g., from a minimum depth 62a to a maximum depth 62b). The angle 70 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 5°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, or 80° (e.g., between 20° and 40°). As shown, the ramp 54 is defined by a plurality of steps 74 having a rise and depth such that the ramp has any of the sloped portions 66 described above, however, in other embodiments, the ramp can be defined by a single flat surface.

[0041] The droplet generation region 38 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 expansion section, or an expansion section upstream of a fixed section. Also, in other embodiments, the droplet generation region 38 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 networks 10 can include multiple chip inlet ports, and 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).

[0042] Due at least in part to the geometry of droplet generation region 38, droplets generated within droplet generation region 38 may have a relatively small volume, such as, for example, any one 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 a volume between any two of 10,000, 5,000, 1,000, 500, 400, 300, 200, 100, 75, or 25 pL (e.g., 25-500 pL). Each droplet can have a diameter of, for example, any one of 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 100, 95, 90, 85, 80, 75, 70, 65, or 60 μm (e.g., 60-85 μm). A relatively small amount of droplets can, for example, facilitate 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 encapsulated microorganisms in the droplets can be relatively high due to the small droplet volume, which can allow their detection without requiring prolonged incubation to grow the microorganisms.

[0043] Droplets from droplet generation region 38 may flow into test volume 22, which may have a droplet capacity to accommodate sufficient droplets for analysis. For example, test volume 22 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, or between any two of 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 droplets (e.g., 13,000-25,000 droplets). To do so, test volume 22 can have a length and width, 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 and width can each be any one 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, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mm, 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 test volume 22 can accommodate droplets while mitigating droplet stacking (e.g., without compressing the droplets), and 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 15, 30, 45, 60, 75, 90, 105, or 120 μm (e.g., 15-90 μm, 65-85 μm, etc.) (e.g., substantially the same as maximum depth 62b of expansion region 50), and optionally can be substantially the same throughout test volume 22.

[0044] 2A-2D, to load a microfluidic network (e.g., 10) (e.g., any of those described herein) of a microfluidic chip (e.g., 100), some methods include placing an aqueous liquid (e.g., 76) (e.g., a liquid containing a sample for analysis, such as urine, saliva, blood, soft tissue, mucus, etc. from a patient) into a port (e.g., 14) thereof. As described above, the aqueous liquid in the port can flow into a channel (e.g., 18) of the network that is in fluid communication with the port (FIGS. 2A-2B).

[0045] Some methods include introducing one or more, optionally two or more, reagents into the aqueous liquid, each of which is contained in a respective one of one or more chambers (e.g., 30) of the microfluidic device. For example, the aqueous liquid can contain one or more microorganisms, and each of the reagents can include a drug, such as an antibiotic (e.g., an antibacterial agent or an antifungal agent), so that the microfluidic chip can be used to evaluate the ability of an antibiotic to kill or inhibit the growth of a microorganism. Vacuum loading can be used to introduce the reagents into the aqueous liquid. In vacuum loading, some methods include, for each of the chambers (e.g., 30) in fluid communication with the port (e.g., 14), reducing the pressure at the port (e.g., 14) such that gas flows out of the chamber (e.g., through the aqueous liquid disposed therein) and out of the port. The pressure at the port can be reduced below ambient pressure. For example, the pressure can be reduced so that the pressure at the inlet port is any one 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, or between any two of 0.5, 0.4, 0.3, 0.2, 0.1, or 0 atm. A larger pressure drop can increase the amount of gas exhausted from each of the chambers.

[0046] Then, for each of the chambers in fluid communication with the port, the pressure at the port can be increased (e.g., to ambient pressure) such that at least a portion of the aqueous liquid flows from the port into the chamber (FIG. 2B). For example, as described above, for each of the chambers, a portion of the aqueous liquid can flow from the port (e.g., 14), along one or more channels (e.g., 18), through a passage (e.g., 96) into the chamber. By venting 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 venting gas (e.g., without reducing the pressure at the port before increasing the pressure at the port).

[0047] The portion of the aqueous liquid received in the chamber containing the reagent can completely fill the chamber and 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 chambers can be omitting the reagent so that a control experiment can be performed.

[0048] As shown, multiple chambers and test volumes can be loaded simultaneously so that multiple reagents (e.g., multiple antibiotics) can be evaluated along with a control. Some methods for doing so include further loading a microfluidic network (e.g., 10) of a microfluidic chip (e.g., 100) (e.g., any of those described herein) with a crosslinkable material (e.g., 80) (e.g., a material including a monomer, a crosslinker, and an initiator, as described above) at a port (e.g., 14) of the microfluidic network along with an aqueous sample. As described above, the crosslinkable material in the port can be directed to a channel (e.g., 18) of the network that is in fluid communication with the port, as with the aqueous sample (FIGS. 2A-2B). Directing the crosslinkable material can include directing the crosslinkable material from the port into at least one of the channels. As illustrated in FIG. 2D, once directed into the channel of the network, the crosslinkable material can be crosslinked such that none of the test volumes (e.g., 22) are in fluid communication with the port when a portion of the liquid is in the test volume (e.g., 92).

[0049] In some cases, the crosslinkable material is disposed above the aqueous liquid in the port and has a density lower than that of the liquid such that the crosslinkable material flows into the channel after the aqueous liquid flows into the channel. After one or more portions of the aqueous liquid are loaded into the chamber to completely fill the chamber, the portion of the aqueous liquid in the channel and the crosslinkable material flowing behind it meet at the entrance to the chamber because the crosslinkable material is immiscible with the aqueous liquid and does not enter the chamber because the chamber is filled with the aqueous liquid, but instead remains in the channel connecting to the chamber. Where the crosslinkable material meets the aqueous material at the entrance to the channel, an activator, such as ultraviolet light, visible light, or heat, can be applied to crosslink the crosslinkable material, thereby occluding the channel (e.g., 92) (FIG. 2D). In some methods, the crosslinking occurs before one portion of the liquid flows into one of the test volumes, but after one portion of the liquid flows into one of the chambers.

[0050] The localized occlusion of the channel seals a chamber that is normally in fluid communication with the network from the network such that the chamber is no longer in fluid communication with the port when a portion of the aqueous liquid is within the chamber, allowing for the simultaneous evaluation of multiple reagents in multiple chambers and test volumes without interference or crosstalk between different reagents contained in different chambers.

[0051] Some methods include generating droplets of the aqueous liquid for each of the chambers. Droplet generation can include, for each of the chambers, directing the aqueous liquid in the chamber to a reservoir containing a non-aqueous liquid. Similar to reagent introduction, droplet generation can be achieved by vacuum loading, and methods describing such droplet generation are described, for example, in U.S. Provisional Patent Application No. 63 / 227,303, which is incorporated by reference in its entirety. At least a portion of the aqueous liquid (e.g., 76) and at least a portion of the non-aqueous liquid (e.g., 88) flow from the reservoir through the droplet generation region (FIG. 2C). The aqueous liquid can form droplets (e.g., 76) as it passes through the droplet generation region and then enter the test volume (e.g., 22) 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, for example, 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 1.3, 1.4, 1.5, 1.6, or 1.7 (e.g., 1.5 or more). In vacuum loading, as the aqueous and non-aqueous liquids enter the test volume, the pressure within the test volume can increase until it reaches substantially ambient pressure.

[0052] 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 chip returns to ambient pressure. To mitigate that ejection, conventionally loaded chips may require a seal or other retention mechanism to hold the droplets 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 chip (e.g., to ambient pressure), the position of the droplets 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 chip 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.

[0053] After the droplets are generated and placed in the test volumes of the microfluidic device, some methods include capturing an image of the liquid (e.g., the droplets) in the test volume for each of the test volumes. The aqueous liquid may contain a fluorescent compound, such as a viability indicator (e.g., resazurin, the active compound in ALAMARBLUE®), that may have a specific fluorescence that changes over time in the presence of microorganisms. For example, in droplets that encapsulate microorganisms, the microorganisms 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 microorganisms encapsulated in the droplets, and fewer droplets exhibiting a fluorescent signature relative to droplets in a control test volume may demonstrate the effectiveness of the antibiotic. EXAMPLES

[0054] The present invention will be described in more detail with specific examples.The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce essentially the same results.

[0055] Separation of chambers and test volumes in microfluidic networks using the crosslinkable materials disclosed herein was demonstrated using an aqueous liquid containing the fluorescent dye ALAMARBLUE®.

[0056] In the first experiment, a crosslinkable material was placed on top of an aqueous liquid containing a fluorescent dye, and the crosslinkable material and aqueous liquid were introduced into a port of the microfluidic network, with the aqueous liquid entering the network before the crosslinkable material. The fluorescent fluid was flowed through the channel of the network into the chamber of the network to fill the chamber, while the flow of the crosslinkable material in the channel followed the flow of the fluorescent fluid from the channel to the inlet of the chamber. The crosslinkable material was then activated (e.g., crosslinked) to block the channel at the inlet of the chamber to prevent the aqueous liquid containing the fluorescent dye from flowing back out of the chamber into the channel. Vacuum loading was then used to direct the aqueous fluorescent dye-containing liquid into the non-aqueous liquid-containing reservoir of the network, and droplet generation was achieved when a portion of the aqueous fluorescent dye-containing liquid and a portion of the non-aqueous liquid flowed through the droplet generation region of the network. Finally, the droplets containing the fluorescent dye were allowed to accumulate in the test volume of the network.

[0057] To observe the flow of aqueous fluorescent dye-containing liquid through the network and droplet accumulation within the test volume, time-lapse images were taken to visualize the liquid within the network. In all images taken, the aqueous fluorescent dye-containing liquid was perfectly localized in the individual chambers, without any backflow of the fluorescent liquid into the channel (data not shown).

[0058] A second experiment was performed in which the network was loaded with aqueous fluid containing no fluorescent dye and then certain chambers of the network were injected with concentrated fluorescent dye instead. The second experiment was otherwise identical to the first. The results of the second experiment are shown in Figures 3 and 4.

[0059] Droplets generated from chambers 1, 3, 5, 7, 10, 12, 14, and 16 of the network in which the fluorescent dye was injected showed strong fluorescent signals in overlapping fluorescent and bright field images of droplets collected in the corresponding test volumes (Figure 3A). In contrast, droplets generated from chambers 2, 4, 6, 8, 9, 11, 13, and 15 of the network in which the fluorescent dye was not injected had negligible fluorescent signals in overlapping fluorescent and bright field images of droplets collected in the corresponding test volumes (Figure 3B). Figure 4 shows the average fluorescent intensity in the region of interest from all 16 test volumes to quantitatively show that the fluorescent dye did not pass back into the network from the chambers in which the fluorescent dye was injected into the chambers in which the fluorescent dye was not injected. These results demonstrate that despite stresses on the chambers and / or test volumes due to pressure changes or other mechanical forces exerted on the chambers and / or test volumes during droplet generation, the chambers, test volumes, and the liquids therein can be completely isolated from each other in the network due to the occlusion of channels upon activation of the crosslinkable material.

[0060] The ability of the crosslinking material to be utilized in biological assays, reactions, and / or manipulations was demonstrated using the culture of bacterial strains with the crosslinking material in 96-well plates. As shown in Figure 5A (left panel), the crosslinking material solution was placed on top of an aqueous bacterial solution containing a fluorescent dye to simulate the conditions in which a corresponding aqueous bacterial solution and the crosslinking material are introduced at the port of a microfluidic network.

[0061] As illustrated in Figure 5A (right panel), three bacterial solutions containing three bacterial strains, media, and ALAMARBLUE® to assay bacterial cell viability were prepared and each cultured in triplicate. The bacteria in the bacterial solutions included Klebsiella pneumoniae 87 (rows A and E of the 96-well plate), Pseudomonas aeruginosa 262 (rows B and F of the 96-well plate); or Staphylococcus aureus 29213 (rows C and G of the 96-well plate). A solution containing only ALAMARBLUE® was also included as a control (rows D and H of the 96-well plate).

[0062] As further illustrated in Figure 5A (right panel), four crosslinkable material solutions containing Tris buffer and increasing concentrations of crosslinkable material were tested: Tris buffer + 0 mg / mL crosslinkable material (rows A-D, columns 1-3 of the 96-well plate); Tris buffer + 10 mg / mL crosslinkable material (rows A-D, columns 4-6 of the 96-well plate); Tris buffer + 20 mg / mL crosslinkable material (rows A-D, columns 7-9 of the 96-well plate); and Tris buffer + 40 mg / mL crosslinkable material (rows A-D, columns 10-12 of the 96-well plate). Solutions without Tris buffer or crosslinkable material were also included as controls (rows E-H, columns 1-3 of the 96-well plate).

[0063] After 24 h of incubation of bacteria and the crosslinking solution, color images of the 96-well plate were taken to colorimetrically visualize bacterial cell viability (Figure 5B). All wells containing the crosslinking material showed the same color indicators and similar sized bacterial clusters as the control for all three bacterial species, indicating that the crosslinking material does not impair bacterial growth and survival. These results suggest that the crosslinking material is compatible with biological samples and can be used in microfluidic applications without interfering with biological assays, reactions, and / or manipulations.

[0064] 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.

[0065] 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 "means for" or "step for," respectively.

Claims

1. A method for loading a microfluidic chip, comprising the steps of: one or more test volumes; One or more channels placing a liquid into a port of a microfluidic network comprising: flowing each of the one or more portions of the liquid from the port through at least one of the channels into a respective one of the test volumes; and Directing a photocrosslinkable material and / or a thermally crosslinkable material into at least one of the channels and crosslinking the crosslinkable material such that none of the test volumes are in fluid communication with the port when the portion of the liquid is within the test volume.

2. the one or more test volumes comprises two or more test volumes; the one or more portions of the liquid include two or more portions of the liquid; and and directing and cross-linking the cross-linkable material is performed such that when the portion of the liquid is within the test volume, the test volume is not in fluid communication with any other of the test volumes.

2. The method of claim 1.

3. 3. The method of claim 1 or 2, comprising the step of introducing a reagent into each of said portions of said liquid.

4. The method of any one of claims 1 to 3, wherein directing the crosslinkable material comprises directing the crosslinkable material from the port into at least one of the channels.

5. 5. The method of claim 4, wherein the crosslinkable material is disposed in the port above the liquid prior to the flowing step.

6. The method of any one of claims 1 to 5, wherein the density of the crosslinkable material is less than the density of the liquid.

7. The method of any one of claims 1 to 6, wherein the cross-linking is performed before a portion of the liquid flows into one of the test volumes.

8. The method of any one of claims 1 to 7, wherein the microfluidic network comprises, for each of the test volumes, a chamber through which a fluid must pass before entering the test volume.

9. the microfluidic network includes a droplet generation region for each of the test volumes; and The flowing step comprises, for each of the portions of liquid: the portions flow through respective ones of the droplet generation regions to generate droplets; and The droplet flows into the test volume. It is carried out as follows: The method according to any one of claims 1 to 8.

10. The method of any one of claims 1 to 9, wherein the liquid comprises an aqueous liquid.

11. The crosslinkable material is monomer; A cross-linking agent; and Initiator The method of any one of claims 1 to 10, comprising:

12. the monomers include monomethacrylate-terminated poly(dimethylsiloxane), 3-[tris(trimethylsiloxy)silyl]propyl methacrylate, and / or 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate; the crosslinking agent comprises polydimethylsiloxane-diacrylamide, poly(propylene glycol) diacrylate, poly(propylene glycol) dimethacrylate, ethylene glycol dimethacrylate, 2,2,3,3,4,4,5,5-octafluoro-1,6-hexyl diacrylate; and the initiator comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, and / or 1-hydroxycyclohexyl phenyl ketone; 12. The method of claim 11.

13. Ports and one or more test volumes; one or more channels through which fluid must pass from said port to said test volume; A microfluidic chip defining a microfluidic network comprising: a photocrosslinkable material and / or a thermally crosslinkable material is disposed within the microfluidic network such that the crosslinkable material is flowable through the channels; The microfluidic chip.

14. The microfluidic chip of claim 13 , wherein the one or more test volumes comprises two or more test volumes.

15. The microfluidic chip of claim 13 or 14, wherein the microfluidic network comprises, for each of the test volumes, a chamber through which a fluid must pass before entering the test volume.

16. 16. The microfluidic chamber of claim 15, wherein each of the chambers contains a reagent.

17. 16. The microfluidic chip of claim 13, wherein the microfluidic network includes, for each of the test volumes, a droplet generation region configured to generate droplets from liquid received from the port by the droplet generation region such that droplets flow into the test volumes.

18. The crosslinkable material is monomer; A cross-linking agent; and Initiator The microfluidic chip of any one of claims 13 to 17, comprising:

19. the monomers include monomethacrylate-terminated poly(dimethylsiloxane), 3-[tris(trimethylsiloxy)silyl]propyl methacrylate, and / or 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate; the crosslinking agent comprises polydimethylsiloxane-diacrylamide, poly(propylene glycol) diacrylate, poly(propylene glycol) dimethacrylate, ethylene glycol dimethacrylate, 2,2,3,3,4,4,5,5-octafluoro-1,6-hexyl diacrylate; and the initiator comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, and / or 1-hydroxycyclohexyl phenyl ketone; 20. The microfluidic chip of claim 18.

20. 20. A method comprising exposing a crosslinkable material of the microfluidic chip of any one of claims 13 to 19 to light and / or heat to crosslink said material therein, thereby blocking said channels.

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