Segmented fluid mixing

Abstract

Mixing a liquid sample and at least one reagent in a microchannel of a microfluidic device includes preparing a section of the liquid sample in contact with the reagent within the microchannel. The liquid section has a volume of 2 [mu] L or less and defines a proximal gas-liquid interface and a distal liquid-gas interface. A gas of the proximal gas-liquid interface is in gas communication with a distal liquid-gas interface of another amount of liquid disposed proximal to a liquid section within a microchannel of the microfluidic channel. The gas of the distal liquid-gas interface is disposed distal of the liquid section within the microfluidic channel. The gas of the distal liquid-gas interface and / or the gas of the proximal gas-liquid interface oscillates at an audio frequency to induce mixing of the liquid and the reagent with the liquid of the liquid section in contact with the reagent within the microchannel.

Description

Technical Field

[0001] The present invention relates to methods and systems for mixing liquids and reagents within microfluidic channels.

[0002] Related Applications

[0003] This application claims the benefit and priority of U.S. Patent Application No. 63 / 414,667, entitled "Segmented Fluidicmixing", filed on October 10, 2022, and U.S. Patent Application No. 63 / 441,114, entitled "Segmented Fluidicmixing", filed on January 25, 2023, each of which is incorporated herein by reference in its entirety. This application also relates to International Patent Application No. PCT / GB2023 / 050189, entitled "Microfluidic Devices", filed on January 27, 2023 ("the '189 application"); International Patent Application No. PCT / US2021 / 013325, entitled "Fluid Control inmicrofluidicDevices", filed on January 13, 2021 ("the '325 application"); each of which is incorporated herein by reference in its entirety. Background Art

[0004] Microfluidic devices can be used to combine liquid samples with reagents disposed within the microchannels of the device to assay for targets present in the liquid samples. Mixing materials, such as liquid samples and reagents, within the microchannels of microfluidic devices presents challenges not often faced in macroscopic volumes. For example, it may be difficult to achieve efficient mixing within microchannels where slow diffusion processes may dominate. Summary of the Invention

[0005] In an embodiment, the method includes preparing a liquid segment in a microchannel of a microfluidic device that contacts and / or contains at least one reagent, wherein (i) the liquid segment defines a proximal gas-liquid interface and a distal liquid-gas interface, (ii) the gas of the proximal gas-liquid interface is a separation gas between the liquid segment and a liquid-gas interface of a quantity of liquid proximal to the liquid segment disposed within the microchannel, and (iii) the gas of the distal liquid-gas interface is a distal gas distal to the liquid segment within the microfluidic channel. The method may also include mixing the liquid of the liquid segment with the reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas to form a first mixture.

[0006] The oscillation may include oscillating the pressure of the distal gas. The oscillation may consist of oscillating the pressure of the separated gas. Alternatively, the oscillation may include simultaneously oscillating the pressures of both the distal gas and the separated gas. Wherein the oscillating step includes oscillating the pressures of the distal gas and the separated gas in phase with each other. The pressures of the distal gas and the separated gas may oscillate out of phase with each other or in phase with each other. Oscillating the pressures of the distal gas and the separated gas may be performed at the same frequency and / or different frequencies during at least a portion of the oscillation.

[0007] The pressure oscillation of the distal gas and / or the separated gas pressure may be performed at a frequency of about 2000 Hz or less, about 1500 Hz or less, about 1250 Hz or less, about 1000 Hz or less, about 900 Hz or less, or about 800 Hz or less. The pressure oscillation of the distal gas and / or the separated gas pressure may be performed at a frequency of at least about 250 Hz, at least about 500 Hz, or at least about 600 Hz.

[0008] Oscillating the pressure of the distal gas may be performed by oscillating the internal spacing between a first inner wall and a second inner wall of a region of the microchannel occupied by the distal gas. The region may be a chamber in gas communication with the distal liquid-gas interface within the microchannel. The oscillating region may be spaced from the distal liquid-gas interface of the liquid section along the microchannel. Oscillating the pressure of the separated gas may be performed by oscillating the internal spacing between a first inner wall of a region of the microchannel and a second inner wall of the region occupied by the separated gas. The region may be a chamber in gas communication with the proximal gas-liquid interface within the microchannel. The oscillating region may be spaced from the proximal gas-liquid interface of the liquid section along the microchannel. The separated gas and the distal gas are separated and isolated from each other by the liquid section.

[0009] During the oscillating step, the distal liquid-gas interface and / or the proximal gas-liquid interface of the liquid section may occupy a position where the cross-sectional area of the channel is at least about 0.01 mm 2 、at least about 0.02 mm 2 、at least about 0.03 mm 2 、at least about 0.04 mm 2 、at least about 0.05 mm 2 、at least about 0.06 mm 2 or at least about 0.07 mm 2 . During the oscillating step, the distal liquid-gas interface and / or the proximal gas-liquid interface may occupy a position where the cross-sectional area of the channel is about 0.15 mm 2 or less, about 0.125 mm 2 or less, about 0.1 mm 2 or less, about 0.09 mm 2 or less, or about 0.08 mm 2or a smaller position. During the oscillation step, the distal liquid-gas interface and / or the proximal gas-liquid interface may occupy a position where the cross-sectional area of the channel is from about 0.01 mm 2 to about 0.15 mm 2 such as from about 0.01 mm 2 to about 0.125 mm 2 from about 0.01 mm 2 to about 0.1 mm 2 from about 0.01 mm 2 to about 0.09 mm 2 from about 0.01 mm 2 to about 0.08 mm 2 from about 0.04 mm 2 to about 0.15 mm 2 such as from about 0.04 mm 2 to about 0.125 mm 2 from about 0.04 mm 2 to about 0.1 mm 2 from about 0.04 mm 2 to about 0.08 mm 2 from about 0.07 mm 2 to about 0.15 mm 2 such as from about 0.07 mm 2 to about 0.125 mm 2 from about 0.07 mm 2 to about 0.1 mm 2 from about 0.07 mm 2 to about 0.09 mm 2 or from about 0.07 mm 2 to about 0.08 mm 2 .

[0010] During the oscillation step, the liquid segment can have a volume of at least about 0.2 μL or greater, at least about 0.3 μL or greater, at least about 0.4 μL or greater, or at least about 0.5 μL or greater. During the oscillation step, the liquid segment can have a volume of about 2 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less. During the oscillation step, the liquid segment can have a volume of from about 0.2 μL to about 2 μL, from about 0.2 μL to about 1.75 μL, from about 0.2 μL to about 1.5 μL, from about 0.2 μL to about 1.25 μL, from about 0.2 μL to about 1 μL, from about 0.2 μL to about 0.75 μL, from about 0.2 μL to about 0.5 μL, from about 0.3 μL to about 2 μL, from about 0.3 μL to about 1.75 μL, from about 0.3 μL to about 1.5 μL, from about 0.3 μL to about 1.25 μL, from about 0.3 μL to about 1 μL, from about 0.3 μL to about 0.75 μL, from about 0.3 μL to about 0.5 μL, from about 0.4 μL to about 2 μL, from about 0.4 μL to about 1.75 μL, from about 0.4 μL to about 1.25 μL, from about 0.5 μL to about 2 μL, from about 0.5 μL to about 1.75 μL, from about 0.5 μL to about 1.5 μL, from about 0.5 μL to about 1.25 μL, from about 0.5 μL to about 1 μL, from about 0.5 μL to about 0.75 μL, or about 0.5 μL.

[0011] The method can include, prior to the step of preparing the liquid segment, introducing a volume of liquid into a microchannel of a microfluidic device and separating the liquid segment from the remainder of the introduced liquid. The remainder of the introduced liquid is then the amount of liquid within the microchannel proximal to the liquid segment. The step of separating the liquid segment can be performed by introducing a separation gas into the microchannel at a location occupied by the volume of the introduced liquid.

[0012] The step of introducing a volume of liquid can include applying the liquid to an application zone of the microfluidic device. The introduction can further include moving the introduced liquid along the microchannel, for example by capillary action, until the distal sample liquid-gas interface of the introduced liquid contacts a capillary stop within the microchannel. The step of moving the liquid by capillary action can include moving the liquid along the microchannel until the distal sample liquid-gas interface reaches and moves beyond the location where the separation gas is to be introduced. The capillary stop can include one or more vent openings that provide gas communication between the microfluidic channel and a volume of gas disposed external to the microfluidic channel. The volume of gas can be the gas of the ambient air surrounding the microfluidic device. The capillary stop can optionally or additionally include one or more hydrophobic features within the microfluidic channel, such as a hydrophobic layer, which can be in the form of hydrophobic strips extending across at least some or all of the channel width.

[0013] The application zone of the microfluidic device may include a porous membrane configured to separate the liquid of the particulate-containing liquid from its particulates. For example, the particulate-containing liquid may be whole blood, the separated liquid may be plasma separated from the red blood cells of the whole blood, and the microfluidic device may be configured to facilitate the detection of at least one target. The at least one target may include any target suitable for determination in a plasma sample, such as HbA1c or a cardiac marker, such as troponin I or troponin C. The particulates are retained on the upper surface of and / or within the porous membrane. The device may prepare a liquid section from a liquid separated from the particulate-containing liquid.

[0014] In an embodiment, the method includes contacting the volume of the introduced liquid with at least one reagent disposed in the microchannel prior to the step of separating the liquid section from the remainder of the introduced liquid. For example, the introducing step may include moving the introduced liquid along the microchannel, e.g., by capillary action, until the distal liquid-gas interface of the introduced liquid crosses at least one reagent disposed in the microchannel, and then stopping the movement of the introduced liquid. Stopping may be performed, for example, by using a capillary stop and / or stopping the force acting on the introduced liquid. The force may be, for example, a reduced pressure of the gas at the distal liquid-gas interface of the introduced liquid. Subsequently, the liquid section is separated from the remainder of the introduced liquid to prepare a liquid section in contact with at least one reagent within the microchannel. The liquid section includes all of the introduced liquid that is in contact with and / or contains the at least one reagent. Then a step of mixing the liquid of the liquid section with the reagent by oscillating the pressure of the distal gas and / or the pressure of the separating gas is performed on the liquid section. Then the liquid section in contact with and / or containing at least one reagent may be moved along the microchannel until the liquid of the liquid section contacts at least one additional reagent. The step of mixing the liquid of the liquid section with the reagent by oscillating the pressure of the distal gas and / or the pressure of the separating gas may then be performed on the liquid section in contact with and / or containing at least one additional reagent.

[0015] In an embodiment, the method includes first preparing a liquid segment that does not contact and does not contain the at least one reagent, and then contacting the one or more reagents with the liquid segment to prepare a liquid segment in the microchannel that contacts the at least one reagent. For example, the introducing step may include moving the introduced liquid along the microchannel, for example by capillary action, and then stopping the movement of the introduced liquid before the distal liquid-gas interface of the introduced liquid contacts the one or more reagents disposed in the microchannel. The stopping may be performed by, for example, using a capillary stopper and / or stopping the force acting on the introduced liquid. The force may be, for example, a reduced pressure of the gas at the distal liquid-gas interface of the introduced liquid. The liquid segment is then separated from the remainder of the introduced liquid and moved along the microchannel until the liquid of the liquid segment contacts the one or more reagents. The liquid segment includes all of the introduced liquid that contacts or contains the at least one reagent. Then, a step of mixing the liquid of the liquid segment with the reagent by oscillating the pressure of the distal gas and / or the pressure of the separating gas is performed on the liquid segment. The liquid segment that contacts and / or contains the at least one reagent may be moved along the microchannel until the liquid of the liquid segment contacts at least one additional reagent. The step of mixing the liquid of the liquid segment with the reagent by oscillating the pressure of the distal gas and / or the pressure of the separating gas may then be performed on the liquid segment that contacts and / or contains at least one additional reagent.

[0016] In an embodiment, after introducing a volume of liquid into a microfluidic channel, a distal gas occupies a chamber of the microfluidic device that is isolated from the ambient gas surrounding the microfluidic device. The ambient gas may be air. Before introducing a volume of liquid into the microfluidic channel, the microfluidic channel and optionally one or more vent ports provide the only route for gas communication between the distal gas and the exterior of the microfluidic device. After introducing a volume of liquid into the microfluidic channel, a distal gas may occupy a chamber of the microfluidic device that is isolated from the ambient gas surrounding the microfluidic device. For example, the presence of the introduced liquid may impede the passage of the distal gas or the ambient gas along the microchannel and, if present, through one or more vent ports.

[0017] In an embodiment, a method includes separating a liquid segment (which may also be referred to as a liquid slug) from a total amount of liquid disposed within a microchannel of a microfluidic device. Separating the liquid segment includes introducing a separation gas into the microchannel from a gas introduction opening, the gas introduction opening being disposed at a location within the microchannel occupied by the liquid of the total amount of liquid (i.e., after the liquid has been introduced into the microchannel). Introducing the separation gas into the microchannel (e.g., via the gas introduction opening) forms an asymmetric bubble that separates the liquid segment from the remaining liquid of the total amount of liquid. The gas of the asymmetric bubble and the liquid of the liquid segment form a first gas-liquid interface having a radius of curvature r1. The gas of the asymmetric bubble and the remaining liquid of the total amount of liquid form a second gas-liquid interface having a radius of curvature r2. The asymmetric bubble is asymmetric because the radius of curvature r1 is different from the radius of curvature r2 (e.g., such that the two ends of the asymmetric bubble in contact with the corresponding liquid are asymmetric with respect to each other). After forming the asymmetric bubble, the volume of the liquid segment can have the same volume as the volume of the liquid segment described herein (e.g., as described in the method of preparing a liquid segment above). In an embodiment, the volume of the liquid segment is between about 0.75 μL and about 4 μL, such as between about 1.5 μL and about 3 μL, such as about 2 μL. The volume of the asymmetric bubble disposed within the microchannel between the first gas-liquid interface and the second gas-liquid interface can be, for example, between about 200 nL and about 750 nL, such as about 350 nL.

[0018] In an embodiment, r2 > r1. For example, the ratio r2 / r1 can be at least about 1.25, such as between about 1.5 and 3.5, such as about 2. The ratio of the larger of the width (w2) and height (h2) of the microchannel at the location of the second gas-liquid interface to the larger of the width (w1) and height (h1) of the microchannel at the location of the first gas-liquid interface can be substantially the same as the ratio r2 / r1, i.e., w2 / w1 or h2 / h1 can be substantially the same as r2 / r1. Alternatively or in combination, the ratio of the cross-sectional area (A2) of the separation channel (e.g., the location of the asymmetric bubble within the microchannel) at the location of the second gas-liquid interface to the cross-sectional area (A1) of the separation channel at the location of the first gas-liquid interface can be substantially the same as the ratio r2 / r1, i.e., A2 / A1 can be substantially the same as r2 / r1.

[0019] In an embodiment, the microfluidic device includes a liquid application zone, such as a sample application zone, through which a total amount of liquid is introduced into the microfluidic channel. The first gas-liquid interface can be disposed along the microfluidic channel distal to the second gas-liquid interface and the liquid application zone.

[0020] In an embodiment, the distance along the microchannel between the first gas-liquid interface and the gas introduction opening is distance d1, the distance along the microchannel between the second gas-liquid interface and the gas introduction opening is distance d2, and the ratio d2 / d1 is at least about 2.25, such as between about 2.25 and 10, such as about 4.5. For example, d1 can be between about 250 μm and 1000 μm, such as about 500 μm, and d2 can be between about 1000 μm and about 2750 μm, such as about 2000 μm.

[0021] The liquid introduced into the microfluidic device, the remaining volume of liquid, and / or the liquid section can include any liquid to be mixed with one or more reagents. Exemplary suitable liquids include biological samples, such as nasal samples, nasopharyngeal samples, saliva samples, urine, blood-based samples, such as blood, plasma, or serum. The liquid can be derived from an environmental sample. For example, an environmental sample can be obtained by swabbing a surface such as within a food preparation, storage location, or medical facility. The environmental sample can include a soil or water sample. The liquid can include such biological samples combined in a liquid reagent, such as one or more buffers, lysis media, universal transport media (UTM), viral transport media (VTM), or combinations thereof.

[0022] The liquid introduced into the device can also contact a reagent within the microfluidic device, such as within the application zone or in a supply channel extending distally therefrom. In this case, the liquid dissolves the reagent to prepare a liquid mixture containing the introduced liquid and the dissolved reagent. A liquid portion is then prepared from this liquid mixture. The liquid section can contact or include one or more additional reagents as disclosed herein.

[0023] In an embodiment, the microfluidic device includes a generally planar substrate that includes a sample application zone and a microfluidic network, the microfluidic network including microfluidic channels extending from intersections between the sample application zone and the microfluidic channels. A porous membrane can cover the sample application zone. The lower inner surface and the inner sidewalls of the sample application zone can be defined by the substrate. The upper inner surface of the sample application zone can be defined by the lower surface of the porous membrane. Additionally or in combination, the lower inner surface, the upper inner surface, and the first and second opposing inner sidewalls of the microfluidic channels are defined by the substrate. A portion of the substrate that defines the upper inner surface of the microfluidic channel can protrude beyond the intersection between the sample application zone and the microfluidic channel and at least partially protrude into the sample application zone.

[0024] Substantially all, such as all, of the portion of the substrate that protrudes beyond the intersection into the sample application zone can be under the porous membrane.

[0025] The intersection between the sample application region and the microfluidic channel can define the width between the first and second opposing inner walls of the microfluidic channel. The width can be taken along a direction that is generally perpendicular to the longitudinal axis of the microfluidic channel and parallel to the plane defined by the generally planar substrate. In some embodiments, the width of the intersection is at least about 0.75 mm, at least about 1.0 mm, at least about 1.25 mm, at least about 1.5 mm, such as about 1.5 mm, and less than about 2.5 mm, less than about 2.0 mm, such as less than about 1.75 mm.

[0026] In some embodiments, the portion of the substrate that protrudes beyond the intersection into the sample application region covers and can extend beyond the entire width of the intersection. In other embodiments, the portion of the substrate that protrudes into the sample application region beyond the intersection is narrower than the width of the intersection. For example, at the intersection, the width of the protruding portion of the substrate can be between about 20% and 75% of the width of the intersection.

[0027] The portion of the substrate that protrudes beyond the intersection into the sample application region can taper from a first width that covers the intersection to a second smaller width that is located within the sample application region.

[0028] The portion of the substrate that protrudes beyond the intersection into the sample application region can extend beyond the intersection into the sample application region by at least about 0.25 mm and less than about 1 mm, such as about 0.25 mm or about 0.5 mm.

[0029] The height of the microfluidic channel taken along an axis perpendicular to the plane of the substrate can be at least about 50 μm and less than about 250 μm, such as about 110 μm, the height being between the lower inner surface and the upper inner surface of the microfluidic channel at a position 5 mm distal to the intersection along the longitudinal axis of the microfluidic channel. The height between the lower surface of the sample application region and the lower surface of the portion of the substrate that defines the upper inner surface of the microfluidic channel, where the upper inner surface of the microfluidic channel protrudes beyond the intersection and into the sample application region, can be less than the height of the microfluidic channel. For example, the ratio of the height of the sample application region to the height of the channel can be less than about 0.9. The ratio can be at least about 0.5, such as at least about 0.75.

[0030] In some embodiments, the substrate of the microfluidic device includes a lower layer and an upper layer, the lower layer defining the lower inner surface of the sample application region and the lower inner surface of the microfluidic channel, the upper layer defining the upper inner surface of the microfluidic channel and including a portion of the substrate that defines the upper inner surface of the microfluidic channel, the portion of the substrate protruding beyond the intersection and into the sample application region. The substrate can include an intermediate layer, where the upper layer and the lower layer are spaced apart by the intermediate layer and fixed (e.g., adhered) to the intermediate layer. The intermediate layer can define the inner sidewalls of the sample application region and the first and second opposing walls of the microfluidic channel.

[0031] The middle layer may include middle layer holes that define the inner wall of the sample application area, and wherein the upper layer may include upper layer holes that cover the middle layer holes. The diagonal dimension of the upper layer holes is typically greater than the diagonal dimension of the middle layer holes. The peripheral portion of the lower surface of the porous membrane may be adhered to the upper surface of the middle layer, the upper surface being adjacent to the middle layer holes and exposed through the upper layer holes (e.g., exposed outside the microfluidic device).

[0032] In some embodiments, each of the upper layer, middle layer, and lower layer is a separate layer, wherein the upper layer and the lower layer are relatively fixed and separated by the middle layer. In other embodiments, the middle layer is integral with one of the upper layer or the lower layer. For example, the substrate may include two layers, the first of the two layers defining the inner wall of the sample application area and the microfluidic channel, and the lower inner surface of the sample application area and the microfluidic channel. The second of the two layers defines the upper inner surface of the microfluidic channel and the substrate portion that defines the upper inner surface of the microfluidic channel, the upper inner surface of the microfluidic channel protruding beyond the intersection into the sample application area. As another example, the substrate may include two layers, the first of the two layers defining the inner wall of the sample application area and the microfluidic channel, the upper inner surface of the microfluidic channel and the portion of the substrate that defines the upper inner surface of the microfluidic channel, the upper inner surface protruding beyond the intersection into the sample application area. The second of the two layers defines the lower inner surface of the sample application area and the microfluidic channel. In each of these examples, at least the first of the two layers may be, for example, a molded layer, such as an injection molded layer.

[0033] A microfluidic device including a porous membrane may be configured to receive a particle-containing liquid applied to the upper surface of the porous membrane and separate at least some of the liquid from the particles in order to detect and / or measure at least one target in the separated liquid. For example, the particle-containing liquid may be whole blood, the separated liquid may be plasma separated from the red blood cells of the whole blood, and the at least one target may include any target suitable for assay in a plasma sample such as HbA1c or a cardiac marker such as troponin I or troponin C. The particles remain on the upper surface of the porous membrane and / or within the porous membrane. The separated liquid, e.g., plasma, enters the sample application area below the porous membrane through the porous membrane. In an embodiment, the separated liquid enters the microfluidic channel through the intersection. The movement of the separated liquid through the intersection and along the microfluidic channel may be by capillary action and / or by applying a force to the separated liquid, e.g., by reducing the pressure of a gas disposed distally of the separated liquid within the microfluidic channel in order to provide motive power for moving the separated liquid.

[0034] In an embodiment, the microfluidic device includes a venting channel extending from a venting intersection between a sample application zone and a venting channel. A distal portion of the venting channel is in gaseous communication with the ambient gas around the substrate such that the gas in the sample application zone can leave the sample application zone via the venting channel and / or the ambient gas around the substrate can enter the sample application zone via the venting channel. The lower inner surface, the upper inner surface, and the first and second opposite inner sidewalls of the venting channel are defined by the substrate. A portion of the substrate defining the upper inner surface of the venting channel protrudes beyond the venting intersection into the sample application zone.

[0035] In some embodiments, the microfluidic device is configured to receive a whole blood sample via the sample application zone such that the whole blood sample contacts the upper surface of the porous membrane, wherein red blood cells of the whole blood sample can be entrained on and / or within the upper surface of the porous membrane, and plasma separated from the whole blood sample through the porous membrane can contact the lower surface of the porous membrane. At least some of the plasma can contact a portion of the substrate defining the upper inner surface of the microfluidic channel, the upper inner surface of the microfluidic channel protruding beyond the intersection into the sample application zone.

[0036] In an embodiment, the microfluidic device includes a generally planar substrate that includes a sample application zone and a microfluidic network that includes a microfluidic channel extending from an intersection between the sample application zone and the microfluidic channel. A porous membrane can cover the sample application zone. A lower layer of the substrate defines the lower inner surface and the inner sidewalls of the sample application zone. The lower surface of the porous membrane defines the upper inner surface of the sample application zone. The lower layer of the substrate can define the lower inner surface of the microfluidic channel and the first and second opposite inner sidewalls. The upper surface of the lower layer of the substrate can include an adhesive surface. An upper layer of the substrate covers the lower layer of the substrate and is fixed to its adhesive surface and defines the upper inner surface of the microfluidic channel. A portion of the substrate defining the upper inner surface of the microfluidic channel protrudes beyond the intersection into the sample application zone. In an embodiment, the lower layer of the substrate is formed of at least a first lower layer and a second lower layer. The first lower layer can be an adhesive layer that spaces and bonds the upper layer and the second lower layer together. Typically, at least some portions of the first lower layer are absent such that the inner sidewalls of the microfluidic channel are defined.

[0037] In an embodiment, the microfluidic device includes a substantially planar substrate that includes a sample application region and a microfluidic network. The microfluidic network includes microfluidic channels extending from intersections between the sample application region and the microfluidic channels. A lower layer of the substrate defines inner sidewalls of a perimeter of the sample application region and opposite inner sidewalls of the microfluidic channels. An upper layer of the substrate covers the lower layer of the substrate. A lower surface of the upper layer of the substrate defines an upper inner surface of a distal portion of the microfluidic channels. The upper layer of the substrate includes an opening surrounding the inner sidewalls of the perimeter of the sample application region. The opening exposes a perimeter portion of an upper surface of the lower layer of the substrate. The perimeter portion surrounds the inner sidewalls of the perimeter of the sample application region. A proximal portion of the microfluidic channels is disposed adjacent to the intersection between the sample application region and the microfluidic channels. In the proximal portion of the microfluidic channels, a portion of the upper layer of the substrate has a width narrower than a width of the microfluidic channels, where each width is oriented substantially parallel to the substantially planar substrate and perpendicular to a longitudinal axis of the microfluidic channels in their proximal portion. For example, a ratio of the width of the portion of the upper layer of the substrate to the width of the microfluidic channels in the proximal portion may be less than about 0.8, less than about 0.7, less than about 0.6, for example about 0.5. The ratio may be at least about 0.3, at least about 0.4, for example about 0.5. The width of the microfluidic channels in the proximal portion may be between about 0.75 mm and 2.5 mm, for example between about 1 mm and 2 mm, for example about 1.5 mm. A length of the proximal portion of the microfluidic channels where the portion of the upper layer has the narrower width may be between about 0.75 mm and 3 mm, between about 1 mm and 2 mm, for example about 1 mm, where the length is taken along the longitudinal axis of the proximal portion of the microfluidic channels. The portion of the upper layer of the substrate may extend beyond the intersection into the application region. For example, the upper layer of the substrate may extend beyond the intersection by a length between about 0.25 mm and about 1.5 mm, for example about 0.25 mm or about 0.5 mm.

[0038] A porous membrane may cover the sample application region. A lower surface of the porous membrane defines an upper inner surface of the sample application region. A perimeter portion of the lower surface of the porous membrane is fixed to the exposed perimeter portion of the upper surface of the lower layer of the substrate. A portion of the porous membrane may cover the proximal portion of the microfluidic channels such that the lower surface of the porous membrane defines a portion of the upper inner surface of the proximal portion of the microfluidic channels. A lower surface of the narrower portion of the upper layer of the substrate defines a remaining portion of the upper inner surface of the proximal portion of the microfluidic channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a top plan view of a microfluidic device according to an embodiment described herein, the microfluidic device configured to prepare liquid segments from a volume of liquid introduced into the device and mix the liquid of the liquid segments with one or more reagents to facilitate determination of one or more targets within the liquid of the liquid segments.

[0040] Figure 2is of a microfluidic device according to an embodiment described herein Figure 1 Planar top view of a microfluidic device, where the electrical components of the microfluidic device are not shown for clarity, and where liquid has been introduced into the microchannels of the microfluidic device via an application port and has moved along the microchannels by capillary action until the distal liquid-gas interface of the sample liquid reaches a capillary stop.

[0041] Figure 3 is of a microfluidic device according to an embodiment described herein Figure 2 Planar top view of a microfluidic device, where a separation gas has been introduced into the microchannels to separate the liquid segment of the liquid from the remaining amount of sample liquid introduced into the microchannels.

[0042] Figure 4 is of a microfluidic device according to an embodiment described herein Figure 3 Planar top view of a microfluidic device, where the liquid segment has moved distally along the microchannels compared to the position shown in Figure 3 .

[0043] Figure 5 Partial planar top view of another embodiment of a microfluidic network of a microfluidic device according to an embodiment described herein, the microfluidic device being configured to prepare a liquid segment from a volume of liquid introduced into the microchannels of the device and to mix the liquid of the liquid segment with one or more reagents, where its electrical components and gas chambers are not shown for clarity, and where liquid has been introduced into its microchannels via an application port and has moved along the microchannels by capillary action until the distal liquid-gas interface of the sample liquid reaches a capillary stop.

[0044] Figure 6 is of a microfluidic device according to an embodiment described herein Figure 5 Partial planar top view of a microfluidic network, where a separation gas has been introduced into the gas separation portion of the microchannels to prepare an initial bubble for separating the liquid segment from the remainder of the sample liquid.

[0045] Figure 7 is of a microfluidic device according to an embodiment described herein Figure 5 and Figure 6 Partial planar top view of a microfluidic network, where an additional amount of separation gas has been introduced into the gas separation portion of the microchannels to prepare an asymmetric bubble for separating the liquid segment from the remainder of the sample liquid.

[0046] Figure 8 is an enlarged partial planar top view of the gas separation portion of a microchannel of a microfluidic network as shown in Figure 7 according to an embodiment described herein.

[0047] Figure 9is an enlarged plan top partial view of the gas separation portion of a microchannel of a microfluidic network as shown in Figure 8 and also shows the radius of curvature of the gas-liquid interface of an asymmetric bubble within the gas separation portion of the microchannel.

[0048] Figure 10 is a plan top view of an embodiment of a microfluidic device according to an embodiment described herein, the microfluidic device being configured to form a first section including a sample liquid and a second section including a diluent and then mix the first section and the second section, where as shown, a blood sample has been introduced into the microfluidic network and forms the first section.

[0049] Figure 11 is a plan top view of an embodiment of a microfluidic device according to an embodiment described herein, the microfluidic device being configured to form a first section including a sample liquid and a second section including a diluent and then mix the first section and the second section, where Figure 11 as shown, a blood sample has been introduced into the microfluidic network and forms the first section.

[0050] Figure 12a shows a Figure 11 microfluidic device according to an embodiment described herein, where Figure 11 compared to, a first section of blood has moved proximally within the microfluidic network.

[0051] Figure 12b shows an enlarged view of a section combination area of a Figure 12a microfluidic device according to an embodiment described herein.

[0052] Figure 13 shows a Figure 11 microfluidic device according to an embodiment described herein, where a diluent has been introduced into the device and the proximal gas-liquid interface of the first section of blood has been placed in contact with the distal gas-liquid interface of the diluent.

[0053] Figure 14 shows a Figure 13 microfluidic device according to an embodiment described herein, where a section of the diluent has been separated from the remainder of the diluent.

[0054] Figure 15 shows a Figure 14 microfluidic device according to an embodiment described herein, where the first section of blood and the section of the diluent have been combined into a mixture by the oscillatory action of gas pressure.

[0055] Figure 16 shows a Figure 15A microfluidic device in which the mixture has moved distally within the microfluidic network to the detection region.

[0056] Figure 17 FIG. 4 is a plan top view of a microfluidic device according to an embodiment herein, the microfluidic device being configured to separate a liquid from particulates to form segments of the separated liquid, to mix the segments of the separated liquid with one or more reagents to facilitate detection of one or more targets in a sample liquid with an electrical component (not shown for clarity) of the microfluidic device.

[0057] Figure 18 is according to an embodiment herein Figure 17 exploded view of a microfluidic device, showing its three-layer structure.

[0058] Figure 19 According to one embodiment herein, Figure 17 detailed plan top view of the sample application region of a microfluidic device, the porous membrane covering the sample application region has been removed for clarity. DETAILED DESCRIPTION

[0059] Reference Figure 1 , the microfluidic device 10 is configured to receive a sample liquid, contact a portion (less than all) of the received sample liquid with one or more reagents disposed in a microchannel, prepare a liquid segment comprising substantially all or all of the portion of the received sample liquid that has contacted and / or contains the reagent, mix the liquid and the reagent of the liquid segment, and determine the presence of one or more targets in the liquid of the liquid segment. As used herein, the term "microchannel" may be used interchangeably with the term "microfluidic channel". The step of contacting the portion of the received sample liquid with one or more reagents may be performed before and / or after preparing the liquid segment. For example, the preparation of the liquid segment may be performed while the sample liquid is in contact with one or more of the reagents. Once prepared, the liquid segment includes all of the received sample liquid that has contacted or has been contacted with one or more reagents. Such a liquid segment may move along the microchannel and contact one or more additional reagents. Alternatively, the liquid segment may be prepared without first contacting the reagent with its liquid, and subsequently, the liquid segment may move along the microchannel and contact one or more reagents.

[0060] The prepared liquid segment has a predetermined volume defined between a proximal gas-liquid interface and a distal liquid-gas interface. One or more reagents are placed in the microchannel in a predetermined amount. Thus, the concentration(s) of the reagent(s) in the mixture formed by subjecting the liquid segment to a mixing step is known, regardless of whether the liquid segment is prepared from a received sample liquid that has contacted one or more reagents, or whether the liquid segment contacts such a reagent (or both) after the liquid segment is prepared. In the absence of forming a liquid segment defined by such interfaces, the overall movement within the sample liquid caused by the mixing step may result in one or more reagents being distributed within an indeterminate volume of the sample liquid, such that the concentration of the one or more reagents is not known with the same precision as in the liquid segment. Due to the higher precision of the reagent concentration within the liquid segment, one or more targets therein can be determined with a higher precision than can be achieved for such targets in a liquid where the reagent(s) within the liquid segment is not confined by a pair of interfaces (e.g., the proximal and distal gas-liquid interfaces of the liquid segment).

[0061] The microfluidic device 10 includes a microfluidic network 12. Starting from the sample application zone 14 and proceeding from proximal to distal, the microfluidic network 12 includes microfluidic channels having a supply channel portion 16, a segment separation channel portion 28, an analysis channel portion 18 including a reagent zone 18' and a detection zone 18", and a gas chamber 20. The terms supply channel portion, segment separation channel portion, and analysis channel portion are used interchangeably with the terms supply channel, segment separation channel, and analysis channel, respectively. The analysis channel 18 also includes a vent 32 that operates as a capillary stop, and one or more reagents 60 configured to facilitate the determination (e.g., detection) of one or more targets in the liquid of the liquid segment. As an alternative to or in combination with the vent 32, the capillary stop may include a hydrophobic layer that extends partially or completely through the analysis channel 18, and / or a hydrophobic layer that extends along and within the vent 32. The microfluidic device 10 also includes a separation gas chamber 26 that is arranged to be in gas communication with the segment separation channel 28 via a separation gas channel 30 that intersects the separation channel 28 at a gas introduction opening 30'.

[0062] The microfluidic device 10 can be constructed similarly to the microfluidic strip of the '325 application. For example, the device 10 can consist of a lower substrate (e.g., a flexible polymer layer) and an upper substrate (e.g., a flexible polymer layer), which are adhesively adhered to each other. The area occupied by the adhesive layer is less than all of the area of the opposing surfaces between the upper and lower substrates, and includes sidewalls to define a microfluidic network 12 therebetween. The upper and lower substrates and the adhesive layer can have the same properties (e.g., thickness, composition, and mechanical properties) as those disclosed for the upper and lower substrates and the adhesive layer of the microfluidic strip disclosed in the '325 application. The internal height of the microfluidic network 12 between the inner surfaces of the upper and lower substrates is typically between about 50 μm and about 200 μm, e.g., about 110 μm.

[0063] The gas chamber 20 includes a plurality of spaced-apart locations 22a, 22b, 22c, which are arranged to be in gas communication with each other and in gas communication with the analysis channel 18 via the gas chamber opening 24. Each of the spaced-apart locations 22a, 22b, 22c is separated from an adjacent spaced-apart location by an inner sidewall that allows the upper layer covering each spaced-apart location to be compressed / decompressed and / or oscillated independently of the upper layer covering the other spaced-apart locations. The gas chamber 20 and the spaced-apart locations 22a, 22b, 22c can be configured, arranged, and operated as disclosed in the '858 application. The separation gas chamber 26 is configured to introduce a separation gas through the separation gas channel 30 and into the section separation channel 28 via the gas introduction opening 30' to separate a liquid section from a certain amount of sample liquid present in the microfluidic network 12. The separation gas chamber 26 can be configured, arranged, and operated as disclosed as a gas chamber in the '325 application. The term gas chamber can be used interchangeably with the term airbag.

[0064] Before introducing a liquid sample into the microfluidic device 10, the ambient gas (e.g., air) around the microfluidic device can enter and exit the microfluidic network 12 via the application zone 14 and the vent 32. There is no other route through which gas can enter or exit the microfluidic network 12. Thus, the microfluidic network 12 (e.g., its channels and chambers) is occupied by this ambient gas.

[0065] Reagent 60 includes one or more reagents configured to facilitate the detection of a target, such as a reagent configured to bind to the target and labeled to allow detection of such a reagent. The reagent may also include magnetic particle reagents to allow magnetic capture of the reagent. The one or more reagents may include, for example, any reagent disclosed in the '325 application. The one or more reagents may be set in a dry state in the microchannel and configured to be movable when contacted by a liquid. The one or more reagents may be set within a single location or in two or more spaced-apart locations. For example, the microchannel may include reagents set in each of at least two locations spaced a certain distance along the microchannel, the distance being sufficient to allow contact and mixing of the reagents at the first location without contacting the reagents at the second location. Subsequently, a liquid (e.g., a liquid segment) moves along the channel to the second location to allow contact and mixing of the reagents therein.

[0066] The microfluidic device 10 further includes electrodes that are arranged and configured to allow a reader to monitor the proper filling of the device 10 with a sample liquid, the proper movement of the sample liquid and the liquid segments therein, and to monitor the operation (e.g., the compressed state) of each of the spaced-apart locations 22a, 22b, 22c of the gas chamber 20 and the separation gas chamber 26. The electrodes may be arranged, configured, and operated generally as disclosed in the '325 application and / or the '858 application. For example, the device 10 includes a supply electrode 21 connected to a contact 23 via a lead 21' and a first fill electrode 25 and a second fill electrode 33 each connected to a contact 27 via a common lead 25'. When the device 10 is fully inserted into the reader, the contacts 23 and 27 engage corresponding contacts within the reader. The engaged contacts allow the reader to transmit and / or receive electrical signals to and from the supply electrode 21 and the fill electrodes 25 and / or the second fill electrode 33 to determine the presence of a liquid sample at the respective locations of the fill electrodes 25 and the fill electrode 33, as disclosed in the '325 application. The microfluidic device 10 further includes electrodes configured to perform both a liquid sensing function and a mechanical sensing function. For example, a lead 31 extends from a contact 41 through the gas chamber 26 to a third fill electrode 29. A lead 35 extends from a contact 37 into the gas chamber 26. The gas chamber 26 also includes a bridge contact 39. When the gas chamber 26 is fully compressed, the bridge contact 39 electrically connects the lead 31 and the lead 35, which is sensed by the reader via the contacts 37 and 41. When the leads 31, 35 are not electrically connected (i.e., when the gas chamber 26 is not in a fully compressed state), the reader operating the microfluidic device 10 may also use the third fill electrode 29 to sense the presence of liquid in the analysis channel 18. The spaced-apart locations 22a, 22b, 22c also include corresponding electrodes and corresponding bridge contacts to allow the reader to determine when the locations are in a fully compressed state in the same manner as the gas chamber 26.

[0067] Reference Figures 2-4 describes a method for forming a liquid segment and mixing the liquid of the liquid segment with a reagent. The method begins by inserting the microfluidic device 10 into a reader (not shown), as disclosed in Application '325. The reader compresses the spaced-apart positions 22a, 22b, 22c of the gas chamber 20 to an operable fully compressed state, thereby discharging the gas therein through the application zone 14 and the vent 32. The separation gas chamber 26 remains in an uncompressed state. The method continues by introducing the sample liquid 50 into the microfluidic network 12 via the application zone 14. The sample liquid 50 moves along the supply channel 16 by capillary action, passes through the segment separation channel 28 and enters the analysis channel 18 until the distal liquid-gas interface 52 of the sample liquid 50 reaches the vent 32, which serves as a capillary stop to halt further movement of the liquid sample 50. The sample liquid 50 then occupies the microfluidic network 12, which extends from the gas-liquid interface 52' formed at the application zone 14 to the distal gas-liquid interface 52 located at the vent 32 ( Figure 2 ).

[0068] The gas chamber 20 (including the spaced-apart positions 22a, 22b, 22c) and the portion of the analysis channel 18 remote from the distal liquid-gas interface 52 are occupied by the gas 54. The separation gas chamber 26 and the separation gas channel 30 are occupied by the gas 56. As an example, the gases 54 and 56 may consist of the remaining ambient gas (e.g., air) that had occupied the microfluidic network prior to the introduction of the sample liquid 50. Since the sample liquid 50 occupying the microfluidic network 12 blocks the vent 32 and the application zone 14, the gases 54 and 56 are isolated from each other and from the ambient gas surrounding the microfluidic device 10.

[0069] As Figure 2 shown, the sample liquid 50 contacts the reagent 60 within the analysis channel 18. The sample liquid begins to move the reagent. Since the sample liquid 50 stops moving along the microchannel and is not subject to active mixing at this stage, the mixing of the sample liquid 50 and the reagent 60 is driven by slow diffusion. Thus, the reagent 60 does not distribute within the sample liquid 50 in an amount sufficient to create the volume uncertainties described above.

[0070] Reference Figure 3 , the separation gas chamber 26 is compressed, increasing the pressure of the gas 56 therein and forcing the gas 56 through the separation gas channel 30 and into the separation channel 28 via the gas introduction opening 30'. Introducing the gas 56 into the separation channel 28 separates the liquid segment 55 from the remainder 57 of the sample liquid 50 ( Figure 2as shown). The liquid section 55 includes a proximal gas-liquid interface 58, all of the sample liquid 50 that contacts and / or contains the reagent(s) 60, and a distal liquid-gas interface 52. That is, the remainder 57 of the sample liquid 50 does not include any reagent 60. Instead, all of the reagent 60 is contained in and / or contacts the liquid of the liquid section 55. The remaining liquid portion 57 includes a distal liquid-gas interface 59. The gas 56 can occupy at least a portion of the supply channel 16 and the separation channel 28 and is isolated from the ambient gas surrounding the device 10 by the remaining liquid portion 57 (which blocks the application zone 14), and is isolated from such ambient gas and the gas 54 by the liquid section 55 (which blocks the vent 32 and separates the gases 54 and 56). As an alternative to introducing the separation gas by compressing the separation gas chamber, the chamber containing the separation gas can be heated, thereby increasing the pressure of the separation gas and introducing the separation gas into the microchannel containing the sample liquid and separating the liquid section from the remaining liquid. The heating can be performed, for example, by using a resistive conductor within the chamber.

[0071] The microfluidic network 12 can also include reagents disposed at different locations along the analysis channel 18, and / or reagents disposed at locations different from the analysis channel 18, e.g., at locations of the microfluidic network near the analysis channel 18. For example, the application zone and / or the supply channel 16 can include one or more reagents to facilitate the determination of the target, e.g., to lyse and / or disrupt cells within the sample liquid to release the contents of the cells and / or one or more reagents, such as heparin, to reduce the clotting of a blood-based sample liquid. In such an embodiment, such proximally disposed reagents can contact a significant portion, e.g., most or substantially all, of the introduced sample liquid such that the concentration of such reagents is not known with the same precision as when in contact with a fixed volume of the liquid section. However, the liquid section can be prepared from such reagent-containing sample liquid and contacted with additional reagents before and / or after the preparation of the liquid section. In such a case, as disclosed herein, the concentration(s) of the additional reagent(s) in the liquid section will be known with a higher precision.

[0072] After separating the liquid section 55 from the remainder 57 of the sample liquid 50, the reader can actuate an oscillation of the pressure of the gas 54 within the gas chamber 20, and / or an oscillation of the pressure of the gas 56 within the gas chamber 26. The oscillation can be performed, for example, as disclosed in the '325 application and the '858 application, including via the use of a piezoelectric actuator. For example, compression, decompression, and / or oscillation of each spaced-apart location 22a, 22b, 22c of the gas chamber 20 and the gas chamber 26 can be performed using a respective piezoelectric actuator having an actuating foot to independently compress, decompress, and / or oscillate (e.g., synchronously) covering each spaced-apart location 22a, 22b, 22c and / or the upper wall of the gas chamber 26. Oscillating the upper wall at each spaced-apart location causes the volume occupied by the gas 54 within the gas chamber 20 to oscillate (and thus the pressure of the gas 54) by oscillating the spacing between the opposing inner walls at each spaced-apart location. As an alternative, or in combination with the oscillation of the pressure of the gas 54, the pressure of the gas 56 can oscillate, for example, synchronously at the same frequency, out of phase but at the same frequency as the oscillation frequency of the gas 54, or at a frequency different from the oscillation frequency of the gas 54.

[0073] Oscillating the pressure of the distal gas 54 causes a bulk movement of the liquid within the liquid section 55, thereby distributing the reagent 60 throughout the liquid section and establishing a uniform concentration distribution therein. The bulk movement of the liquid causes the liquid within the liquid section 55 to mix with the reagent faster than can be achieved by diffusion alone. The proximal gas-liquid interface 58 and the distal liquid-gas interface 52 of the liquid section 55 prevent the reagent from leaving the liquid section during oscillatory mixing. Thus, mixing within the liquid section 55 provides a mixture of the reagent(s) 60 having a known uniform concentration. The volume of the liquid section 55 is determined by the volume of the microchannel between (i) the intersection of the gas introduction opening 30' of the separation gas channel 30 and the separation channel 28 and (ii) a capillary stop (e.g., the vent 32). The aforementioned volume of the microchannel is determined by the internal dimensions of the microchannel (e.g., cross-sectional area) and the distance along the microchannel between (i) the intersection of the gas introduction opening 30' of the separation gas channel 30 and the separation channel 28 and (ii) the exhaust hole 32 and / or the hydrophobic layer (if present). Typically, the dimensions and distances are selected to provide a liquid section having a volume, for example, between about 0.2 μL and about 2.5 μL, such as between about 0.2 μL and about 0.750 μL, such as about 0.35 μL.

[0074] By reducing the pressure of the gas 54 acting on the distal liquid-gas interface 52, the liquid section 55 can move distally along the microchannel, e.g., within the analysis channel 18. For example, the pressure of the gas 54 can be reduced by reducing the compression of one or more of the spaced-apart locations 22a, 22b, 22c within the gas chamber 20. Reducing the compression can be performed, e.g., as disclosed in the '325 application and the '858 application, such as via the use of a piezoelectric actuator in contact with the outer wall of the microfluidic device 10 covering the spaced-apart locations. Reducing the compression increases the volume of the gas chamber 20 by increasing the spacing between the opposing inner walls at each of the spaced-apart locations 22a, 22b, 22c.

[0075] The distal movement of the liquid section 55 can be performed to, e.g., bring the liquid of the liquid section 55 into contact with other reagents disposed within the analysis channel 18 and / or to cause the liquid section to enter the detection zone 18” therein.

[0076] After the liquid section 55 has been brought into contact and mixed with all the (one or more) reagents necessary for performing the assay (e.g., detection) of the target, the liquid section is subjected to a detection step for such an assay. Such a detection step can include, e.g., fluorescence or other optical detection and / or electrochemical detection. Any detection step disclosed in the '325 application can be performed. For example, the reagent can include magnetic microparticles, and the detection step can include subjecting the liquid section to a magnetic field to capture the magnetic microparticles. Then, the detection step can include compressing one or more (e.g., all) of the spaced-apart locations 22a, 22b, 22c or the gas chamber 26 to increase the pressure of the gas 54, thereby removing (e.g., expelling) the liquid of the liquid section 55 in contact with the captured magnetic particles from the detection zone 18” prior to detection (e.g., separating the liquid of the liquid section 55 from the captured magnetic particles). Alternatively, the detection step can then include decompressing one or more (e.g., all) of the spaced-apart locations 22a, 22b, 22c or the gas chamber 26 to reduce the pressure of the gas 54, thereby removing (e.g., withdrawing) the liquid of the liquid section 55 in contact with the captured magnetic microparticles from the detection zone 18”. As another example, the detection step can then include, on the one hand, decompressing one or more (e.g., all) of the spaced-apart locations 22a, 22b, 22c or the gas chamber 26 and, on the other hand, compressing another one or more (e.g., all) of the spaced-apart locations 22a, 22b, 22c or the gas chamber 26, thereby removing the liquid of the liquid section 55 by a combination of expelling and pumping out the gas pressure.

[0077] In an embodiment, the microfluidic device 10 includes two or more microfluidic networks 12, each of which may be configured to receive a sample liquid, mix with one or more reagents, and independently perform an analysis on the sample liquid to, for example, detect the presence or absence of a target in the sample liquid. In some embodiments, each microfluidic network includes the same components as those described herein for the microfluidic network 12, which includes, for example, a microfluidic channel having a supply channel portion 16, a segment separation channel portion 28, an analysis channel portion 18 including a reagent zone 18' and a detection zone 18", and a gas chamber 20. In some embodiments, the supply channel portion of each microfluidic network branches from a common channel that extends from an application zone. Thus, two or more microfluidic networks are configured to analyze the sample liquid simultaneously.

[0078] Two or more microfluidic networks may be positioned adjacent to each other on the microfluidic device. In an embodiment, at least some of the microfluidic networks have the same reagents in order to provide redundant analysis of the sample liquid. This redundancy can be used as a check in the event of any contamination or failure in a given microfluidic network and further provides higher precision and adjustability for any sensitivity of the sample liquid.

[0079] In some cases, at least some of the microfluidic networks have one or more reagents that are different from each other, thereby allowing the detection of one or more different targets in the sample liquid by the microfluidic device. This detection of different targets can be performed simultaneously.

[0080] Reference Figures 5-9, depicts another exemplary microfluidic network 112 of a microfluidic device (only a part of the microfluidic network is shown), where the microfluidic network includes a tapered-section separation channel 128 for separating a liquid section from a liquid introduced into the microfluidic network. The microfluidic network 112 includes a supply channel 16 extending from a sample application zone 14, a section separation channel 128, and an analysis channel 118 including a reagent zone 118' and a detection zone 118". A hydrophobic layer in the form of vents 132 and / or hydrophobic strips 133 may be located on the inner surface of the analysis channel 118 and may serve as a hydrophobic stopper. The vents 132 are typically between about 75 μm and about 300 μm, such as about 150 μm, along the width w4 of the analysis channel 118. The microfluidic network 112 also includes a separation gas channel 130 having an opening 130' connected to the section separation channel 128. The separation gas channel 130 leads to a separation airbag, which may be configured to operate as described for the separation airbag 26 of the microfluidic network 10. The distal portion of the analysis channel 118 includes a gas chamber opening 124 connected to a gas chamber, which may be configured to operate as described for the separation airbag 26 of the microfluidic network 10. The microfluidic network 112 further includes electrical features (not shown) as discussed for the microfluidic device 10. The internal height of the microfluidic network 112 is typically between about 50 μm and about 200 μm, such as about 110 μm.

[0081] The section separation channel 128 has a length between a proximal separation starting point 135 and a distal separation ending point 137. The length of the section separation channel 128 is typically between about 1.5 mm and about 4.5 mm, such as about 3 mm. The width of the section separation channel 128 is defined by a first tapered sidewall 128' and a second tapered sidewall 128”. The width between the sidewalls 128', 128” tapers from the width w2 at the proximal separation starting point 135 to a narrower width w1 at the distal separation ending point w1. The width w2 is typically between about 800 μm and 2200 μm, such as about 1650 μm. The width w1 is typically between about 400 μm and 1200 μm, such as about 800 μm. The ratio of the widths w2 / w1 is typically at least about 1.25, such as between about 1.5 and 3.5, such as about 2. From the proximal separation starting point 135 to the distal separation ending point 137, the width of the section separation channel 128 typically decreases at an average rate of about 12%mm -1 to about 25%mm -1 such as about 17.5%mm -1。The height of the section separation channel 128 is generally constant or substantially constant. As used herein, the term "substantially" can refer to a variation of less than or equal to + / - 1%, + / - 2%, + / - 3%, + / - 4%, + / - 5%, + / - 6%, + / - 7%, + / - 8%, + / - 9%, + / - 10%, + / - 11%, + / - 12%, + / - 14%, or + / - 15%. Thus, the cross-sectional area (not shown) of the tapered gas separation channel 128 tapers from a first cross-sectional area A2 at the proximal separation starting point 135 to a smaller cross-sectional area A1 at the distal separation ending point 137. For example, the cross-sectional area A1 can be between about 0.044 mm 2 and about 0.13 mm 2 , for example about 0.09 mm 2 , and the second cross-sectional area A2 can be between about 0.13 mm 2 and about 0.24 mm 2 , for example about 0.18 mm 2 . The ratio of the cross-sectional areas (area A2 of the proximal separation starting point 135 / area A1 of the distal separation ending point 137) can be the same as the ratio of the widths (w2 / w1), such that for example A2 is approximately equal to A1 × w2 / w1. The cross-sectional area of the section separation channel 128 can decrease from the proximal separation starting point 135 to the distal separation ending point 137 at the same relative rate of decrease as its width.

[0082] The separation opening 130' has a width w5 along the first tapered sidewall 128' of the section separation channel 128. The width w5 is generally between about 75 μm and about 300 μm, for example about 150 μm. The distance d3 ( Figure 6 and Figure 8 ) between the center of the separation opening 130' and the distal separation end 137 is between about 300 μm and about 1000 μm, for example about 750 μm. Moving distally along the section separation channel 128 from the center of the separation opening 130' to the distal separation end 137, the width between the tapered walls 128' and 128" tapers as described above. Moving distally beyond the distal separation end 137, the width of the microchannel widens from the width w1 at the distal separation end 137 to the width w3 within the analysis channel 118. The width w3 is greater than the width w1. The width w3 is generally between 800 μm and about 3000 μm, for example about 2000 μm. The ratio of the width w3 / w1 is generally between about 1 and 3.5, for example about 2.5.

[0083] The reagent region 118' of the analysis channel 118 includes one or more reagents arranged and composed as described for the reagent 60 of the microfluidic device 10. For example, the reagent 60 can include a reagent configured to prepare a sample for sample analysis. For example, the reagent can be a heparin reagent that inhibits blood sample coagulation or a lysis reagent configured to lyse cells to release their contents for analysis. In Figures 5-9 , the microfluidic network 112 is shown to have sample liquid that has contacted and dissolved such a reagent. The microfluidic network is used to prepare a liquid segment containing all such contacted and dissolved reagents.

[0084] Before introducing the liquid sample into the microfluidic network 112, the ambient gas (such as air) around the microfluidic device containing the network 112 can enter and leave the microfluidic network 112 via the application zone 14 and the vent 132. There is no other route through which gas can enter or leave the microfluidic network 112. Thus, the microfluidic network 112 (e.g., its channels and chambers) is occupied by this ambient gas.

[0085] The method for forming a liquid segment using the microfluidic network 112 proceeds in a manner similar to the process described for the microfluidic device 10. The method begins by inserting the microfluidic device containing the microfluidic network 112 into a reader (not shown), as disclosed in the '325 application. The reader compresses the gas chamber (e.g., the distal side of the gas chamber channel 124) of the microfluidic network 112 to an operable fully compressed state, thereby discharging the gas therein through the application zone 14 and the vent 132. The separation gas chamber remains in an uncompressed state. The method continues by introducing the sample liquid 150 into the microfluidic network 112 via the application zone 114. The sample liquid 150 moves along the supply channel 16 by capillary action, passes through the segment separation channel 128, and enters the analysis channel 118 until the distal liquid-gas interface 152 reaches the vent 132 and / or the hydrophobic strip 133, either of which can be used as a capillary stopper to stop the further movement of the liquid sample 150. As Figure 5 shown, the sample liquid 150 then occupies the portion of the microfluidic network 112 that extends from the gas-liquid interface 152' formed at the application zone 14 to the distal gas-liquid interface 152 located at the vent 132. Within the analysis channel 118, the sample liquid 150 can contact one or more reagents therein and mix by diffusion, as described for the sample liquid 50 within the microfluidic network 12 of the microfluidic device 10.

[0086] The gas chamber of the microfluidic network 112 and the portion of the analysis channel 118 remote from the distal liquid-gas interface 152 are occupied by a gas 154 having the same composition and properties as the gas 54. The separation gas chamber and the separation gas channel 130 of the microfluidic network 112 are occupied by a gas 156 having the same composition and properties as the gas 56. The gas 154 and the gas 156 are composed of the remaining ambient gas (e.g., air) that had occupied the microfluidic network 112 prior to the introduction of the sample liquid 150. Since the sample liquid 150 that occupies the microfluidic network 112 blocks the vent 132 and the application zone 14, the gas 154 and the gas 156 are isolated from each other and from the ambient gas surrounding the microfluidic device that includes the microfluidic network 112.

[0087] Reference Figure 6 , the pressure of the gas 156 in the separation gas chamber of the microfluidic network 112 is increased, e.g., by compressing the separation gas chamber as described for the separation gas chamber 56 of the microfluidic device 10, thereby forcing the gas 156 through the separation gas channel 130 and into the separation channel 128 via the gas introduction opening 130'. The introduction of the gas 156 into the separation channel 128 forms an initial bubble 141 that separates a liquid segment 155 within the microfluidic network 112 from the remaining liquid portion 157. The initial bubble 141 and the liquid segment 155 form a gas-liquid interface 158, and the initial bubble 141 and the remaining liquid 157 form a gas-liquid interface 159.

[0088] The liquid of the liquid segment 155 consists essentially of the amount of the sample liquid 150 within the microfluidic network 112 that had been disposed between the gas introduction opening 130' and the distal gas-liquid interface 152. The volume of the liquid segment 155 is typically between about 0.75 μL and about 4 μL, e.g., between about 1.5 μL and about 3 μL, e.g., about 2 μL. A smaller volume of the sample liquid (e.g., between about 150 nL and about 5 μL, e.g., about 350 nL) may enter the vent 132 and is not considered part of the liquid segment 155. The liquid of the remaining liquid portion 157 is the amount of the sample liquid 150 within the microfluidic network 112 that had been disposed between the gas introduction opening 130' and the application zone 14.

[0089] Reference Figures 7-9, the pressure of the gas 156 in the separation gas chamber of the microfluidic network 112 has been further increased, increasing the volume of the initial bubble 141 to the final bubble 141'. Since the walls 128', 128'' gradually narrow distally from the position of the gas-liquid interface 158 (e.g., the width of the separation channel 128 between the walls 128', 128'' gradually decreases distally along the separation channel 128 from the position of the gas-liquid interface 158), the radius of curvature r1 of the gas-liquid interface 158 will have to decrease in order for the gas-liquid interface 158 to move distally when additional gas is introduced from the separation gas chamber through the opening 130'. The radius of curvature r1 is defined in a plane parallel to the longitudinal axis of the separation channel and the larger of the width or height of the separation channel at the position of the gas-liquid interface 158. In Figures 5-9 the embodiment, the radius of curvature r1 is in a plane parallel to the longitudinal axis of the separation channel and parallel to the width of the separation channel because the width is greater than the height at the position of the gas-liquid interface 158. In an embodiment, the radius of curvature of each interface is measured along a radial axis that is aligned with the longitudinal axis of the channel and perpendicular to the width of the channel at the position of the interface, and wherein the radius of curvature of the interface can also be parallel to the plane of the substantially planar microfluidic device.

[0090] Moving the liquid segment 155 distally along the separation channel will decrease the radius of curvature of the gas-liquid interface 158, thereby increasing the surface tension and energy of that interface. Thus, the gas-liquid interface 158 resists distal movement and remains substantially at the same position along the segment separation channel 128 as additional separation gas is introduced. Since the gas-liquid interface 158 remains substantially in the same position, the liquid segment 155 also remains substantially in the same position and substantially no additional liquid enters the vent 132.

[0091] However, the gas-liquid interface 159 can move proximally in the segment separation channel 128 because the width of the channel 128 continues to expand proximally, thereby increasing the radius of curvature of the interface 159 until the interface 159 forms an interface 159' with a radius of curvature r2, where r2 > r1. The radius of curvature r2 can be defined in the same plane as the radius of curvature r1. The different radii of curvature impart an asymmetric shape to the bubble 141'. The ratio r2 / r1 is approximately the same as the ratio of the widths of the channel 128 at the corresponding positions of the gas-liquid interfaces 158, 159' of the bubble 141'. For example, when the interfaces are at the positions w2 and w1, the ratio r2 / r1 can be approximately the same as the ratio w2 / w1, as Figure 7As shown. Compared with the gas-liquid interface 159 of the initial bubble 141, increasing the radius of curvature of the interface 159' reduces the surface tension and energy of the interface 159'. Thus, in some cases, the pressure or energy required for the separated gas in the segment separation channel 128 to move the liquid segment 155 will be greater than the pressure or energy required for the remaining liquid 157, due to the smaller cross-sectional area of the gas-liquid interface 158 exposed to the separated gas pressure or energy compared to the larger cross-sectional area of the gas-liquid interface 159'. Thus, the incoming separated gas will preferentially move the remaining liquid 157 proximally rather than the liquid segment 155 distally. The distance along the microchannel between the gas-liquid interface 158 and the gas introduction opening 130' is distance d1, the distance along the microchannel between the gas-liquid interface 159' and the gas introduction opening 130' is distance d2, and the ratio d2 / d1 is at least about 2.25, such as between about 2.25 and 10, such as about 4.5. For example, d1 can be between about 250 μm and 1000 μm, such as about 500 μm, and d2 can be between about 1000 μm and about 2750 μm, such as about 2000 μm.

[0092] After separating the liquid segment 155 from the remainder 157 of the sample liquid 150 and forming the bubble 141', the reader actuates the oscillation of the pressure of the gas 154 in the gas chamber of the microfluidic network 112 to facilitate the mixing of the reagent in contact with the liquid segment 155. The oscillation can be performed, for example, as disclosed for the device 10 and the '325 application and the '858 application, for example, including via the use of a piezoelectric actuator. Then the pressure of the gas 154 can be reduced, the liquid segment 155 can be aspirated distally and brought into contact with additional reagent 160 within the detection region 118” of the analysis channel 118. The additional reagent 160 typically includes one or more reagents configured to bind to a target within the sample. For example, the reagent 160 can include one or more different microparticles, which include binding agents, such as antibodies for the target. The microparticles can include, for example, magnetic microparticles and fluorescent microparticles, and the microparticles can be configured to form a detectable sandwich with the target. Then the target can be detected, for example, optically or electrochemically within the detection region 118”.

[0093] In an embodiment, the microfluidic device 210 includes two or more microfluidic networks 112, each of which may be configured to receive a sample liquid, mix with one or more reagents, and independently perform an analysis on the sample liquid to, for example, detect the presence or absence of a target in the sample liquid. In some embodiments, each microfluidic network includes the same components as those described herein for the microfluidic network 112, which include, for example, a supply channel 16, a tapered-segment separation channel 128, an analysis channel 118 including a reagent zone 118' and a detection zone 118", a gas chamber, a separation gas chamber, a separation gas channel, a vent 132, and / or a hydrophobic layer in the form of a hydrophobic strip 133, the hydrophobic strip 133 being locatable on the inner surface of the analysis channel 118 and serving as a hydrophobic stopper. In some embodiments, the supply-channel portion of each microfluidic network branches from a common channel that extends from an application zone 14. Thus, two or more microfluidic networks are configured to analyze the sample liquid simultaneously.

[0094] Two or more microfluidic networks may be positioned adjacent to each other on the microfluidic device. In an embodiment, at least some of the microfluidic networks have the same reagents to provide redundant analysis of the sample liquid. This redundancy can serve as a check in the event of any contamination or failure in a given microfluidic network and further provide higher precision and adjustability for any sensitivity of the sample liquid.

[0095] In some cases, at least some of the microfluidic networks have one or more reagents that are different from each other, thereby allowing detection of one or more different targets in the sample liquid by the microfluidic device. This detection of different targets can be performed simultaneously.

[0096] Referring Figures 10-16 to, the microfluidic device 210 is configured to combine two liquid segments each having a known precise volume to prepare a mixture of two liquids having a known concentration. In the illustrated embodiment, one of the liquids is a biological sample, such as blood, and the other liquid is a diluent, such as a buffer. Other liquids may be used. The microfluidic device 210 includes a microfluidic network having a sample-introduction zone 214, a primary sample-introduction channel 216, a hematocrit-introduction channel 216", a hematocrit-detection chamber 217, a secondary sample-introduction channel 216', a segment-combination chamber 228, an analysis channel 218, a diluent-application zone 219, and a diluent-introduction channel 221.

[0097] A first gas chamber 220 communicates with a distal portion of the analysis channel 218. The gas chamber 220 may be configured as the gas chamber 20 of the device 10. A combined gas chamber 226 communicates with the segment-combination chamber 228 at a segment-gas opening 241' via a segment-gas channel 241, the segment-gas opening 241' having the same dimensions as the opening 130' of the microfluidic network 112.

[0098] Device 210 also includes electrical features to monitor the presence of the sample and dilution liquid and to monitor the compression state of gas chambers 220 and 226. Signal emission lead 229 includes signal emission electrodes 229', 229", 229"', 229"" disposed within the microfluidic network to be in electrical communication with the liquid present at corresponding locations of each electrode. Signal emission lead 229 and the corresponding electrodes are configured to emit time-varying signals, as disclosed in the '325 application. Device 210 includes inductive sensing features including sensing electrodes 251, sensing electrode 227, sensing electrode 223, sensing electrode 255, sensing electrode 231, sensing electrode 233, and sensing electrode 257. The foregoing sensing electrodes cooperate with the emission electrodes to detect the presence of liquid at the location of each sensing electrode, as disclosed in the '325 application. Each of the emission electrodes and sensing electrodes is disposed on the inner surface of the microchannel network of device 210. In addition, the microfluidic network 210 includes hydrophobic strips covering or located above sensing electrode 223 and hydrophobic strips covering or located below emission electrode 229"". The sensing electrodes or emission electrodes in the figure obscure the hydrophobic strips. Each hydrophobic strip cooperates with sensing electrode 223 and emission electrode 229"" to create a hydrophobic barrier, such as a capillary stop, that prevents liquid from flowing distally or proximally by capillary action beyond the location.

[0099] The operation of strip 210 proceeds as follows. Prior to operation, gas chamber 226 is compressed as discussed for gas chamber 26 of device 10. As Figure 10 shown, a blood sample is applied to application zone 214 and flows by capillary action along primary sample introduction channel 216. A portion of the blood flows along hematocrit introduction channel 216" and fills hematocrit detection chamber 217. The proper filling of the blood within chamber 217 is confirmed by sensing, via sensing electrode 257, the signal emitted by emission electrode 229'. A second portion of the blood flows along secondary sample introduction channel 216 and forms a blood segment 261 within the volume of segment combination chamber 228 disposed between sensing electrode 223 (and the corresponding hydrophobic strip) and emission electrode 229"" (and the corresponding hydrophobic strip). The length of chamber 228 along sensing electrode 223 and emission electrode 229"" is between about 500 μm and 2000 μm, such as about 1400 μm. The width of chamber 228 between sensing electrode 223 and emission electrode 229"" is between about 500 μm and 1750 μm, such as about 1000 μm. The height within the chamber is between about 50 μm and about 200 μm, such as about 110 μm. The volume of blood segment 261 is determined by the above dimensions and is typically between about 75 nL and 500 nL, such as about 150 nL. The proper filling of the blood is confirmed by sensing, via sensing electrode 233, the signal emitted by emission electrode 229"".

[0100] Reference Figure 11 As a result, the gas pressure within the gas chamber 220 increases, for example, as discussed with respect to gas chamber 20. The increased gas pressure forces the blood segment 261 proximally within chamber 228 beyond the emission electrode 229”” (and corresponding hydrophobic strip) until the proximal gas-liquid interface 271 of the blood segment 261 contacts the sensing electrode 227, thereby electrically connecting the sensing electrode 227 with the emission electrode 229””. When the presence of the blood segment 261 is sensed at the sensing electrode 227, compression of the gas chamber 220 is stopped.

[0101] Reference Figure 12a 、 12b A diluent 267 (e.g., buffer) is introduced into the diluent application zone 219. The diluent 267 flows by capillary action along the diluent introduction channel 221 until the distal gas-liquid interface of the diluent 267 moves distally beyond the vent 232. As best shown in Figure 12b , where, for clarity, the sensing electrode 227 has been shown as partially transparent, the proximal interface 271 of the blood segment 261 and the distal interface 269 of the diluent 267 are separated by a small amount of gas (e.g., air). The gas pressure within the gas chamber 226 is decreased, thereby aspirating the gas and a small amount of the diluent 267 that separates the proximal interface 271 and the interface 269 through the opening 241' into the channel 241, thereby allowing the blood segment 261 to contact and / or combine with the diluent 267. When the gas pressure within the gas chamber 226 is decreased, the hydrophobic strip and the emission electrode 229”” prevent the proximal interface 271 of the blood segment 261 from moving proximally, thereby maintaining the volume and position of the blood segment 261. In contrast, the distal interface 269 of the diluent 267 moves distally along the segment combination chamber 228 until the interface 269 combines with the interface 271 of the blood segment 261.

[0102] Reference Figure 13 The gas pressure within the gas chamber 220 is decreased, drawing the blood segment 261 and the diluent 267 distally along the analysis channel 218 until the distal gas-liquid interface 271' establishes electrical connectivity between the emission electrode 229”' and the sensing electrode 255, at which point the presence of the liquid is detected and the operation of the gas chamber 220 is stopped.

[0103] Reference Figure 14 The gas pressure within the separation gas chamber 226 is increased, thereby forcing gas via the channel 241 and the opening 241' into the chamber 228, thereby dividing the diluent 267 into a diluent segment 275 and a remainder 277. The dividing of the diluent 267 into the diluent segment 275 and the remainder 277 is performed after the distal interface 269 of the diluent 267 has been combined with the interface 271 of the blood segment 261.

[0104] ReferenceFigure 15 , the gas pressure in the gas chamber 220 is reduced again, and the blood segment 261 and the diluent segment 275 are drawn distally along the analysis channel 218 until the distal gas-liquid interface 271' of the blood segment 261 contacts the sensing electrode 231, establishing electrical communication between the sensing electrode 231 and the emitting electrode 229. At this time, the presence of liquid is detected and the operation of the gas chamber 220 is stopped. The analysis channel 218 may include a reagent facilitating the determination of the target. For example, a reagent for determining the HbA1c content of the blood segment 261. After stopping the movement of the blood segment 261 and the diluent segment 275, the pressure of the gas in the chamber 220 and / or the separation gas chamber 226 oscillates as discussed for the device 10, so as to mix the blood segment 261, the diluent segment 275 and such a reagent to form a sample segment 279. Oscillation may also be performed during at least a portion (e.g., most or all) of the time when the blood segment 261 and the diluent segment 275 move distally. Exemplary suitable reagents and methods for determining HbA1c are disclosed in U.S. Application No. 17 / 409,279 filed on August 23, 2021, which is incorporated herein by reference in its entirety.

[0105] Reference Figure 16 , the gas pressure in the gas chamber 220 is reduced again, and the sample segment 279 is pulled distally along the analysis channel 218 until the distal gas-liquid interface 271” of the sample segment 279 contacts the sensing electrode 233, thereby establishing electrical communication between the sensing electrode 233 and the emitting electrode 229”’. At this time, the presence of liquid is detected and the operation of oscillating the gas chamber 220 is stopped. The analysis channel 218 may include additional reagents disposed between the sensing electrodes 231 and 233, and such additional reagents are contacted and moved by the sample segment 279. The gas pressure in the gas chamber 220 may oscillate to enhance the mixing of such reagents and the sample segment 279. Subsequently, the presence of the target (e.g., HbA1c) is detected.

[0106] Reference Figures 17-19 , according to some embodiments, the microfluidic device 310 is configured to receive a liquid containing particles, separate at least some of the liquid from the particles, form a segment containing some of the liquid separated from the particles, and determine whether at least one target is present in the liquid of the separated segment. For example, the liquid containing particles may be whole blood, the separated liquid may be plasma separated from the red blood cells of whole blood, and at least one target may be a cardiac marker, such as troponin I or troponin C.

[0107] The microfluidic device 310 includes a substrate 311 having a generally planar surface in which a microfluidic network 312 is defined. Starting from a sample application zone 314 and proceeding from proximal to distal, the microfluidic network 312 may include microfluidic channels having a supply channel portion 316, a segment separation channel portion 328, an analysis channel portion 318 including a reagent zone 318' and a detection zone 318", a gas chamber 320, a separation gas chamber 326, or any combination thereof. A porous membrane 319 covers the sample application zone 314. The microfluidic device may also include i) electrical features such as electrodes for determining the compression of the gas chambers 320, 326 and the presence of a liquid sample within the microfluidic network 312, and / or ii) vents and capillary stops as disclosed for the microfluidic devices 10, 210 and the microfluidic network 112. For clarity, these features are not shown in Figure 17 , Figure 18 . The dimensions (e.g., width, height, and length) as well as the functions and operations of the elements of the microfluidic network 312 (e.g., the supply channel portion 316, the segment channel portion 328, the analysis channel 318, the gas chambers 320, 326, and the separation gas channel 330) may be similar, e.g., the same, as the corresponding elements of the microfluidic devices 10, 210 and the microfluidic network 112.

[0108] Referring to Figure 18 , the substrate 311 may include an upper layer 313 and a lower layer 315 adhesively bonded to each other through an intermediate layer 317. The upper layer 313 and the lower layer 315 may be formed of a polymer (e.g., polyester), and each may have a typical total thickness between about 80 μm and 130 μm (e.g., about 100 μm). The intermediate layer 317 may be formed of an intermediate layer of a polymer (e.g., polypropylene) having an upper adhesive layer and a lower adhesive layer. The intermediate layer 317 may have a typical total thickness between about 80 μm and 150 μm (e.g., about 110 μm).

[0109] The lower inner surface of the microfluidic channels and the application zone 314 may be defined by the upper surface 315' of the lower layer 315. The upper inner surface of the microfluidic network 312 including the microfluidic channels 316 may be defined by the lower surface 313' of the upper layer 313. The upper inner surface of the application zone 314 may be defined by the lower surface 319' of the porous membrane. The inner sidewalls 314' of the application zone 314 and the opposing inner sidewalls 312', 312" of the microfluidic channels 316 may be defined by the intermediate layer 317.

[0110] The upper layer 313 includes a first application zone aperture 321 defined by sidewalls 313", and has a maximum diagonal d1 along an axis parallel to the plane of the substrate 311 ( Figure 18 , 19). The intermediate layer 317 includes a second application zone hole that defines an inner sidewall 314' and has a maximum diagonal d2 along an axis parallel to the plane of the substrate 311 ( Figure 18 , 19 ). The first application zone hole d1 of the upper layer 313 is larger than the second application zone hole d2 of the intermediate layer 317 such that the first application zone hole d1 exposes a peripheral portion 325 of the adhesive upper surface 317'. The peripheral portion of the lower surface 319' of the porous membrane adheres to the peripheral portion 325 of the adhesive upper surface 317'. For example, the porous membrane 319 can be fixed to the substrate 311 by positioning the porous membrane 319 such that the peripheral portion of the lower surface 319' of the porous membrane contacts and covers the peripheral portion 325 of the adhesive upper surface 317'. Pressure and / or heat can be applied to fix the porous membrane 319 and the substrate 311 together. The peripheral portion 325 has a radial width d3 sufficient to adhere the porous membrane 319 to the intermediate layer 317 ( Figure 19 ). The width d3 is typically between about 1 mm and 3 mm, such as about 2 mm.

[0111] The sample application zone 314 and the microfluidic channel 316 intersect at an intersection 327 from which the microfluidic channel 316 extends. The protruding portion 331 of the upper layer 313 (whose lower surface 313' defines the upper surface of the microfluidic channel 316) extends beyond the intersection 327 into the sample application zone 314. Substantially all (e.g., all) of the protruding portions 331 are located below the porous membrane 319.

[0112] The intersection 327 defines a width w6 between the first and second opposing inner sidewalls 312', 312'' of the microfluidic channel 316 at the location of the intersection 327. The width w6 is taken along a direction that is generally perpendicular to the longitudinal axis of the microfluidic channel 316 and parallel to the plane defined by the microfluidic device 310 at the location of the intersection 327. The width w6 can be, for example, about 1.5 mm. The microfluidic channel 316 defines a width w9 adjacent to the intersection 327. The ratio of the width w6 / w9 is typically between about 1 and 3, such as about 2.

[0113] The protruding portion 331 extends a distance d5 beyond the sample application zone sidewall 314' into the sample application zone 314. The distance d5 is typically at least about 0.25 mm and is typically less than about 1 mm, such as about 0.25 mm or about 0.5 mm. The protruding portion 331 has a total length d4 intercepted from the sidewall 313'' of the first application zone hole 321 to the tip 361 of the protruding portion 331.

[0114] The width w7 of the protruding portion 331 at the position of the intersection 327 is narrower than the width w6 of the intersection 327. For example, at the intersection 327, the width w7 of the protruding portion 331 is typically between about 20% and 75% of the width w6 of the intersection 327. The protruding portion 331 tapers from the width w7 at the intersection 327 to a smaller width w8 disposed within the sample application zone 314. The ratio of the width w8 / w7 is typically between about 0.5 and 0.9, such as about 0.75.

[0115] In an embodiment, the protruding portion aids in the alignment of the porous membrane. For example, the porous membrane can help prevent or reduce the risk of the porous membrane blocking or at least partially blocking the opening leading to the microfluidic channel 316.

[0116] The microfluidic device 310 includes a ventilation channel 350 extending from a ventilation intersection 351 between the sample application zone 314 and the ventilation channel 350. The distal portion 352 of the ventilation channel 350 is in gaseous communication with the ambient gas around the substrate, such that the gas disposed within the sample application zone 314 can leave the sample application zone 314 via the ventilation channel 350 and / or the ambient gas around the microfluidic device 310 can enter the sample application zone 314 via the ventilation channel 350. The lower inner surface of the ventilation channel 350 is defined by the upper surface 315' of the lower layer 315. The upper inner surface of the ventilation channel 350 is defined by the lower surface 313' of the upper layer 313. The opposing inner sidewalls of the ventilation channel 350 are defined by the intermediate layer 317. The protruding portion 353 of the upper layer 313 that defines the upper inner surface of the ventilation channel 350 protrudes beyond the ventilation intersection 351 into the sample application zone 314.

[0117] The microfluidic device 310 can be operated as follows. A liquid containing microparticles (e.g., blood) is applied to the upper surface of the porous membrane 319. The microparticles (e.g., red blood cells) are retained on the upper surface of the porous membrane 319 and / or within the porous membrane 319. The separated liquid, e.g., plasma, passes through the porous membrane 319 into the sample application zone 314 therebelow. The separated liquid enters the microfluidic channel 316 through the intersection 327. Ambient gas enters and exits the sample application zone 314 via the ventilation port 352, thereby balancing the pressure therein as the separated liquid enters and exits the sample application zone 314. At least some of the separated liquid enters the segment separation channel portion 328. Segments of the separated liquid are formed, e.g., as disclosed for Figures 1-9 the embodiments of. One or more targets are detected within the liquid of the liquid segment.

[0118] numbered embodiments

[0119] Example 1: A microfluidic device includes: a generally planar substrate including a microfluidic network, the microfluidic network including i) a distal gas chamber configured to regulate the pressure of a distal gas within at least a portion of the microfluidic network; ii) a microfluidic channel extending distally from an application zone to the distal gas chamber, the application zone configured to receive a sample liquid therein; and iii) a separation gas chamber in communication with the microfluidic channel via a separation gas channel that intersects the microfluidic channel at a separation gas introduction location, the separation gas chamber configured to regulate the pressure of a separation gas within at least a portion of the microfluidic network; wherein the sample liquid is configured to flow from the application zone through at least a portion of the microfluidic channel to a capillary stop distal to the separation gas introduction location such that the separation gas forms a separation bubble within the microfluidic channel after being introduced into the microfluidic channel, the separation bubble separating the sample liquid therein into i) a liquid segment distal to the separation bubble, and ii) a remaining volume of the sample liquid proximal to the separation bubble, the liquid segment forming i) a distal gas-liquid interface disposed between the distal gas and the liquid segment, and ii) a proximal gas-liquid interface disposed between the separation gas and the liquid segment.

[0120] Example 2: The microfluidic device according to claim 1, wherein the microfluidic channel includes a reagent zone distal to the separation gas introduction location and configured to include one or more reagents.

[0121] Example 3: The microfluidic device according to claim 2, wherein the reagent is configured to dissolve with the sample liquid when in contact with the sample liquid.

[0122] Example 4: The microfluidic device according to claim 2 or 3, wherein the reagent is configured to enable detection of a target in the sample liquid when in contact with the sample liquid.

[0123] Example 5: The microfluidic device according to claim 4, wherein the microfluidic channel includes a detection zone distal to the reagent zone and configured to detect the presence or absence of a target within the sample liquid.

[0124] Example 6: The microfluidic device according to any one of claims 1 to 5, wherein the distal gas chamber includes a first inner wall, a second inner wall, and a distal chamber spacing therebetween, the distal chamber spacing configured to be occupied by the distal gas.

[0125] Example 7: The microfluidic device according to claim 6, wherein an outer wall of the distal gas chamber is configured to contact an oscillating member so as to oscillate and / or regulate the distal chamber spacing and thereby oscillate and / or regulate the pressure of the distal gas.

[0126] Example 8: The microfluidic device according to claim 7, wherein adjusting the pressure of the distal gas promotes the movement of the liquid segment within the microfluidic channel.

[0127] Example 9: The microfluidic device according to claim 7, wherein the oscillating member is configured to contact an outer wall of the distal gas chamber at a location spaced apart from the distal liquid-gas interface along the microfluidic channel by at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

[0128] Example 10: The microfluidic device according to any one of claims 1 to 9, wherein the separation gas chamber is configured to heat the separation gas therein in order to pressurize the separation gas and facilitate its introduction into the microfluidic channel to form separation bubbles.

[0129] Example 11: The microfluidic device according to any one of claims 1 to 9, wherein the separation gas chamber includes a third inner wall, a fourth inner wall, and a separation gas chamber spacing therebetween, the separation gas chamber spacing being configured to be occupied by the separation gas.

[0130] Example 12: The microfluidic device according to claim 11, wherein an outer wall of the separation gas chamber is configured to contact a second oscillating member in order to oscillate and / or adjust the separation gas chamber spacing, thereby oscillating and / or adjusting the pressure of the separation gas.

[0131] Example 13: The microfluidic device according to any one of claims 1 to 13, wherein the separation bubble defines a volume of the separation gas disposed between a proximal gas-liquid interface and a gas-liquid interface (RVSL gas-liquid interface) between the remaining volume of sample liquid and the separation bubble, the separation bubble being disposed within a separation zone of the microfluidic channel.

[0132] Example 14: The microfluidic device according to claim 13, wherein the separation zone of the microfluidic channel tapers distally from a larger cross-sectional area to a smaller cross-sectional area.

[0133] Example 15: The microfluidic device according to claim 13 or 14, wherein the proximal gas-liquid interface occupies a portion of the microfluidic channel having a cross-sectional area A1, wherein the proximal gas-liquid interface occupies a portion of the microchannel having a cross-sectional area A2, and wherein A2 is greater than A1 such that the separation bubble includes an asymmetric shape.

[0134] Example 16: The microfluidic device according to claim 15, wherein when the separation gas is introduced into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is less than the radius of curvature of the RVSL gas-liquid interface, such that further introduction of the separation gas causes the RVSL gas-liquid interface to move proximally while keeping the proximal gas-liquid interface at the same position or substantially at the same position within the microfluidic channel, such that the liquid segment i) remains at the same position or substantially at the same position within the microfluidic channel, and / or ii) has the same or substantially the same volume within the microfluidic channel.

[0135] Example 17: The microfluidic device according to claim 15 or 16, wherein the ratio of A2 to A1 (“R A ”) is at least about 1.25.

[0136] Example 18: The microfluidic device according to any one of claims 15 to 17, wherein the cross-sectional area A1 is between about 0.04 mm 2 and about 0.13 mm 2 .

[0137] Example 19: The microfluidic device according to any one of claims 15 to 18, wherein the distance between the RSVL and the proximal gas-liquid interface along the longitudinal axis of the microfluidic channel is between about 1 mm and 3 mm.

[0138] Example 20: The microfluidic device according to any one of claims 14 to 19, wherein the cross-sectional area of the microfluidic channel between the RSVL and the proximal gas-liquid interface decreases at an average rate between about 12% mm -1 and about 25% mm -1 .

[0139] Example 21: The microfluidic device according to any one of claims 14 to 20, wherein the distance between the proximal gas-liquid interface and the separation gas introduction position along the microfluidic channel is distance d1, wherein the distance between the proximal gas-liquid interface and the separation gas introduction position along the microfluidic channel is distance d2, and wherein the ratio d2 / d1 is between about 2.25 and 10.

[0140] Example 22: The microfluidic device according to any one of claims 14 to 21, wherein the proximal gas-liquid interface is disposed in the separation zone, and wherein the sample liquid of the liquid segment is substantially disposed in the reagent zone or the detection zone of the microfluidic channel that is distal to the separation zone, and the reagent zone or the detection zone has a cross-sectional area A3 that is greater than the cross-sectional area A1.

[0141] Example 23: The microfluidic device according to any one of claims 1 to 22, wherein the substantially planar substrate comprises an upper layer, a lower layer, and an intermediate layer disposed between the upper layer and the lower layer.

[0142] Example 24: The microfluidic device according to claim 23, wherein the intermediate layer comprises an upper surface having an adhesive adhered to the upper layer, a lower surface having an adhesive adhered to the lower layer, or both.

[0143] Example 25: The microfluidic device according to claim 23 or 24, wherein the upper surface of the lower layer defines the lower inner surface of the microfluidic channel and the application area.

[0144] Example 26: The microfluidic device according to any one of claims 23 to 25, wherein the lower surface of the upper layer defines the upper inner surface of the microfluidic channel.

[0145] Example 27: The microfluidic device according to any one of claims 23 to 26, wherein the upper layer, the lower layer, the intermediate layer, or any combination thereof comprises a hole defining the application area.

[0146] Example 28: The microfluidic device according to any one of claims 23 to 27, further comprising a porous membrane configured to cover the application area.

[0147] Example 29: The microfluidic device according to claim 28, wherein the porous membrane is configured to separate the sample liquid from one or more microparticles.

[0148] Example 30: The microfluidic device according to any one of claims 23 to 29, further comprising a protrusion extending from the upper layer and / or the intermediate layer into the application area.

[0149] Example 31: The microfluidic device according to any one of claims 1 to 30, wherein the microfluidic device further comprises one or more additional microfluidic networks, wherein each of the one or more additional microfluidic networks is configured to independently receive a portion of the sample liquid from the application area and analyze the sample liquid to detect a target therein.

[0150] Example 32: The microfluidic device according to claim 31, wherein two or more of the microfluidic channels and the one or more additional microfluidic channels are configured to analyze the sample liquid simultaneously or sequentially.

[0151] Example 33: The microfluidic device according to claim 31 or 32, wherein at least one of the one or more additional microfluidic channels is configured in the same manner as the microfluidic network according to any one of claims 1 to 30.

[0152] Example 34: A method comprising: a) introducing a sample liquid into a microfluidic channel within a microfluidic device, the microfluidic channel containing a distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal liquid-gas interface therebetween; b) introducing a separation gas into the microfluidic channel at a location occupied by the sample liquid, thereby separating a segment of the sample liquid from the remaining volume of the sample liquid introduced into the microfluidic channel, the liquid segment comprising (i) the distal liquid-gas interface, (ii) a portion of the sample liquid introduced into the microfluidic channel, and (iii) a proximal gas-liquid interface between the separation gas and the portion of the sample liquid, wherein the separation gas separates the proximal gas-liquid interface of the liquid segment from the remaining volume of the sample liquid; c) moving the liquid segment to a reagent zone of the microfluidic channel by reducing the pressure of the distal gas, the reagent zone comprising at least one reagent disposed therein; and d) mixing the portion of the sample liquid of the liquid segment with the at least one reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas, thereby forming a first mixture; wherein oscillating the pressure of the distal gas and / or the pressure of the separation gas is performed i) before, ii) simultaneously with, and / or iii) after reducing the pressure of the distal gas.

[0153] Example 35: A method for analyzing a sample liquid to detect at least one target material therein, the method comprising: a) introducing a sample liquid into a microfluidic channel within a microfluidic device, the microfluidic channel containing a distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal liquid-gas interface therebetween; b) moving the sample liquid along the microfluidic channel until at least some of the sample liquid contacts at least one reagent disposed within a reagent zone of the microfluidic channel; c) introducing a separation gas into the microfluidic channel at a location occupied by the sample liquid, thereby separating a segment of the sample liquid from the remaining volume of the sample liquid introduced into the microfluidic channel, the liquid segment comprising (i) the distal liquid-gas interface, (ii) a portion of the sample liquid that contacts the at least one reagent, and (iii) a proximal gas-liquid interface disposed between the separation gas and the portion of the sample liquid, wherein the separation gas separates the proximal gas-liquid interface of the liquid segment from the remaining volume of the sample liquid; and d) mixing the portion of the sample liquid of the liquid segment with the at least one reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas, thereby forming a first mixture.

[0154] Example 36: The method according to claim 35, wherein moving the sample liquid is via capillary action.

[0155] Example 37: The method according to claim 35, wherein moving the sample liquid is via reducing the pressure of the distal gas.

[0156] Example 38: The method according to claim 37, wherein the pressure of the oscillating distal gas and / or the separating gas is carried out i) before, ii) simultaneously with, and / or iii) after reducing the pressure of the distal gas.

[0157] Example 39: The method according to any one of claims 34 to 38, wherein the pressure of the oscillating distal gas and / or the separating gas is carried out at a frequency of about 2000 Hz or less, about 1500 Hz or less, about 1250 Hz or less, about 1000 Hz or less, about 900 Hz or less, about 800 Hz or less, about 5 Hz to about 2500 Hz, or about 10 Hz to about 2000 Hz.

[0158] Example 40: The method according to any one of claims 34 to 39, wherein the oscillating step comprises oscillating the pressure of the distal gas, and during the oscillating step, the distal liquid-gas interface occupies a position in the microfluidic channel having a cross-sectional area of at least about 0.01 mm 2 , at least about 0.02 mm 2 , at least about 0.03 mm 2 , at least about 0.04 mm 2 , at least about 0.05 mm 2 , at least about 0.06 mm 2 or at least about 0.07 mm 2 .

[0159] Example 41: The method according to any one of claims 34 to 40, wherein the oscillating step comprises oscillating the pressure of the distal gas, and during the oscillating step, the distal liquid-gas interface occupies a position in the channel having a cross-sectional area of about 0.15 mm 2 or less, about 0.125 mm 2 or less, about 0.1 mm 2 or less, about 0.09 mm 2 or less, or about 0.08 mm 2 or less.

[0160] Example 42: The method according to any one of claims 34 to 41, wherein during the oscillating step, the liquid segment has a volume of at least about 0.2 μL or greater, at least about 0.3 μL or greater, at least about 0.4 μL or greater, or at least about 0.5 μL or greater.

[0161] Example 43: The method according to any one of claims 34 to 42, wherein during the oscillating step, the liquid segment has a volume of about 2 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less.

[0162] Example 44: The method according to any one of claims 34 to 43, wherein introducing the sample liquid comprises moving the sample liquid along the microfluidic channel by capillary action until the distal sample liquid-gas interface contacts a capillary stop within the microfluidic channel.

[0163] Example 45: The method according to claim 44, wherein introducing the sample liquid comprises moving the sample liquid along the microfluidic channel by capillary action until the distal liquid-gas interface reaches and moves beyond the location where the separation gas will be introduced.

[0164] Example 46: The method according to claim 43 or 44, wherein the capillary stop comprises one or more vent holes that provide gas communication between the microfluidic channel and a volume of gas disposed outside the microfluidic channel.

[0165] Example 47: The method according to claim 46, wherein the volume of gas comprises ambient air surrounding the microfluidic device.

[0166] Example 48: The method according to any one of claims 34 to 47, wherein after introducing the sample liquid into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device, the chamber being isolated from the ambient gas surrounding the microfluidic device.

[0167] Example 49: The method according to any one of claims 34 to 45 or 48, wherein prior to introducing the liquid sample into the microfluidic channel, the microfluidic channel provides the only route for gas communication between the distal gas and the exterior of the microfluidic device.

[0168] Example 50: The method according to any one of claims 48 or 49, wherein after introducing the liquid sample into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device, the chamber being isolated from the ambient gas surrounding the microfluidic device.

[0169] Example 51: The method according to claim 46, wherein prior to introducing the liquid sample into the microfluidic channel, the microfluidic channel and the one or more vent holes provide the only path for gas communication between the distal gas and the exterior of the microfluidic device.

[0170] Example 52: The method according to any one of claims 48 to 51, wherein the ambient gas surrounding the microfluidic device is ambient air surrounding the microfluidic device.

[0171] Example 53: The method according to any one of claims 34 to 52, wherein oscillating the pressure of the distal gas comprises oscillating the internal spacing between a first inner wall and a second inner wall of a region of the microfluidic channel occupied by the distal gas, the region being distal to the liquid section.

[0172] Example 54: The method according to claim 53, wherein the region is the distal gas chamber of the microfluidic device, and the first inner wall and the second inner wall are the inner walls of the chamber.

[0173] Example 55: The method according to claim 54, wherein oscillating the internal spacing between the first wall and the second wall of the distal gas chamber includes oscillating the internal spacing at a location of the distal gas chamber spaced apart from the distal liquid-gas interface along the microfluidic channel.

[0174] Example 56: The method according to claim 55, wherein oscillating includes contacting the outer wall of the distal gas chamber with the oscillating member.

[0175] Example 57: The method according to claim 56, wherein the oscillating member contacts the outer wall of the distal gas chamber at a location spaced apart from the distal liquid-gas interface along the microfluidic channel by at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

[0176] Example 58: The method according to any one of claims 34 to 57, wherein after introducing a liquid sample into the microfluidic channel, the separation gas occupies the separation gas chamber of the microfluidic device, and the separation gas chamber is sealed relative to the ambient gas surrounding the microfluidic device.

[0177] Example 59: The method according to claim 58, wherein the ambient gas is air.

[0178] Example 60: The method according to claim 58 or 59, wherein introducing the separation gas includes heating the separation gas in the separation gas chamber.

[0179] Example 61: The method of any one of claims 34 to 60, wherein the step of introducing the separation gas includes increasing the pressure of the separation gas in the separation gas chamber.

[0180] Example 62: The method according to claim 61, wherein the method further includes reducing the pressure of the distal gas during at least a portion of the step of increasing the pressure of the separation gas in the separation gas chamber.

[0181] Example 63: The method according to any one of claims 34 to 62, wherein the method further includes operating the microfluidic device with an instrument, and wherein the method is performed without introducing any gas from the gas source of the instrument into the microfluidic channel.

[0182] Example 64: The method according to any one of claims 34 to 63, wherein the only gas present in the microfluidic device before introducing the sample liquid is ambient air, and during the execution of the method, the distal gas and the separation gas consist of ambient air present in the microfluidic device before introducing the sample liquid.

[0183] Example 65: The method according to any one of claims 34 to 64, wherein the microfluidic channel is a sample microfluidic channel, and the step of introducing the separation gas includes introducing the separation gas through a separation gas microfluidic channel that intersects the sample microfluidic channel at a location occupied by the sample liquid.

[0184] Example 66: The method according to one of claims 34 to 65, wherein the oscillating step includes simultaneously oscillating the pressures of the distal gas and the separation gas.

[0185] Example 67: The method according to claim 66, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas in phase with each other.

[0186] Example 68: The method according to claim 66, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas out of phase with each other.

[0187] Example 69: The method according to any one of claims 66 to 68, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas at the same frequency and / or different frequencies during at least a portion of the oscillation.

[0188] Example 70: The method according to any one of claims 34 to 69, wherein the step of introducing the separation gas into the microchannel displaces at least some of the liquid sample from the separation zone within the microfluidic channel and forms a separation bubble therein, the separation bubble being disposed between the liquid segment and the remaining volume of the sample liquid.

[0189] Example 71: The method according to claim 70, wherein the volume of the separation bubble is at least about 0.2 μL or greater, at least about 0.3 μL or greater, at least about 0.4 μL or greater, or at least about 0.5 μL or greater.

[0190] Example 72: The method according to claim 70, wherein the volume of the separation bubble is about 5 μL or less, about 3.5 μL or less, about 2.75 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less.

[0191] Example 73: The method according to claim 71 or 72, wherein the volume of the separated bubble defines the volume of the separated gas that separates the proximal gas-liquid interface of the liquid section from the gas-liquid interface ("RSVL gas-liquid interface") between the remaining volume of the sample liquid and the separated gas.

[0192] Example 74: The method according to any one of claims 70 to 73, wherein the separation zone of the microfluidic channel tapers distally from a larger cross-sectional area to a smaller cross-sectional area.

[0193] Example 75: The method according to any one of claims 70 to 74, wherein the proximal gas-liquid interface occupies a portion of the microfluidic channel having a cross-sectional area A1, wherein the proximal gas-liquid interface occupies a portion of the microchannel having a cross-sectional area A2, and wherein A2 is greater than A1 such that the separated bubble comprises an asymmetric shape.

[0194] Example 76: The method according to claim 75, wherein, when introducing the separated gas into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is less than the radius of curvature of the RSVL gas-liquid interface such that further introduction of the separated gas moves the RSVL gas-liquid interface proximally while keeping the proximal gas-liquid interface at the same position or substantially at the same position, such that the liquid section i) remains at the same position or substantially the same position within the microfluidic channel, and / or ii) has the same or substantially the same volume within the microfluidic channel.

[0195] Example 77: The method according to claim 76, wherein mixing a portion of the sample liquid with the at least one reagent comprises mixing the same or substantially the same volume with the at least one reagent to provide a decisive reagent concentration within the portion of the sample liquid.

[0196] Example 78: The method according to any one of claims 75 to 77, wherein the ratio of A2 to A1 ("R A ") is at least about 1.25.

[0197] Example 79: The method according to any one of claims 75 to 78, wherein the cross-sectional area A1 is between about 0.04 mm 2 and about 0.13 mm 2 .

[0198] Example 80: The method according to any one of claims 75 to 79, wherein the distance between the RSVL and proximal gas-liquid interfaces along the longitudinal axis of the microfluidic channel is between about 1 mm and 3 mm.

[0199] Embodiment 81: The method of any one of claims 74 to 80, wherein the cross-sectional area of ​​the microfluidic channel between the RSVL and the proximal air-liquid interface is about 12% mm -1 and about 25% mm -1 The average rate between them decreases.

[0200] Example 82: A method according to any one of claims 74 to 81, wherein the distance along the microfluidic channel between the RSVL gas-liquid interface and the location where the separation gas is introduced into the microfluidic channel is distance d1, wherein the distance along the microfluidic channel between the proximal gas-liquid interface and the location where the separation gas is introduced into the microfluidic channel is distance d2, and wherein the ratio d2 / d1 is between approximately 2.25 and 10.

[0201] Example 83: A method according to any one of claims 74 to 82, wherein the proximal gas-liquid interface is disposed in the separation zone, and wherein a portion of the sample liquid of the liquid segment is substantially disposed in an analysis zone of the microfluidic channel located distal to the separation zone, and the cross-sectional area A3 of the analysis zone is greater than the cross-sectional area A1.

[0202] Embodiment 84: The method according to any one of claims 34 to 83 further comprises analyzing a portion of the sample liquid to detect the presence or absence of a target therein.

[0203] Example 85: A method according to any one of claims 34 to 84, wherein at least one reagent includes a binding reagent capable of specifically binding to a target within a portion of the sample liquid, and wherein the portion of the sample liquid is mixed with the at least one reagent so that the binding reagent and the target can bind to detect and / or determine the amount of the binding reagent bound to the target.

[0204] Embodiment 86: The method according to any one of claims 34 to 85, wherein at least one reagent is disposed in a dry state within the microfluidic channel before the sample liquid is introduced into the microfluidic channel.

[0205] Embodiment 87: The method of claim 86, wherein at least one reagent is configured to dissolve with the sample liquid via contact with the sample liquid.

[0206] Example 88: The method according to any one of claims 34 to 87, wherein the microfluidic device further comprises one or more additional microfluidic channels, wherein each of the one or more additional microfluidic channels is configured to perform the method according to any one of claims 35 to 87 so as to individually analyze the sample liquid.

[0207] Example 89: The method according to claim 88, wherein two or more of the microfluidic channel and one or more additional microfluidic channels are configured to analyze the sample liquid simultaneously or sequentially.

[0208] Example 90: The method according to claim 88 or 89, wherein at least one of the one or more additional microfluidic channels includes a respective separation gas chamber configured to (i) introduce a respective separation gas into a respective additional microfluidic channel of the one or more additional microfluidic channels, and (ii) oscillate a respective pressure of the respective separation gas.

[0209] Example 91: The method according to any one of claims 88 to 90, wherein at least one of the one or more additional microfluidic channels includes a respective distal gas chamber, the respective distal gas chamber being configured to (i) reduce a pressure of the respective distal gas to move a respective liquid segment distally along a respective additional microfluidic channel of the one or more additional microfluidic channels, and (ii) oscillate a respective pressure of the respective distal gas.

[0210] Example 92: A method comprising: a) introducing a volume of sample liquid into a microfluidic channel within a microfluidic device, the microfluidic channel containing a distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal sample liquid-gas interface therebetween; b) introducing a separation gas into the microfluidic channel at a location occupied by the introduced volume of sample liquid, thereby separating a segment of the sample liquid from a remaining volume of the introduced volume of sample liquid, the liquid segment including (i) a distal liquid-gas interface and (ii) a proximal gas-liquid interface, wherein the gas of the proximal gas-liquid interface is the separation gas and the separation gas separates the proximal gas-liquid interface of the liquid segment from a liquid-gas interface of the remaining volume of the introduced volume of sample liquid; c) moving the liquid segment to a reagent zone of the microfluidic channel by reducing a pressure of the distal gas, the reagent zone including at least one reagent disposed therein; and d) mixing the sample liquid of the liquid segment with the at least one reagent by oscillating a pressure of the distal gas and / or a pressure of the separation gas, thereby forming a first mixture; wherein oscillating the pressure of the distal gas and / or the separation gas is performed (i) before, (ii) simultaneously with, and / or (iii) after reducing the pressure of the distal gas.

[0211] Example 93: A method for detecting at least one target material in a sample liquid, the method comprising: a) introducing a volume of the sample liquid into a microfluidic channel within a microfluidic device, the microfluidic channel containing a distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal sample liquid-gas interface therebetween; b) moving the sample liquid along the microfluidic channel until at least some of the sample liquid contacts at least one reagent disposed within a reagent region of the microfluidic channel; c) introducing a separation gas into the microfluidic channel at a location occupied by the volume of the sample liquid, thereby separating a segment of the sample liquid from the remaining volume of the introduced volume of the sample liquid, the liquid segment comprising (i) a distal liquid-gas interface, (ii) the sample liquid in contact with at least one reagent, and (iii) a proximal gas-liquid interface, wherein the gas of the proximal gas-liquid interface is the separation gas and the separation gas separates the proximal gas-liquid interface of the liquid segment from the liquid-gas interface of the remaining volume of the introduced volume of the sample liquid; d) mixing the sample liquid of the liquid segment with at least one reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas, thereby forming a first mixture; wherein oscillating the pressure of the distal gas and / or the pressure of the separation gas is performed i) before, ii) simultaneously with, and / or iii) after reducing the pressure of the distal gas.

[0212] Example 94: The method of any one of Examples 92 to 93, wherein oscillating the pressure of the distal gas and / or the pressure of the separation gas is performed at a frequency of about 2000 Hz or less, about 1500 Hz or less, about 1250 Hz or less, about 1000 Hz or less, about 900 Hz or less, about 800 Hz or less, about 5 Hz to about 2500 Hz or about 10 Hz to about 2000 Hz.

[0213] Example 95: The method of any one of Examples 92 to 94, wherein the oscillating step comprises oscillating the pressure of the distal gas, and during the oscillating step, the distal liquid-gas interface occupies a position of at least about 0.01 mm 2 、at least about 0.02 mm 2 、at least about 0.03 mm 2 、at least about 0.04 mm 2 、at least about 0.05 mm 2 、at least about 0.06 mm 2 or at least about 0.07 mm 2 of the cross-sectional area of the channel.

[0214] Example 96: The method of any one of Examples 92 to 95, wherein the oscillating step comprises oscillating the pressure of the distal gas, and during the oscillating step, the distal liquid-gas interface occupies a position of about 0.15 mm 2 or less, about 0.125 mm 2or less, about 0.1 mm 2 or less, about 0.09 mm 2 or less or about 0.08 mm 2 or at a position less than that.

[0215] Example 97: The method according to any one of Examples 92 to 96, wherein during the oscillation step, the liquid segment has a volume of at least about 0.2 μL or greater, at least about 0.3 μL or greater, at least about 0.4 μL or greater, or at least about 0.5 μL or greater.

[0216] Example 98: The method according to any one of Examples 92 to 97, wherein during the oscillation step, the liquid segment has a volume of about 2 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less.

[0217] Example 99: The method according to any one of Examples 92 to 98, wherein the step of introducing the volume of sample liquid comprises moving the sample liquid along the microfluidic channel by capillary action until the distal sample liquid - gas interface contacts a capillary stop within the microfluidic channel.

[0218] Example 100: The method according to any one of Examples 92 to 99, wherein the step of introducing the sample liquid comprises moving the sample liquid along the microfluidic channel by capillary action until the distal sample liquid - gas interface reaches and moves beyond the position where the separation gas is to be introduced.

[0219] Example 101: The method according to any one of Examples 92 to 100, wherein the capillary stop comprises one or more air vents that provide gas communication between the microfluidic channel and a volume of gas disposed outside the microfluidic channel.

[0220] Example 102: The method according to any one of Examples 92 to 101, wherein the volume of gas is ambient air around the microfluidic device.

[0221] Example 103: The method according to any one of Examples 92 to 102, wherein after introducing a volume of liquid sample into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device that is isolated from the ambient gas around the microfluidic device.

[0222] Example 104: The method according to any one of Examples 92 to 103, wherein before introducing a volume of liquid sample into the microfluidic channel, the microfluidic channel provides the only route for gas communication between the distal gas and the exterior of the microfluidic device.

[0223] Example 105: The method of any one of Examples 92 to 104, wherein after introducing a volume of a liquid sample into the microfluidic channel, a distal gas occupies a chamber of the microfluidic device, and the chamber is isolated from the ambient gas surrounding the microfluidic device.

[0224] Example 106: The method according to any one of Examples 92 to 105, wherein before introducing a volume of a liquid sample into the microfluidic channel, the microfluidic channel and one or more vent ports provide the only path for gas communication between the distal gas and the exterior of the microfluidic device.

[0225] Example 107: The method of any one of Examples 92 to 106, wherein the ambient gas surrounding the microfluidic device is ambient air surrounding the microfluidic device.

[0226] Example 108: The method of any one of Examples 92 to 107, wherein oscillating the pressure of the distal gas includes oscillating an internal spacing between a first inner wall and a second inner wall of a region of the microchannel occupied by the distal gas.

[0227] Example 109: The method of any one of Examples 92 to 108, wherein the region is a chamber of the microfluidic device, and the first inner wall and the second inner wall are inner walls of the chamber.

[0228] Example 110: The method of any one of Examples 92 to 109, wherein oscillating the internal spacing between the first wall and the second wall of the chamber includes oscillating the internal spacing at a location along the microchannel spaced apart from the distal liquid-gas interface.

[0229] Example 111: The method of any one of Examples 92 to 110, wherein oscillating includes bringing an outer wall of the chamber into contact with an oscillating member.

[0230] Example 112: The method of any one of Examples 92 to 111, wherein the oscillating member contacts the outer wall of the chamber at a location spaced apart from the distal liquid-gas interface along the microchannel by at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

[0231] Example 113: The method of any one of Examples 92 to 112, wherein after introducing the volume of the liquid sample into the microfluidic channel, a separation gas occupies a chamber of the microfluidic device sealed with respect to the ambient gas surrounding the microfluidic device.

[0232] Example 114: The method of any one of Examples 92 to 113, wherein the ambient gas is air.

[0233] Example 115: The method of any one of Examples 92 to 116, wherein introducing the separation gas includes heating the separation gas in the chamber occupied by the separation gas.

[0234] Example 116: The method of any one of Examples 92 to 115, wherein the step of introducing the separation gas includes increasing the pressure of the separation gas, and the method includes reducing the pressure of the distal gas during at least a portion of the step of increasing the pressure of the separation gas.

[0235] Example 117: The method of any one of Examples 92 to 116, wherein the method includes operating the microfluidic device with an instrument, and the method is performed without introducing any gas from a gas source of the instrument into the microfluidic channel.

[0236] Example 118: The method of any one of Examples 92 to 117, wherein the only gas present in the microfluidic device before introducing the sample liquid is ambient air, and during the execution of the method, the distal gas and the separation gas are composed of the ambient air present in the microfluidic device before introducing the sample liquid.

[0237] Example 119: The method as described in any one of Examples 92 to 118, wherein the microfluidic channel is a sample microfluidic channel, and the step of introducing the separation gas includes introducing the separation gas through a separation gas microfluidic channel that intersects the sample microfluidic channel at a location occupied by the sample liquid.

[0238] Example 120: The method of any one of Examples 92 to 119, wherein the oscillating step includes simultaneously oscillating the pressures of the distal gas and the separation gas.

[0239] Example 121: The method of any one of Examples 92 to 120, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas in phase with each other.

[0240] Example 122: The method of any one of Examples 92 to 121, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas out of phase with each other.

[0241] Example 123: The method of any one of Examples 92 to 122, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas at the same frequency and / or different frequencies during at least a portion of the oscillation.

[0242] Example 124: The method of any one of Examples 92 to 123, wherein the step of introducing the separation gas into the microchannel displaces at least some of the liquid sample from the separation zone within the microchannel, and the volume of the separation gas occupying the microchannel after introduction is at least about 0.2 μL or more, at least about 0.3 μL or more, at least about 0.4 μL or more, or at least about 0.5 μL or more.

[0243] Example 125: The method of any one of Examples 92 to 124, wherein the step of introducing the separation gas into the microchannel displaces at least some of the liquid sample from the separation zone within the microchannel, and the volume of the separation gas occupying the microchannel after introduction is about 5 μL or less, about 3.5 μL or less, about 2.75 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less.

[0244] Example 126: The method of any one of Examples 92 to 125, wherein the volume of the separation gas occupying the microchannel after introduction defines the volume of the separation gas that spaces the liquid-gas interface of the remaining volume of the sample liquid of the introduced volume from the proximal gas-liquid interface of the liquid segment.

[0245] Example 127: The method as described in any one of Examples 92 to 126, wherein at least one reagent comprises a binding reagent capable of specifically binding to a target, and the method further comprises binding the binding reagent to the target and detecting the amount of the binding reagent bound to the target.

[0246] Example 128: The method as described in any one of Examples 92 to 127, wherein at least one reagent is provided in a dry state within the microchannel prior to introduction of the volume of the sample liquid.

[0247] Example 129: A method comprising: preparing a liquid segment in contact with and / or comprising at least one reagent within a microchannel of a microfluidic device, wherein (i) the liquid segment defines a proximal gas-liquid interface and a distal liquid-gas interface, (ii) the gas at the proximal gas-liquid interface is a separation gas disposed between the liquid segment and the liquid-gas interface of an amount of liquid disposed within the microchannel adjacent to the liquid segment, and (iii) the gas at the distal liquid-gas interface is a distal gas disposed within the microfluidic channel remote from the liquid segment; and mixing the liquid of the liquid segment with the reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas to form a first mixture.

[0248] Example 130: The method of Example 129, wherein the amount of liquid disposed within the microchannel adjacent to the liquid segment is the remaining amount of liquid remaining from a first amount of liquid, and the method comprises, prior to the mixing step, introducing the first amount of liquid into the microchannel and separating the liquid segment from the remaining amount of liquid.

[0249] Example 131: The method of any one of Examples 129 to 130, wherein the step of separating the liquid segment from the remaining amount of liquid comprises introducing a separation gas into the microchannel through a separation gas microchannel that intersects the microfluidic channel at a location occupied by the first amount of liquid.

[0250] Example 132: The method of any one of Examples 129 to 131, wherein the step of introducing the sample liquid includes moving a first volume of liquid along the microfluidic channel by capillary action until at least some of the first volume of liquid reaches and moves past the position where the separation gas microfluidic channel intersects the microfluidic channel.

[0251] Example 133: The method of any one of Examples 129 to 132, wherein the step of introducing the first volume of liquid into the microchannel includes moving the first volume of liquid along the microfluidic channel by capillary action until the distal liquid-gas interface of the first volume of liquid contacts a capillary stop within the microfluidic channel.

[0252] Example 134: The method of any one of Examples 129 to 133, wherein the capillary stop includes one or more vent holes that provide gas communication between the microfluidic channel and a volume of gas disposed outside the microfluidic channel.

[0253] Example 135: The method of any one of Examples 129 to 134, wherein the volume of gas disposed outside the microfluidic channel is ambient air surrounding the microfluidic device.

[0254] Example 136: The method as described in any one of Examples 129 to 135, wherein prior to introducing a volume of liquid sample into the microfluidic channel, the microfluidic channel provides the only route for gas communication between the distal gas and the exterior of the microfluidic device.

[0255] Example 137: The method as described in any one of Examples 129 to 136, wherein after introducing a volume of liquid sample into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device, and the chamber is isolated from the ambient gas surrounding the microfluidic device.

[0256] Example 138: The method as described in any one of Examples 129 to 137, wherein prior to introducing a volume of liquid sample into the microfluidic channel, the microfluidic channel and one or more vent holes provide the only route for gas communication between the distal gas and the exterior of the microfluidic device.

[0257] Example 139: A method, comprising: disposing a liquid in a microchannel of a microfluidic device; forming a bubble within the liquid in the microchannel, thereby separating the liquid into a first portion and a second portion, wherein a first gas-liquid interface between the bubble and the first portion of the liquid occupies a portion of the microchannel having a cross-sectional area A1, and a second gas-liquid interface between the bubble and the second portion of the liquid occupies a portion of the microchannel having a cross-sectional area A2, and the ratio A2 / A1 = R Ais at least about 1.25, such as between about 1.5 and 3.5, such as about 2.

[0258] Example 140: The method as described in Example 139, wherein the cross-sectional area A1 is between about 0.04 mm 2 and about 0.13 mm 2 , such as about 0.09 mm 2 , and the second cross-sectional area A2 is equal to R A ×A1.

[0259] Example 141: The method of any one of Examples 139 to 140, wherein the distance along the longitudinal axis of the microchannel between the first and second gas-liquid interfaces is between about 1 and 3 mm, such as about 2 mm.

[0260] Example 142: The method of any one of Examples 139 to 141, wherein the volume of the bubble disposed within the microchannel between the first and second gas-liquid interfaces is between about 200 nL and about 750 nL, such as about 350 nL.

[0261] Example 143: The method of any one of Examples 139 to 142, wherein along the longitudinal axis of the microchannel, advancing from the position of the first gas-liquid interface to the position of the second gas-liquid interface, the cross-sectional area of the first microchannel gradually decreases from the first area to the second smaller area.

[0262] Example 144: The method of any one of Examples 139 to 143, wherein the cross-sectional area of the microchannel between the first and second gas-liquid interfaces decreases at an average rate of about 12% mm -1 to about 25% mm -1 , such as about 17.5% mm -1 .

[0263] Example 145: The method of any one of Examples 139 to 144, wherein the microchannel has an internal height h1 and an internal width w1 at the position where the microchannel is occupied by the first gas-liquid interface, wherein w1 > h1.

[0264] Example 146: The method of any one of Examples 139 to 145, wherein the microchannel has an internal height h2 and an internal width w2 at the position occupied by the second gas-liquid interface, wherein w2 > w1 > h2.

[0265] Example 147: The method of any one of Examples 139 to 146, wherein the ratio w2 / w1 is substantially the same as the ratio R A of the second cross-sectional area to the first cross-sectional area.

[0266] Example 148: The method according to any one of Examples 139 to 147, wherein h1 and h2 are substantially the same, for example, substantially identical, for example, h1 and h2 can each be between about 50 μm and about 200 μm, for example, about 110 μm.

[0267] Example 149: The method according to any one of Examples 139 to 148, wherein w1 is between about 400 μm and 1200 μm, for example, about 800 μm.

[0268] Example 150: The method according to any one of Examples 139 to 149, wherein w2 is about R A ×w1 μm.

[0269] Example 151: The method according to any one of Examples 139 to 150, wherein w1 is about 800 μm and w2 is about 1600 μm.

[0270] Example 152: The method of any one of Examples 139 to 151, wherein the first portion of the liquid includes a distal gas-liquid interface disposed distal to the first gas-liquid interface within the microchannel.

[0271] Example 153: The method of any one of Examples 139 to 152, wherein the gas of the distal gas-liquid interface is enclosed within the distal portion of the microchannel of the microfluidic device.

[0272] Example 154: The method of any one of Examples 139 to 153, wherein the second portion of the liquid includes a proximal gas-liquid interface, wherein the gas of the gas-liquid interface is the gas of the ambient atmosphere surrounding the microfluidic device, such as air.

[0273] Example 155: The method of any one of Examples 139 to 154, wherein the microchannel is a first microchannel, and the step of forming the bubble includes introducing the gas of the bubble from a second microchannel that intersects the first microchannel at a gas introduction opening occupied by the liquid.

[0274] Example 156: The method of any one of Examples 139 to 155, wherein the distance along the microchannel between the first gas-liquid interface and the gas introduction opening is distance d1, the distance along the microchannel between the second gas-liquid interface and the gas introduction location is distance d2, and the ratio d2 / d1 is at least about 2.25, for example, between about 2.25 and 10, for example, about 4.5.

[0275] Example 157: The method of any one of Examples 139 to 156, wherein disposing the liquid in the microchannel includes, before forming the bubble, introducing the liquid into the application region of the microchannel and causing the liquid to flow along the microchannel by capillary action until the distal gas-liquid interface of the liquid reaches a capillary stopper located distal to the gas introduction opening within the microchannel.

[0276] Example 158: The method of any one of Examples 139 to 157, wherein causing the liquid to flow by capillary action includes causing the distal gas-liquid interface of the liquid to flow along the distal portion of the microchannel disposed distal to the gas introduction opening, and wherein the width w3 of the distal portion of the microchannel is greater than the width w1.

[0277] Example 159: The method as described in any one of Examples 139 to 158, wherein the ratio w3 / w1 is between about 2 and about 6, such as about 3.

[0278] Example 160: The method of any one of Examples 139 to 159, wherein the distal portion of the microchannel has a cross-sectional area A3, and wherein the ratio A3 / A1 is between about 2 and about 6, such as about 3.

[0279] Example 161: The method as described in any one of Examples 139 to 160, wherein the distal portion of the microchannel has a height h3, wherein h3, h2, and h1 are substantially the same, such as essentially the same, such as h3, h2, and h1 can each be between about 50 μm and about 200 μm, such as about 110 μm.

[0280] Example 162: The method of any one of Examples 139 to 161, wherein the step of forming the bubble includes introducing an initial amount of the gas of the bubble into the microchannel through the gas introduction opening, thereby forming an initial bubble having first and second gas-liquid interfaces, wherein the distance along the microchannel between the first gas-liquid interface of the initial bubble and the gas introduction opening is distance d1', the distance along the microchannel between the second gas-liquid interface of the initial bubble and the gas introduction position is distance d2', and the ratio d2' / d1' is about 1.

[0281] Example 163: The method of any one of Examples 139 to 162, wherein the step of forming the bubble includes introducing additional gas into the initial bubble through the gas introduction opening such that the distance along the microchannel between the second gas-liquid interface of the bubble and the gas introduction opening increases relative to the distance along the microchannel between the first gas-liquid interface of the bubble and the gas introduction opening until the distance along the microchannel between the second gas-liquid interface of the bubble and the gas introduction opening is d2 and the distance along the microchannel between the first gas-liquid interface of the bubble and the gas introduction opening is d1.

[0282] Example 164: For any one of Examples 1 to 163, wherein the liquid comprises a sample liquid obtained from a mammal such as a human.

[0283] Example 165: For any one of Examples 1 to 164, wherein the sample liquid comprises blood, serum, plasma, saliva, urine, sputum, or material obtained from a swab such as a nasopharyngeal swab.

[0284] Example 166: For any one of Examples 1 to 165, wherein the liquid is a mixture comprising the sample liquid and an additional liquid such as a buffer.

[0285] Example 167: For any one of Examples 1 to 166, wherein the liquid of the first portion of the liquid comprises a reagent configured to facilitate determination of one or more targets present in the first portion of the liquid.

[0286] Example 168: For any one of Examples 1 to 167, wherein the method comprises contacting the liquid of the first portion of the liquid with the reagent after the step of forming the bubbles.

[0287] Example 169: For any one of Examples 1 to 168, wherein the method comprises contacting the liquid of the first portion of the liquid with the reagent before the step of forming the bubbles.

[0288] Example 170: For any one of Examples 1 to 169, wherein the method comprises contacting the liquid of the first portion of the liquid with a different additional reagent after the step of forming the bubbles.

[0289] Example 171: For any one of Examples 1 to 170, wherein the reagent contacted before the step of forming the bubbles is a reagent configured to inhibit blood sample coagulation, such as a reagent comprising heparin.

[0290] Example 172: For any one of Examples 1 to 171, wherein the different additional reagent comprises one or more reagents configured to bind to the target.

[0291] Example 173: For any one of Examples 1 to 172, wherein the different additional reagent comprises one or more different microparticles, the microparticles comprising a binding agent for the target, such as an antibody.

[0292] Example 174: For any one of Examples 1 to 173, wherein the microparticles comprise magnetic microparticles and fluorescent microparticles, and the microparticles are configured to form a detectable sandwich with the target.

Claims

1. A microfluidic device, comprising: a. A generally planar substrate including a microfluidic network therein, the microfluidic network including: i. A distal gas chamber configured to regulate the pressure of distal gas within at least a portion of the microfluidic network; ii. A microfluidic channel extending distally from an application zone to the distal gas chamber, the application zone configured to receive a sample liquid therein; and iii. A separation gas chamber in communication with the microfluidic channel via a separation gas channel that intersects the microfluidic channel at a separation gas introduction location, the separation gas chamber configured to regulate the pressure of separation gas within at least a portion of the microfluidic network; wherein the sample liquid is configured to flow from the application zone through at least a portion of the microfluidic channel to a capillary stop located distal to the separation gas introduction location such that after the separation gas is introduced into the microfluidic channel, a separation bubble is formed within the microfluidic channel, the separation bubble separating the sample liquid therein into i) a liquid segment located distal to the separation bubble, and ii) a remaining volume of the sample liquid located proximal to the separation bubble, the liquid segment forming i) a distal liquid-gas interface disposed between the distal gas and the liquid segment, and ii) a proximal liquid-gas interface disposed between the separation gas and the liquid segment.

2. The microfluidic device according to claim 1, wherein the microfluidic channel comprises a reagent region, the reagent region being distal to the separation gas introduction position and configured to comprise one or more reagents.

3. The microfluidic device according to claim 2, wherein the reagent is configured to dissolve with the sample liquid when in contact with the sample liquid.

4. The microfluidic device according to claim 2 or 3, wherein the reagent is configured to enable detection of a target in the sample liquid when in contact with the sample liquid.

5. The microfluidic device according to claim 4, wherein the microfluidic channel comprises a detection region, the detection region being distal to the reagent region and configured to detect the presence or absence of the target in the sample liquid.

6. The microfluidic device according to any one of claims 1 to 5, wherein the distal gas chamber comprises a first inner wall, a second inner wall, and a distal chamber spacing between the first inner wall and the second inner wall, the distal chamber spacing being configured to be occupied by the distal gas.

7. The microfluidic device according to claim 6, wherein an outer wall of the distal gas chamber is configured to contact an oscillating member so as to oscillate and / or adjust the distal chamber spacing, thereby oscillating and / or adjusting the pressure of the distal gas.

8. The microfluidic device according to claim 7, wherein adjusting the pressure of the distal gas promotes movement of the liquid segment within the microfluidic channel.

9. The microfluidic device according to claim 7, wherein the oscillating member is configured to contact the outer wall of the distal gas chamber at a position spaced apart from the distal liquid-gas interface along the microfluidic channel by at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

10. The microfluidic device according to any one of claims 1 to 9, wherein the separation gas chamber is configured to heat the separation gas therein so as to pressurize the separation gas and promote its introduction into the microfluidic channel to form the separation bubble.

11. The microfluidic device according to any one of claims 1 to 9, wherein the separation gas chamber includes a third inner wall, a fourth inner wall, and a separation gas chamber spacing therebetween, the separation gas chamber spacing being configured to be occupied by the separation gas.

12. The microfluidic device according to claim 11, wherein an outer wall of the separation gas chamber is configured to contact a second oscillating member so as to oscillate and / or adjust the separation gas chamber spacing, thereby oscillating and / or adjusting the pressure of the separation gas.

13. The microfluidic device according to any one of claims 1 to 13, wherein the separation bubble defines a volume of the separation gas disposed between a proximal gas-liquid interface and a gas-liquid interface (RVSL gas-liquid interface) between a remaining volume of the sample liquid and the separation bubble, the separation bubble being disposed in a separation zone of the microfluidic channel.

14. The microfluidic device according to claim 13, wherein a separation zone of the microfluidic channel tapers distally from a larger cross-sectional area to a smaller cross-sectional area.

15. The microfluidic device according to claim 13 or 14, wherein the proximal gas-liquid interface occupies a portion of the microfluidic channel having a cross-sectional area A1, wherein the proximal gas-liquid interface occupies a portion of the microchannel having a cross-sectional area A2, and wherein A2 is greater than A1 such that the separation bubble includes an asymmetrical shape.

16. The microfluidic device according to claim 15, wherein when the separation gas is introduced into the microfluidic channel, a radius of curvature of the proximal gas-liquid interface is less than a radius of curvature of the RVSL gas-liquid interface such that further introduction of the separation gas moves the RVSL gas-liquid interface proximally while keeping the proximal gas-liquid interface at the same position or substantially at the same position within the microfluidic channel, such that the liquid segment i) remains at the same position or substantially at the same position within the microfluidic channel, and / or ii) has the same or substantially the same volume amount within the microfluidic channel.

17. The microfluidic device according to claim 15 or 16, wherein a ratio of A2 to A1 (“R A ”) is at least about 1.

25.

18. The microfluidic device according to any one of claims 15 to 17, wherein the cross-sectional area A1 is between about 0.04 mm 2 and about 0.13 mm 2 .

19. The microfluidic device according to any one of claims 15 to 18, wherein a distance between the RSVL and proximal gas-liquid interfaces along a longitudinal axis of the microfluidic channel is between about 1 mm and 3 mm.

20. The microfluidic device according to any one of claims 14 to 19, wherein the cross-sectional area of the microfluidic channel between the RSVL and the proximal gas-liquid interface decreases at an average rate between about 12% mm -1 and about 25% mm -1 .

21. The microfluidic device according to any one of claims 14 to 20, wherein the distance along the microfluidic channel between the proximal gas-liquid interface and the separated gas introduction position is distance d1, wherein the distance along the microfluidic channel between the proximal gas-liquid interface and the separated gas introduction position is distance d2, and wherein the ratio d2 / d1 is between about 2.25 and 10.

22. The microfluidic device according to any one of claims 14 to 21, wherein the proximal gas-liquid interface is disposed in the separation zone, and wherein the sample liquid of the liquid segment is substantially disposed in a reagent zone or a detection zone of the microfluidic channel distal to the separation zone, the reagent zone or the detection zone having a cross-sectional area A3 greater than the cross-sectional area A1.

23. The microfluidic device according to any one of claims 1 to 22, wherein the substantially planar substrate comprises an upper layer, a lower layer, and an intermediate layer disposed between the upper layer and the lower layer.

24. The microfluidic device according to claim 23, wherein the intermediate layer comprises an upper surface having an adhesive adhered to the upper layer, a lower surface having an adhesive adhered to the lower layer, or both.

25. The microfluidic device according to claim 23 or 24, wherein the upper surface of the lower layer defines the lower inner surface of the microfluidic channel and the application zone.

26. The microfluidic device according to any one of claims 23 to 25, wherein the lower surface of the upper layer defines the upper inner surface of the microfluidic channel.

27. The microfluidic device according to any one of claims 23 to 26, wherein the upper layer, the lower layer, the intermediate layer, or any combination thereof comprises a hole defining the application zone.

28. The microfluidic device according to any one of claims 23 to 27, further comprising a porous membrane configured to cover the application zone.

29. The microfluidic device according to claim 28, wherein the porous membrane is configured to separate the sample liquid from one or more microparticles.

30. The microfluidic device according to any one of claims 23 to 29, further comprising a protrusion extending from the upper layer and / or the intermediate layer into the application zone.

31. The microfluidic device according to any one of claims 1 to 30, wherein the microfluidic device further comprises one or more additional microfluidic networks, wherein each of the one or more additional microfluidic networks is configured to independently receive a portion of the sample liquid from the application zone and analyze the sample liquid to detect a target therein.

32. The microfluidic device according to claim 31, wherein two or more of the microfluidic channels and the one or more additional microfluidic channels are configured to analyze the sample liquid simultaneously or sequentially.

33. The microfluidic device according to claim 31 or 32, wherein at least one of the one or more additional microfluidic channels is configured in the same manner as the microfluidic network according to any one of claims 1 to 30.

34. A method comprising: a) Introduce a sample liquid into a microfluidic channel within a microfluidic device, the microfluidic channel containing distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal liquid-gas interface therebetween; b) Introduce a separation gas into the microfluidic channel at a location occupied by the sample liquid, thereby separating a segment of the sample liquid from a remaining volume of the sample liquid introduced into the microfluidic channel, the liquid segment including (i) a distal liquid-gas interface, (ii) a portion of the sample liquid introduced into the microfluidic channel, and (iii) a proximal liquid-gas interface between the separation gas and the portion of the sample liquid, wherein the separation gas separates the proximal liquid-gas interface of the liquid segment from the remaining volume of the sample liquid; c) Move the liquid segment to a reagent zone of the microfluidic channel by reducing the pressure of the distal gas, the reagent zone including at least one reagent disposed therein; and d) Mix a portion of the sample liquid of the liquid segment with the at least one reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas, thereby forming a first mixture; wherein oscillating the pressure of the distal gas and / or the separation gas is performed i) before, ii) simultaneously with, and / or iii) after reducing the pressure of the distal gas.

35. A method for analyzing a sample liquid to detect at least one target material therein, the method comprising: a) Introduce the sample liquid into a microfluidic channel within a microfluidic device, the microfluidic channel containing distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal liquid-gas interface therebetween; b) Move the sample liquid along the microfluidic channel until at least some of the sample liquid contacts at least one reagent disposed within a reagent zone of the microfluidic channel; c) introducing a separation gas into the microfluidic channel at a location occupied by the sample liquid, thereby separating a liquid segment of the sample liquid from the remaining volume of the sample liquid introduced into the microfluidic channel, the liquid segment including (i) a distal liquid-gas interface, (ii) a portion of the sample liquid in contact with at least one reagent, and (iii) a proximal gas-liquid interface disposed between the separation gas and the portion of the sample liquid, wherein the separation gas separates the proximal gas-liquid interface of the liquid segment from the remaining volume of the sample liquid; and d) mixing the portion of the sample liquid of the liquid segment with the at least one reagent by oscillating the pressure of the distal gas and / or the pressure of the separation gas, thereby forming a first mixture.

36. The method according to claim 35, wherein moving the sample liquid is via capillary action.

37. The method according to claim 35, wherein moving the sample liquid is via reducing the pressure of the distal gas.

38. The method according to claim 37, wherein oscillating the pressure of the distal gas and / or the separation gas is performed i) before, ii) simultaneously with, and / or iii) after reducing the pressure of the distal gas.

39. The method according to any one of claims 34 to 38, wherein oscillating the pressure of the distal gas and / or the separation gas is performed at a frequency of about 2000 Hz or less, about 1500 Hz or less, about 1250 Hz or less, about 1000 Hz or less, about 900 Hz or less, about 800 Hz or less, about 5 Hz to about 2500 Hz or about 10 Hz to about 2000 Hz.

40. The method according to any one of claims 34 to 39, wherein the oscillating step comprises oscillating the pressure of the distal gas, and during the oscillating step, the distal liquid-gas interface occupies a position in the microfluidic channel having a cross-sectional area of at least about 0.01 mm 2 , at least about 0.02 mm 2 , at least about 0.03 mm 2 , at least about 0.04 mm 2 , at least about 0.05 mm 2 , at least about 0.06 mm 2 or at least about 0.07 mm 2 .

41. The method according to any one of claims 34 to 40, wherein the oscillating step comprises oscillating the pressure of the distal gas, and during the oscillating step, the distal liquid-gas interface occupies a position in the channel having a cross-sectional area of about 0.15 mm 2 or less, about 0.125 mm 2 or less, about 0.1 mm 2 or less, about 0.09 mm 2 or less or about 0.08 mm 2 or less.

42. The method according to any one of claims 34 to 41, wherein during the oscillating step, the liquid segment has a volume of at least about 0.2 μL or greater, at least about 0.3 μL or greater, at least about 0.4 μL or greater, or at least about 0.5 μL or greater.

43. The method according to any one of claims 34 to 42, wherein during the oscillating step, the liquid segment has a volume of about 2 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less.

44. The method according to any one of claims 34 to 43, wherein introducing the sample liquid comprises moving the sample liquid along the microfluidic channel by capillary action until the distal sample liquid-gas interface contacts a capillary stop within the microfluidic channel.

45. The method according to claim 44, wherein introducing the sample liquid comprises moving the sample liquid along the microfluidic channel by capillary action until the distal liquid-gas interface reaches and moves beyond the position where the separation gas will be introduced.

46. The method according to claim 43 or 44, wherein the capillary stop comprises one or more vent holes that provide gas communication between the microfluidic channel and a volume of gas disposed outside the microfluidic channel.

47. The method according to claim 46, wherein the volume of gas comprises ambient air surrounding the microfluidic device.

48. The method according to any one of claims 34 to 47, wherein after introducing the sample liquid into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device, the chamber being isolated from the ambient gas surrounding the microfluidic device.

49. The method according to any one of claims 34 to 45 or 48, wherein before introducing the liquid sample into the microfluidic channel, the microfluidic channel provides the only route for gas communication between the distal gas and the exterior of the microfluidic device.

50. The method according to any one of claims 48 or 49, wherein after introducing the liquid sample into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device, the chamber being isolated from the ambient gas surrounding the microfluidic device.

51. The method according to claim 46, wherein before introducing the liquid sample into the microfluidic channel, the microfluidic channel and the one or more vent openings provide the only route for gas communication between the distal gas and the exterior of the microfluidic device.

52. The method according to any one of claims 48 to 51, wherein the ambient gas surrounding the microfluidic device is ambient air surrounding the microfluidic device.

53. The method according to any one of claims 34 to 52, wherein oscillating the pressure of the distal gas comprises oscillating the internal spacing between a first inner wall and a second inner wall of a region of the microfluidic channel occupied by the distal gas, the region being distal to the liquid segment.

54. The method according to claim 53, wherein the region is a distal gas chamber of the microfluidic device, and the first inner wall and the second inner wall are inner walls of the chamber.

55. The method according to claim 54, wherein oscillating the internal spacing between the first wall and the second wall of the distal gas chamber comprises oscillating the internal spacing at a position of the distal gas chamber spaced apart from the distal liquid-gas interface along the microfluidic channel.

56. The method according to claim 55, wherein the oscillation comprises contacting an outer wall of the distal gas chamber with an oscillating member.

57. The method according to claim 56, wherein the oscillating member contacts the outer wall of the distal gas chamber at a position spaced apart from the distal liquid-gas interface along the microfluidic channel by at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

58. The method according to any one of claims 34 to 57, wherein after introducing the liquid sample into the microfluidic channel, the separation gas occupies a separation gas chamber of the microfluidic device that is sealed with respect to the ambient gas surrounding the microfluidic device.

59. The method according to claim 58, wherein the ambient gas is air.

60. The method according to claim 58 or 59, wherein introducing the separation gas includes heating the separation gas in the separation gas chamber.

61. The method according to any one of claims 34 to 60, wherein the step of introducing the separation gas includes increasing the pressure of the separation gas in the separation gas chamber.

62. The method according to claim 61, wherein the method further includes reducing the pressure of the distal gas during at least a portion of the step of increasing the pressure of the separation gas in the separation gas chamber.

63. The method according to any one of claims 34 to 62, wherein the method further includes operating the microfluidic device with an instrument, and wherein the method is performed without introducing any gas from a gas source of the instrument into the microfluidic channel.

64. The method according to any one of claims 34 to 63, wherein the only gas present in the microfluidic device before introducing the sample liquid is ambient air, and during performance of the method, the distal gas and the separation gas are composed of ambient air that was present in the microfluidic device before introducing the sample liquid.

65. The method according to any one of claims 34 to 64, wherein the microfluidic channel is a sample microfluidic channel, and the step of introducing the separation gas includes introducing the separation gas through a separation gas microfluidic channel that intersects the sample microfluidic channel at a location occupied by the sample liquid.

66. The method according to any one of claims 34 to 65, wherein the oscillating step includes simultaneously oscillating the pressures of the distal gas and the separation gas.

67. The method according to claim 66, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas in phase with each other.

68. The method according to claim 66, wherein the oscillating step includes oscillating the pressures of the distal gas and the separation gas out of phase with each other.

69. The method according to any one of claims 66 to 68, wherein the oscillating step comprises oscillating the pressure of the distal gas and the separated gas at the same frequency and / or different frequencies during at least a portion of the oscillation.

70. The method according to any one of claims 34 to 69, wherein the step of introducing the separated gas into the microchannel displaces at least some of the liquid sample from a separation zone within the microfluidic channel and forms a separation bubble therein, the separation bubble being disposed between the liquid segment and the remaining volume of the sample liquid.

71. The method according to claim 70, wherein the volume of the separation bubble is at least about 0.2 μL or greater, at least about 0.3 μL or greater, at least about 0.4 μL or greater, or at least about 0.5 μL or greater.

72. The method according to claim 70, wherein the volume of the separation bubble is about 5 μL or less, about 3.5 μL or less, about 2.75 μL or less, about 1.75 μL or less, about 1.5 μL or less, about 1.25 μL or less, about 1 μL or less, about 0.75 μL or less, or about 0.5 μL or less.

73. The method according to claim 71 or 72, wherein, The volume of the separation bubble defines the volume of the separation gas, and the volume of the separation gas separates the proximal gas-liquid interface of the liquid segment from the gas-liquid interface ("RSVL gas-liquid interface") between the remaining volume of the sample liquid and the separation gas.

74. The method according to any one of claims 70 to 73, wherein the separation zone of the microfluidic channel tapers distally from a larger cross-sectional area to a smaller cross-sectional area.

75. The method according to any one of claims 70 to 74, wherein the proximal gas-liquid interface occupies a portion of the microfluidic channel having a cross-sectional area A1, wherein the proximal gas-liquid interface occupies a portion of the microchannel having a cross-sectional area A2, and wherein A2 is greater than A1 such that the separation bubble comprises an asymmetric shape.

76. The method according to claim 75, wherein when the separated gas is introduced into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is less than the radius of curvature of the RVSL gas-liquid interface such that further introduction of the separated gas moves the RVSL gas-liquid interface proximally while keeping the proximal gas-liquid interface at the same position or substantially at the same position such that the liquid segment i) remains at the same position or substantially the same position within the microfluidic channel, and / or ii) has the same or substantially the same volume amount within the microfluidic channel.

77. The method according to claim 76, wherein mixing a portion of the sample liquid with the at least one reagent comprises mixing the same or substantially the same volume amount with the at least one reagent so as to provide a decisive reagent concentration within the portion of the sample liquid.

78. The method according to any one of claims 75 to 77, wherein the ratio of A2 to A1 (“R A ”) is at least about 1.

25.

79. The method according to any one of claims 75 to 78, wherein the cross-sectional area A1 is between about 0.04 mm 2 and about 0.13 mm 2 .

80. The method according to any one of claims 75 to 79, wherein the distance between the RSVL and the proximal gas-liquid interface along the longitudinal axis of the microfluidic channel is between about 1 mm and 3 mm.

81. The method according to any one of claims 74 to 80, wherein the cross-sectional area of the microfluidic channel between the RSVL and the proximal gas-liquid interface decreases at an average rate between about 12% mm -1 and about 25% mm -1 .

82. The method according to any one of claims 74 to 81, wherein the distance along the microfluidic channel between the RSVL gas-liquid interface and the location where the separated gas is introduced into the microfluidic channel is distance d1, wherein the distance along the microfluidic channel between the proximal gas-liquid interface and the location where the separated gas is introduced into the microfluidic channel is distance d2, and wherein the ratio d2 / d1 is between about 2.25 and 10.

83. The method according to any one of claims 74 to 82, wherein the proximal gas-liquid interface is disposed in the separation zone, and wherein a portion of the sample liquid of the liquid segment is substantially disposed in the analysis zone distal to the separation zone of the microfluidic channel, and the cross-sectional area A3 of the analysis zone is greater than the cross-sectional area A1.

84. The method according to any one of claims 34 to 83, further comprising analyzing the portion of the sample liquid to detect the presence or absence of a target therein.

85. The method according to any one of claims 34 to 84, wherein the at least one reagent comprises a binding reagent capable of specifically binding to a target in the portion of the sample liquid, and wherein the portion of the sample liquid is mixed with the at least one reagent such that the binding reagent and the target can bind to detect and / or determine the amount of the binding reagent bound to the target.

86. The method according to any one of claims 34 to 85, wherein the at least one reagent is disposed in a dry state within the microfluidic channel before the sample liquid is introduced into the microfluidic channel.

87. The method according to claim 86, wherein the at least one reagent is configured to dissolve with the sample liquid upon contact therewith.

88. The method according to any one of claims 34 to 87, wherein the microfluidic device further comprises one or more additional microfluidic channels, each of the one or more additional microfluidic channels being configured to perform the method according to any one of claims 35 to 87 for separately analyzing the sample liquid.

89. The method according to claim 88, wherein two or more of the microfluidic channels and the one or more additional microfluidic channels are configured to analyze the sample liquid simultaneously or sequentially.

90. The method according to claim 88 or 89, wherein at least one of the one or more additional microfluidic channels comprises a respective separation gas chamber configured to: i) introduce a respective separation gas into a respective one of the one or more additional microfluidic channels, and ii) oscillate a pressure of the respective separation gas.

91. The method according to any one of claims 88 to 90, wherein at least one of the one or more additional microfluidic channels comprises a respective distal gas chamber configured to: i) reduce a pressure of a respective distal gas to move a respective liquid segment distally along a respective one of the one or more additional microfluidic channels, and ii) oscillate a pressure of the respective distal gas.

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