Segmented Fluid Mixing

Abstract

Mixing a liquid sample and at least one reagent in a microchannel of a microfluidic device includes preparing a segment of the liquid sample in contact with the reagent in the microchannel. The liquid segment has a volume of 2 μL or less and defines a proximal gas-liquid interface and a distal liquid-gas interface. The gas at the proximal gas-liquid interface is in gas communication with a distal liquid-gas interface of another volume of liquid disposed proximal to the liquid segment in the microchannel of the microfluidic channel. The gas at the distal liquid-gas interface is disposed distal to the liquid segment in the microfluidic channel. With the liquid in the liquid segment in contact with the reagent in the microchannel, the gas at the distal liquid-gas interface and / or the gas at the proximal gas-liquid interface is vibrated at an acoustic frequency to induce mixing of the liquid and the reagent.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method and system for mixing liquids and reagents in microfluidic channels.

[0002] Related Applications This application claims the benefit of and priority to U.S. Patent Application No. 63 / 414,667, entitled "Segmented Fluidic Mixing," filed October 10, 2022, and U.S. Patent Application No. 63 / 441,114, entitled "Segmented Fluidic Mixing," filed January 25, 2023, each of which is incorporated herein by reference in its entirety. This application is further related to International Patent Application No. PCT / GB2023 / 050189, entitled "Microfluidic Devices," filed January 27, 2023 (the "'189 Application"), and International Patent Application No. PCT / US2021 / 013325, entitled "Fluid Control in Microfluidic Devices," filed January 13, 2021 (the "'325 Application"), each of which is incorporated herein by reference in its entirety. [Background technology]

[0003] background Microfluidic devices may be used to perform testing of targets present in the liquid sample by combining the liquid sample with reagents disposed within the microchannels of the device. Mixing materials, such as liquid samples and reagents, within the microchannels of a microfluidic device poses challenges not often faced with macroscopic quantities. For example, it can be difficult to achieve efficient mixing within microchannels where slow diffusion processes may dominate. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention In embodiments, a method includes preparing a liquid segment in contact with and / or containing at least one reagent in a microchannel of a microfluidic device, where (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 a liquid-gas interface of a quantity of liquid disposed in the microchannel proximal to the liquid segment, and (iii) the gas at the distal liquid-gas interface is a distal gas disposed in the microfluidic channel distal to the liquid segment. The method may further 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, thereby forming a first mixture.

[0005] The oscillating may comprise oscillating the pressure of the distal gas. The oscillating may comprise oscillating the pressure of the separation gas. Alternatively, the oscillating may include simultaneously oscillating the pressures of both the distal gas and the separation gas, and the oscillating step includes oscillating the pressures of the distal gas and the separation gas in phase with each other. The pressures of the distal gas and the separation gas may be oscillated out of phase with each other or in phase with each other. The oscillating of the pressures of the distal gas and the separation gas may be performed at the same frequency and / or at different frequencies during at least a portion of the oscillation.

[0006] The pressure oscillations of the distal gas and / or separation 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 oscillations of the distal gas and / or separation 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.

[0007] The oscillation of the pressure of the distal gas may be performed by oscillating an internal spacing between a first internal wall of the region of the microchannel occupied by the distal gas and a second internal wall of the region. The region may be a chamber in gas communication with a distal liquid-gas interface in the microchannel. The oscillation region may be spaced along the microchannel from the distal liquid-gas interface of the liquid segment. The oscillation of the pressure of the separation gas may be performed by oscillating an internal spacing between a first internal wall of the region of the microchannel and a second internal wall of the region occupied by the separation gas. The region may be a chamber in gas communication with a proximal gas-liquid interface in the microchannel. The oscillation region may be spaced along the microchannel from the proximal gas-liquid interface of the liquid segment. The separation gas and the distal gas are spaced and isolated from each other by the liquid segment.

[0008] During the vibration step, the distal liquid-air interface and / or the proximal air-liquid interface of the liquid segment may be 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 vibration step, the distal liquid-air interface and / or the proximal air-liquid interface may occupy a position in a channel having a cross-sectional area of ​​about 0.15 mm. 2 Below, approximately 0.125mm 2 Below, approximately 0.1mm 2 Below, approximately 0.09mm 2 Less than or equal to 0.08 mm 2 During the vibration step, the distal liquid-air interface and / or the proximal air-liquid interface may occupy a position in a channel having a cross-sectional area of ​​about 0.01 mm. 2 ~about 0.15mm 2 , for example, about 0.01 mm 2 ~about 0.125mm 2 , approximately 0.01 mm 2 ~about 0.1mm 2 , approximately 0.01 mm2 ~approx. 0.09mm 2 , approximately 0.01 mm 2 ~approx. 0.08mm 2 , approximately 0.04 mm 2 ~about 0.15mm 2 , for example, about 0.04 mm 2 ~about 0.125mm 2 , approximately 0.04 mm 2 ~about 0.1mm 2 , approximately 0.04 mm 2 ~approx. 0.09mm 2 , approximately 0.04 mm 2 ~approx. 0.08mm 2 , approximately 0.07 mm 2 ~about 0.15mm 2 , for example, about 0.07 mm 2 ~about 0.125mm 2 , approximately 0.07 mm 2 ~about 0.1mm 2 , approximately 0.07 mm 2 ~approx. 0.09mm 2 , or about 0.07 mm 2 ~approx. 0.08mm 2 The channel may occupy a position having a cross-sectional area of ​​.

[0009] During the shaking step, the liquid segment may have a volume of 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. During the shaking step, the liquid segment may 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 shaking step, the liquid segment may be between about 0.2 μL and about 2 μL, about 0.2 μL and about 1.75 μL, about 0.2 μL and about 1.5 μL, about 0.2 μL and about 1.25 μL, about 0.2 μL and about 1 μL, about 0.2 μL and about 0.75 μL, about 0.2 μL and about 0.5 μL, about 0.3 μL and about 2 μL, about 0.3 μL and about 1.75 μL, about 0.3 μL and about 1.5 μL, about 0.3 μL and about 1.25 μL, about 0.3 μL and about 1 μL, about 0.3 μL and about 0.75 μL, about 0.3 μL and about 0.5 μL. The volume may be about 0.5 μL, about 0.4 μL to about 2 μL, about 0.4 μL to about 1.75 μL, about 0.4 μL to about 1.5 μL, about 0.4 μL to about 1.25 μL, about 0.4 μL to about 1 μL, about 0.4 μL to about 0.75 μL, about 0.4 μL to about 0.5 μL, about 0.5 μL to about 2 μL, about 0.5 μL to about 1.75 μL, about 0.5 μL to about 1.5 μL, about 0.5 μL to about 1.25 μL, about 0.5 μL to about 1 μL, about 0.5 μL to about 0.75 μL, or about 0.5 μL.

[0010] The method may include, prior to the step of preparing the liquid segment, loading a quantity of liquid into a microchannel of the microfluidic device and separating the liquid segment from a remainder of the loaded liquid, which is then a quantity of liquid disposed in the microchannel proximal to the liquid segment. The step of separating the liquid segment may be performed by loading a separation gas into the microchannel at a position occupied by the quantity of loaded liquid.

[0011] The step of depositing a quantity of liquid may include depositing the liquid in an application zone of the microfluidic device. The depositing may further include moving the deposited liquid along the microchannel, e.g., by capillary action, until a distal sample liquid-gas interface of the deposited liquid contacts a capillary stop within the microchannel. The step of moving the liquid by capillary action may include moving the liquid along the microchannel until the distal sample liquid-gas interface reaches and moves beyond a location where a separation gas is deposited. The capillary stop may include one or more vents providing gas communication between the microfluidic channel and a quantity of gas disposed outside the microfluidic channel. The quantity of gas may be ambient air surrounding the microfluidic device. Alternatively or additionally, the capillary stop may include one or more hydrophobic features within the microfluidic channel, such as a hydrophobic layer that may be in the form of a hydrophobic strip extending across at least a portion or all of the channel width.

[0012] The application zone of the microfluidic device may include a porous membrane configured to separate the liquid of the microparticle-containing liquid from the microparticles. For example, the microparticle-containing liquid may be whole blood, and 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 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 microparticles are retained on the upper surface and / or within the porous membrane. The device may prepare a liquid segment from the liquid separated from the microparticle-containing liquid.

[0013] In embodiments, the method includes contacting a certain amount of the loaded liquid with at least one reagent disposed in the microchannel before separating the liquid segment from the remainder of the loaded liquid. For example, the loading step may include moving the loaded liquid along the microchannel, e.g., by capillary action, until the distal liquid-gas interface of the loaded liquid passes over at least one reagent disposed in the microchannel, and then stopping the movement of the loaded liquid. The stopping may be performed, e.g., using a capillary stop and / or by stopping a motive force acting on the loaded liquid. The motive force may be, e.g., a reduced pressure of a gas at the distal liquid-gas interface of the loaded liquid. The liquid segment is then separated from the remainder of the loaded liquid to prepare a liquid segment in contact with at least one reagent in the microchannel. The liquid segment includes all of the loaded liquid that is in contact with and / or contains at least one reagent. 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 separation gas is then performed on the liquid segment. The liquid segment in contact with and / or containing the at least one reagent may then move along the microchannel until the liquid of the liquid segment comes into contact with at least one further reagent. 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 may then be performed on the liquid segment in contact with and / or containing the at least one further reagent.

[0014] In embodiments, the method includes first preparing a liquid segment that is not in contact with and does not contain at least one reagent, and then contacting one or more reagents with the liquid segment to prepare a liquid segment in contact with at least one reagent within the microchannel. For example, the step of introducing may include moving the introduced liquid along the microchannel, e.g., by capillary action, before a distal liquid-air interface of the introduced liquid contacts one or more reagents disposed within the microchannel, and then stopping the movement of the introduced liquid. The stopping may be performed, e.g., using a capillary stop and / or by stopping a motive force acting on the introduced liquid. The motive force may be, e.g., a reduced pressure of a gas at the distal liquid-air interface of the introduced liquid. The liquid segment is then separated from the remainder of the introduced liquid and moves along the microchannel until the liquid in the liquid segment contacts one or more reagents. The liquid segment includes all of the introduced liquid that contacts or contains at least one 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 separation gas may then be performed on the liquid segment. The liquid segment in contact with and / or containing at least one reagent may move 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 separation gas may then be performed on the liquid segment in contact with and / or containing at least one additional reagent.

[0015] In embodiments, after a volume of liquid is introduced into the 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 can be air. Prior to introducing a volume of liquid into the microfluidic channel, the microfluidic channel, and optionally one or more vents, provide the only path for gas communication between the distal gas and the exterior of the microfluidic device. After introducing a volume of liquid into the microfluidic channel, the distal gas can 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 can block the passage of the distal gas or ambient gas along the microchannel and, if present, through one or more vents.

[0016] In an embodiment, a method includes separating a liquid segment (sometimes referred to as a liquid segment) from a total volume of liquid disposed within a microchannel of a microfluidic device. Separating the liquid segment includes injecting a separation gas into the microchannel from a gas input opening located at a position in the microchannel occupied by the total volume of liquid (i.e., after the liquid is introduced into the microchannel). Injecting the separation gas into the microchannel (e.g., via the gas input opening) forms an asymmetric bubble that separates the liquid segment from the remaining liquid in the total volume 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 in the total volume 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., therefore, the two ends of the asymmetric bubble that connect with the corresponding liquids are not symmetrical to each other). The volume of the liquid segment, after forming the asymmetric bubble, may have the same volume as the volume of the liquid segment described herein (e.g., as described in the above method for preparing the liquid segment). In embodiments, the volume of the liquid segment is about 0.75 μL to about 4 μL, e.g., about 1.5 μL to about 3 μL, e.g., about 2 μL. The volume of the asymmetric bubble disposed in the microchannel between the first and second gas-liquid interfaces may be, for example, about 200 nL to about 750 nL, e.g., about 350 nL.

[0017] In an embodiment, r2>r1. For example, the ratio r2 / r1 may be at least about 1.25, e.g., about 1.5 to 3.5, e.g., about 2. The ratio of the larger of the width (w2) and height (h2) of the microchannel at the second gas-liquid interface to the larger of the width (w1) and height (h1) of the microchannel at the first gas-liquid interface may be approximately the same as the ratio r2 / r1, i.e., w2 / w1 or h2 / h1 may be approximately the same as r2 / r1. Alternatively, or in combination, the ratio of the cross-sectional area (A2) of the separation channel at the second gas-liquid interface (e.g., the position of an asymmetric gas bubble in the microchannel) to the cross-sectional area (A1) of the separation channel at the first gas-liquid interface may be approximately the same as the ratio r2 / r1, i.e., A2 / A1 may be approximately the same as r2 / r1.

[0018] In embodiments, the microfluidic device includes a liquid application zone, e.g., a sample application zone, through which a total volume of liquid is introduced into the microfluidic channel. The first air-liquid interface may be located along the microfluidic channel distal to the second air-liquid interface and the liquid application zone.

[0019] In embodiments, the distance along the microchannel between the first gas-liquid interface and the gas input opening is distance d1, the distance along the microchannel between the second gas-liquid interface and the gas input opening is distance d2, and the ratio d2 / d1 is at least about 2.25, e.g., about 2.25 to 10, e.g., about 4.5. For example, d1 can be about 250 μm to 1000 μm, e.g., about 500 μm, and d2 can be about 1000 μm to about 2750 μm, e.g., about 2000 μm.

[0020] The liquid introduced into the microfluidic device, the remaining amount of liquid, and / or the liquid segment may 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 may be derived from an environmental sample. For example, an environmental sample may be obtained by swabbing a surface, such as in food preparation, storage areas, or healthcare facilities. An environmental sample may include a soil sample or a water sample. The liquid may include such a biological sample combined in a liquid reagent, such as one or more buffers, lysis media, universal transport media (UTM), viral transport media (VTM), or a combination thereof.

[0021] The liquid introduced into the device may also contact a reagent within the microfluidic device, such as in an application zone or feed channel extending distally therefrom. In such cases, the liquid solubilizes the reagent to prepare a liquid mixture containing the introduced liquid and the solubilized reagent. A liquid segment is then prepared from such a liquid mixture. The liquid segment may contact or contain one or more additional reagents disclosed herein.

[0022] In embodiments, the microfluidic device includes a substantially planar substrate including a sample application zone and a microfluidic network including a microfluidic channel extending from an intersection between the sample application zone and the microfluidic channel. The porous membrane may overlie the sample application zone. The lower inner surface and an inner sidewall of the sample application zone may be defined by the substrate. The upper inner surface of the sample application zone may be defined by a lower surface of the porous membrane. Additionally, or in combination, the lower inner surface, upper inner surface, and first and second opposing inner sidewalls of the microfluidic channel are defined by the substrate. A portion of the substrate defining the upper inner surface of the microfluidic channel may protrude beyond the intersection between the sample application zone and the microfluidic channel and at least partially into the sample application zone.

[0023] Substantially all, eg, all, of the portion of the substrate that protrudes beyond the intersection into the sample application zone may be under the porous membrane.

[0024] The intersection between the sample application zone and the microfluidic channel may define a width between first and second opposing interior sidewalls of the microfluidic channel. The width may be taken along a direction oriented approximately perpendicular to the longitudinal axis of the microfluidic channel and parallel to a plane defined by the substantially 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, or at least about 1.5 mm, e.g., about 1.5 mm, and is less than about 2.5 mm, less than about 2.0 mm, e.g., less than about 1.75 mm.

[0025] In some embodiments, the portion of the substrate that protrudes beyond the intersection into the sample application zone may be above the intersection and extend beyond the entire width of the intersection. In other embodiments, the portion of the substrate that protrudes beyond the intersection into the sample application zone is narrower than the width of the intersection. For example, at the intersection, the width of the protruding portion of the substrate may be about 20% to 75% of the width of the intersection.

[0026] The portion of the substrate that protrudes beyond the intersection into the sample application zone may taper from a first width above the intersection to a second, smaller width located within the sample application zone.

[0027] The portion of the substrate that protrudes beyond the intersection into the sample application zone may extend beyond the intersection into the sample application zone a distance of at least about 0.25 mm and less than about 1 mm, for example about 0.25 mm or about 0.5 mm.

[0028] The height of the microfluidic channel, taken along an axis perpendicular to the plane of the substrate, between the lower and upper inner surfaces of the microfluidic channel at a location 5 mm distal to the intersection along the longitudinal axis of the microfluidic channel, can be at least about 50 μm and less than about 250 μm, e.g., about 110 μm. The height between the lower surface of the sample application zone and the lower surface of the portion of the substrate that defines the upper inner surface of the microfluidic channel and that protrudes beyond the intersection into the sample application zone can be less than the height of the microfluidic channel. For example, the ratio of the sample application zone height to the channel height can be less than about 0.9. This ratio can be at least about 0.5, e.g., at least about 0.75.

[0029] In some embodiments, the substrate of the microfluidic device includes a lower layer that defines a lower inner surface of the sample application zone and a lower inner surface of the microfluidic channel, and an upper layer that defines an upper inner surface of the microfluidic channel and includes a portion of the substrate that defines the upper inner surface of the microfluidic channel that protrudes beyond the intersection into the sample application zone. The substrate may include an intermediate layer, where the upper and lower layers are separated by the intermediate layer and are fixed, e.g., glued, to the intermediate layer. The intermediate layer may define an interior sidewall of the sample application zone and first and second opposing walls of the microfluidic channel.

[0030] The middle layer may include a middle layer opening that defines an interior sidewall of the sample application zone, and the upper layer may include an upper layer opening above the middle layer opening. The diagonal dimension of the upper layer opening is typically larger than the diagonal dimension of the middle layer opening. A peripheral portion of the lower surface of the porous membrane may be adjacent to the middle layer opening and bonded to the upper surface of the middle layer exposed by the upper layer opening (e.g., exposed to the exterior of the microfluidic device).

[0031] In some embodiments, the upper layer, middle layer, and lower layer are each separate layers, with the upper layer and lower layer fixed to each other and separated by the middle layer. In other embodiments, the middle layer is integral with one of the upper or lower layers. For example, the substrate may include two layers, with the first of the two layers defining the sample application zone and the inner wall of the microfluidic channel, as well as the lower inner surface of the sample application zone and the microfluidic channel. The second of the two layers defining the upper inner surface of the microfluidic channel, with a portion of the substrate defining the upper inner surface of the microfluidic channel that protrudes beyond the intersection into the sample application zone. As another example, the substrate may include two layers, with the first of the two layers defining the sample application zone and the inner wall of the microfluidic channel, with the upper inner surface of the microfluidic channel and a portion of the substrate defining the upper inner surface of the microfluidic channel that protrudes beyond the intersection into the sample application zone. The second of the two layers defining the sample application zone and the lower inner surface of the microfluidic channel. In each of these examples, at least the first of the two layers may be a molded layer, such as, for example, an injection molded layer.

[0032] A microfluidic device including a porous membrane may be configured to receive a microparticle-containing liquid applied to the upper surface of the porous membrane and separate at least a portion of the liquid from the microparticles to detect and / or measure at least one target in the separated liquid. For example, the microparticle-containing liquid may be whole blood, the separated liquid may be plasma separated from red blood cells of the whole blood, and 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 microparticles are retained on the upper surface and / or inside the porous membrane. The separated liquid, e.g., plasma, passes through the porous membrane and enters a sample application zone therebelow. In embodiments, the separated liquid passes through an intersection and enters a microfluidic channel. 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 in a microfluidic channel positioned distal to the separated liquid to provide a motive force for moving the separated liquid.

[0033] In an embodiment, the microfluidic device includes a vent channel extending from a vent intersection between the sample application zone and the vent channel. A distal portion of the vent channel is in gas communication with ambient gas surrounding the substrate such that gas disposed within the sample application zone can exit the sample application zone through the vent channel and / or ambient gas surrounding the substrate can enter the sample application zone through the vent channel. The lower inner surface, upper inner surface, and first and second opposing interior sidewalls of the vent channel are defined by the substrate. A portion of the substrate defining the upper inner surface of the vent channel protrudes beyond the vent intersection into the sample application zone.

[0034] In some embodiments, the microfluidic device is configured to receive a whole blood sample through the sample application zone, thereby contacting the whole blood sample with the upper surface of the porous membrane, where red blood cells of the whole blood sample may be entrained on and / or within the porous membrane, and plasma separated from the whole blood sample by the porous membrane may contact the lower surface of the porous membrane. At least a portion of the plasma may contact a portion of the substrate defining the upper interior surface of the microfluidic channel that protrudes beyond the intersection into the sample application zone.

[0035] In embodiments, the microfluidic device includes a substantially planar substrate including a sample application zone and a microfluidic network including microfluidic channels extending from an intersection between the sample application zone and the microfluidic channels. A porous membrane may overlie the sample application zone. A lower layer of the substrate defines a lower inner surface and an inner sidewall of the sample application zone. A lower surface of the porous membrane defines an upper inner surface of the sample application zone. The lower layer of the substrate may define a lower inner surface and first and second opposing inner sidewalls of the microfluidic channels. An upper surface of the lower layer of the substrate may include an adhesive surface. An upper layer of the substrate overlies the lower layer of the substrate and is secured to the adhesive surface, defining an upper inner surface of the microfluidic channels. A portion of the substrate defining the upper inner surface of the microfluidic channels protrudes beyond the intersection into the sample application zone. In embodiments, the lower layer of the substrate is formed from at least first and second lower layers. The first lower layer may be an adhesive layer that separates and bonds the upper and second lower layers together. Typically, at least some portions of the first sub-layer will be absent, thereby defining the interior sidewalls of the microfluidic channel.

[0036] In an embodiment, the microfluidic device includes a substantially planar substrate including a sample application zone and a microfluidic network including a microfluidic channel extending from an intersection between the sample application zone and the microfluidic channel. A lower layer of the substrate defines a peripheral interior sidewall of the sample application zone and an opposing interior sidewall of the microfluidic channel. An upper layer of the substrate overlies the lower layer of the substrate. A lower surface of the upper layer of the substrate defines an upper interior surface of a distal portion of the microfluidic channel. The upper layer of the substrate includes an opening surrounding the peripheral interior sidewall of the sample application zone. The opening exposes a peripheral portion of the upper surface of the lower layer of the substrate. The peripheral portion surrounds the peripheral interior sidewall of the sample application zone. A proximal portion of the microfluidic channel is disposed adjacent to the intersection between the sample application zone and the microfluidic channel. At the proximal portion of the microfluidic channel, a portion of the upper layer of the substrate has a width narrower than the width of the microfluidic channel, and each width is oriented substantially parallel to the substantially planar substrate and perpendicular to the longitudinal axis of the microfluidic channel at the proximal portion. For example, the ratio of the width of a portion of the upper layer of the substrate to the width of the microfluidic channel at the proximal portion can be less than about 0.8, less than about 0.7, less than about 0.6, e.g., about 0.5. The ratio can be at least about 0.3, at least about 0.4, e.g., about 0.5. The width of the microfluidic channel at the proximal portion can be about 0.75 mm to 2.5 mm, e.g., about 1 mm to 2 mm, e.g., about 1.5 mm. The length of the proximal portion of the microfluidic channel, where the portion of the upper layer has a narrower width, can be about 0.75 mm to 3 mm, about 1 mm to 2 mm, e.g., about 1 mm, taken along the longitudinal axis of the proximal portion of the microfluidic channel. A portion of the upper layer of the substrate can extend beyond the intersection into the application zone. For example, the upper layer of the substrate can extend beyond the intersection for a length of about 0.25 mm to about 1.5 mm, e.g., about 0.25 mm, or about 0.5 mm.

[0037] The porous membrane may overlie the sample application zone. The lower surface of the porous membrane defines an upper inner surface of the sample application zone. A peripheral portion of the lower surface of the porous membrane is secured to an exposed peripheral portion of the upper surface of the lower layer of the substrate. A portion of the porous membrane may overlie a proximal portion of the microfluidic channel such that the lower surface of the porous membrane defines a portion of the upper inner surface of the proximal portion of the microfluidic channel. The lower surface of the narrower portion of the upper layer of the substrate defines the remaining portion of the upper inner surface of the proximal portion of the microfluidic channel. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 is a planar top view of a microfluidic device configured to prepare a liquid segment from a volume of liquid introduced into the device and mix the liquid of the liquid segment with one or more reagents to facilitate determination of one or more targets within the liquid of the liquid segment, according to embodiments described herein. [Figure 2] FIG. 2 is a planar top view of the microfluidic device of FIG. 1 , with its electrical elements not shown for clarity, according to an embodiment described herein, with liquid being introduced into its microchannel via an application port and moving along the microchannel via capillary action until the distal liquid-air interface of the sample liquid reaches a capillary stop. [Figure 3] FIG. 3 is a planar top view of the microfluidic device of FIG. 2 according to an embodiment described herein, in which a separation gas is injected into the microchannel to separate a liquid segment of the liquid from the remaining amount of sample liquid injected into the microchannel. [Figure 4] FIG. 4 is a planar top view of the microfluidic device of FIG. 3 according to an embodiment described herein, with the liquid segment moving distally along the microchannel compared to the position shown in FIG. 3. [Figure 5]FIG. 1 is a planar top partial view of another embodiment of a microfluidic network of a microfluidic device configured to prepare a liquid segment from a quantity of liquid introduced into a microchannel of the device and mix the liquid of the liquid segment with one or more reagents, according to embodiments described herein, wherein its electrical elements and gas chambers are not shown for clarity, and wherein liquid is introduced into its microchannel through an application port and moves along the microchannel via capillary action until the distal liquid-gas interface of the sample liquid reaches a capillary stop. [Figure 6] FIG. 6 is a planar top partial view of the microfluidic network of FIG. 5 according to an embodiment described herein, in which a separation gas is injected into a gas separation portion of the microchannel to prepare an initial gas bubble to separate the liquid segment from the remainder of the sample liquid. [Figure 7] FIG. 7 is a planar top partial view of the microfluidic network of FIGS. 5 and 6 according to an embodiment described herein, in which an additional amount of separation gas is introduced into the gas separation portion of the microchannel to prepare an asymmetric bubble that separates the liquid segment from the remainder of the sample liquid. [Figure 8] FIG. 8 is an enlarged plan top partial view of a gas separation portion of a microchannel of the microfluidic network shown in FIG. 7 according to embodiments described herein. [Figure 9] An enlarged planar top partial view of the gas separation portion of the microchannel of the microfluidic network shown in Figure 8, also showing the radius of curvature of the gas-liquid interface of an asymmetric bubble within the gas separation portion of the microchannel, according to an embodiment described herein. [Figure 10] FIG. 1 is a planar top view of an embodiment of a microfluidic device configured to form a first segment containing a sample liquid and a second segment containing a diluent, and then mix the first and second segments, according to embodiments described herein, where a blood sample is introduced into the microfluidic network to form the first segment, as shown. [Figure 11]FIG. 12 is a planar top view of an embodiment of a microfluidic device configured to form a first segment containing a sample liquid and a second segment containing a diluent, and then mix the first and second segments, according to embodiments described herein, where a blood sample is introduced into the microfluidic network to form the first segment, as shown in FIG. 11 . [Figure 12a] 12 illustrates the microfluidic device of FIG. 11 according to an embodiment described herein, with a first segment of blood moving proximally within the microfluidic network compared to FIG. 11. [Figure 12b] 12b shows a close-up view of the segment attachment region of the microfluidic device of FIG. 12a according to embodiments described herein. [Figure 13] 12 illustrates the microfluidic device of FIG. 11 according to an embodiment described herein, with a diluent introduced into the device and the proximal air-liquid interface of the first segment of blood positioned in contact with the distal air-liquid interface of the diluent. [Figure 14] 14 illustrates the microfluidic device of FIG. 13, where a segment of diluent is separated from the remainder of the diluent, according to an embodiment described herein. [Figure 15] 15 illustrates the microfluidic device of FIG. 14, according to an embodiment described herein, where a first segment of blood and a segment of diluent are coupled to the mixture by the oscillating action of gas pressure. [Figure 16] 16 illustrates the microfluidic device of FIG. 15 with the mixture moving proximally within the microfluidic network to a detection region, according to an embodiment described herein. [Figure 17] FIG. 1 is a planar top view of a microfluidic device configured to separate a liquid from particulates, form a separated liquid segment, and mix the separated liquid segment with one or more reagents to facilitate detection of one or more targets in the sample liquid with electrical elements of the microfluidic device (not shown for clarity), according to one embodiment of the present disclosure. [Figure 18]FIG. 18 is an exploded view of the microfluidic device of FIG. 17 showing its three-layer structure, according to embodiments herein. [Figure 19] FIG. 18 is a detailed planar top view of the sample application zone of the microfluidic device of FIG. 17, with the porous membrane overlying the sample application zone removed for clarity, according to embodiments herein. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description Referring to FIG. 1 , a 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 within a microchannel, prepare a liquid segment that includes essentially all or all of the portion of the received sample liquid and that is in contact with and / or includes the reagents, mix the liquid of the liquid segment with the reagents, and determine the presence of one or more targets within 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 a portion of the received sample liquid with one or more reagents may be performed before and / or after preparing the liquid segment. For example, preparation of the liquid segment may be performed by contacting the sample liquid with one or more reagents. Once prepared, the liquid segment includes all of the received sample liquid that is or has been in contact 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 a reagent with the liquid, after which the liquid segment may move along the microchannel and contact one or more reagents.

[0040] The prepared liquid segment has a predetermined volume defined between a proximal air-liquid interface and a distal liquid-air interface. One or more reagents are disposed in a predetermined amount within the microchannel. Therefore, the concentration of the reagents in the mixture formed by subjecting the liquid segment to a mixing process is known regardless of whether the liquid segment is prepared from the sample liquid in contact with one or more reagents, or whether the liquid segment contacts such reagents after preparation (or both). Without forming a liquid segment defined by such an interface, bulk movement within the sample liquid induced by the mixing process may cause one or more reagents to be distributed in uncertain amounts within the sample liquid, resulting in the concentration of the one or more reagents not being known with the same accuracy as the liquid segment. Because the concentration of the reagents within the liquid segment is highly accurate, determination of one or more targets therein can be performed with greater accuracy than determination of such targets in a liquid in which the reagents are not confined within the liquid segment by a pair of interfaces (e.g., the proximal air-liquid interface and the distal air-liquid interface of the liquid segment).

[0041] The microfluidic device 10 includes a microfluidic network 12. Beginning with a sample application zone 14 and proceeding from proximal to distal, the microfluidic network 12 includes a microfluidic channel having a supply channel portion 16, a segmented 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, segmented separation channel portion, and analysis channel portion are used interchangeably with the terms supply channel, segmented separation channel, and analysis channel, respectively. The analysis channel 18 further includes a vent 32 that acts as a capillary stop and one or more reagents 60 configured to facilitate the determination (e.g., detection) of one or more targets within 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 extending partially or entirely across the analysis channel 18 and / or extending along and within the vent 32. The microfluidic device 10 also includes a separation gas chamber 26 disposed in gas communication with the segment separation channel 28 via a separation gas channel 30 that intersects the separation channel 28 at a gas input opening 30'.

[0042] The microfluidic device 10 may be configured similarly to the microfluidic strip of the '325 application. For example, the device 10 may be comprised of a lower substrate, e.g., a flexible polymer layer, and an upper substrate, e.g., a flexible polymer layer, bonded to each other by an adhesive layer. The adhesive layer occupies less than the entire area of ​​the facing surfaces between the upper and lower substrates and includes sidewalls that define a microfluidic network 12 therebetween. The upper and lower substrates and adhesive layer may have the same properties (e.g., thickness, composition, and mechanical properties) as those disclosed for the upper and lower substrates and adhesive layers 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 about 50 μm to about 200 μm, e.g., about 110 μm.

[0043] The gas chamber 20 includes a plurality of spaced apart positions 22a, 22b, 22c disposed in gas communication with each other and with the analysis channel 18 via a gas chamber opening 24. Each of the spaced apart positions 22a, 22b, 22c is spaced from an adjacent spaced apart position by an interior sidewall, thereby allowing the upper layer above each spaced apart position to be compressed / decompressed and / or vibrated independently of the upper layer above the other spaced apart positions. The gas chamber 20 and the spaced apart positions 22a, 22b, 22c may be configured, arranged, and operated as disclosed in the '858 application. The separation gas chamber 26 is configured to input a separation gas through the separation gas channel 30 and into the segment separation channel 28 via the gas input opening 30' to separate a liquid segment from a volume of sample liquid present in the microfluidic network 12. The separation gas chamber 26 may be configured, arranged, and operated as disclosed for the gas chamber in the '325 application. The term gas chamber is sometimes used interchangeably with the term gas bladder.

[0044] Prior to the introduction of a liquid sample into microfluidic device 10, ambient gas, e.g., air, surrounding the microfluidic device can enter and exit microfluidic network 12 via application zone 14 and vent 32. There are no other paths by which gas can enter or exit microfluidic network 12. Thus, microfluidic network 12, e.g., its channels and chambers, is occupied by such ambient gas.

[0045] Reagent 60 includes one or more reagents configured to facilitate detection of a target, e.g., a reagent configured to bind to a target and labeled to enable detection of such a reagent. The reagent may also include a magnetic particle reagent to enable magnetic capture of the reagent. The one or more reagents may include, for example, any of the reagents disclosed in the '325 application. The one or more reagents may be disposed within the microchannel in a dry state and configured to assemble upon contact with a liquid. The one or more reagents may be disposed within a single location or at two or more spaced locations. For example, a microchannel channel may include a reagent disposed at each of at least two locations spaced apart along the microchannel by a distance sufficient to allow contact and mixing with a reagent at a first location without contacting a reagent at a second location. A liquid, e.g., a liquid segment, then travels along the channel to a second location to allow contact and mixing with the reagent therein.

[0046] Microfluidic device 10 also includes electrodes positioned and configured to enable the reader to monitor the proper filling of device 10 with sample liquid, the proper movement of sample liquid and liquid segments therein, and the operation (e.g., compression state) of each spaced apart location 22a, 22b, 22c of gas chamber 20 and separation gas chamber 26. The electrodes may generally be positioned, configured, and operated as disclosed in the '325 and / or '858 applications. For example, device 10 includes supply electrode 21 connected to contact 23 via lead 21', and first and second fill electrodes 25 and 33, each connected to contact 27 via common lead 25'. When device 10 is fully inserted into the reader, contacts 23 and 27 engage corresponding contacts in the reader. The engaged contacts, as disclosed in the '325 application, allow the reader to deliver and / or receive electrical signals between source electrode 21 and fill electrode 25 and / or second fill electrode 33 to determine the presence of a liquid sample at fill electrode 25 and fill electrode 33, respectively. Microfluidic device 10 also includes electrodes configured to perform both liquid sensing and mechanical sensing functions. For example, lead 31 extends from contact 41 through gas chamber 26 to third fill electrode 29. Lead 35 extends from contact 37 into gas chamber 26. Gas chamber 26 also includes bridge contact 39. When gas chamber 26 is fully compressed, bridge contact 39 places lead 31 in electrical communication with lead 35, which is sensed by the reader via contacts 37 and 41. When leads 31, 35 are not in electrical communication (i.e., when gas chamber 26 is not in a fully compressed state), a reader operating microfluidic device 10 can also sense the presence of liquid in analysis channel 18 using third fill electrode 29. Spaced apart locations 22a, 22b, 22c also include corresponding electrodes and corresponding bridging contacts to allow the reader to determine when the locations are in a fully compressed state in the same manner as gas chamber 26.

[0047] A method for forming a liquid segment and mixing the liquid of the liquid segment with a reagent is described with reference to FIGS. 2-4. The method begins by inserting the microfluidic device 10 into a reader (not shown), as disclosed in the '325 application. The reader compresses the spaced apart portions 22a, 22b, and 22c of the gas chamber 20 to an operative, fully compressed state, thereby releasing the gas therein through the application zone 14 and the vent 32. The separation gas chamber 26 remains uncompressed. The method continues by introducing a sample liquid 50 into the microfluidic network 12 via the application zone 14. The sample liquid 50 moves by capillary action along the supply channel 16, through the segment separation channel 28, and into the analysis channel 18 until a distal liquid-air interface 52 of the sample liquid 50 reaches the vent 32, which acts as a capillary stop to prevent further movement of the sample liquid 50. The sample liquid 50 then occupies the microfluidic network 12 extending from the gas-liquid interface 52' formed in the application zone 14 to the distal gas-liquid interface 52 located at the vent 32 (FIG. 2).

[0048] Gas chamber 20, including spaced apart locations 22a, 22b, and 22c, and the portion of analysis channel 18 distal to distal liquid-gas interface 52, are occupied by gas 54. Separation gas chamber 26 and separation gas channel 30 are occupied by gas 56. By way of example, gas 54 and gas 56 may comprise residual ambient gas, such as air, that occupied the microfluidic network prior to introduction of sample liquid 50. Because sample liquid 50 occupying microfluidic network 12 blocks vent 32 and application zone 14, gas 54 and gas 56 are isolated from each other and from the ambient gas surrounding microfluidic device 10.

[0049] As shown in Figure 2, the sample liquid 50 comes into contact with the reagent 60 in the analysis channel 18. The sample liquid begins to mobilize the reagent. Because the sample liquid 50 stops moving along the microchannel and is not undergoing active mixing at this stage, the mixing of the sample liquid 50 and the reagent 60 is driven by slow diffusion. Therefore, the reagent 60 does not distribute within the sample liquid 50 in sufficient quantities to cause the quantity uncertainty described above.

[0050] 3, separation gas chamber 26 is compressed, increasing the pressure of gas 56 therein and forcing gas 56 through separation gas channel 30 and into separation channel 28 via gas input opening 30'. The input of gas 56 into separation channel 28 separates liquid segment 55 from the remaining portion 57 of sample liquid 50 (shown in FIG. 2). Liquid segment 55 includes proximal gas-liquid interface 58, all of sample liquid 50 that is in contact with and / or contains reagent 60, and distal liquid-gas interface 52. That is, remaining portion 57 of sample liquid 50 does not contain any of reagent 60. Instead, all of reagent 60 is contained within and / or in contact with the liquid of liquid segment 55. The remaining liquid portion 57 includes distal liquid-gas interface 59. Gas 56 may occupy at least a portion of supply channel 16 and separation channel 28, and is isolated from ambient gas surrounding device 10 by remaining liquid portion 57 (blocking application zone 14) and from such ambient gas and gas 54 by liquid segment 55 (blocking vent 32 and separating gases 54 and 56). As an alternative to injecting the separation gas by compressing the separation gas chamber, the chamber containing the separation gas may be heated, thereby increasing the pressure of the separation gas and injecting it into the microchannel containing the sample liquid, separating the liquid segment from the remaining liquid. Heating may be performed, for example, by using a resistive conductor in the chamber.

[0051] The microfluidic network 12 may also include reagents located at different locations along the analysis channel 18 and / or at locations different from the analysis channel 18, e.g., at a location in the microfluidic network located proximal to the analysis channel 18. For example, the application zone and / or delivery channel 16 may include one or more reagents to facilitate target determination, such as lysing and / or disrupting cells in the sample liquid to release the cellular contents and / or one or more reagents, such as heparin, to reduce clotting in a blood-based sample liquid. In such embodiments, such proximally located reagents may contact a significant portion, e.g., most or essentially all, of the input sample liquid, and as a result, the concentration of such reagents is not known with the same precision as the concentration of a reagent upon contact with a given volume of a liquid segment. However, a liquid segment may be prepared from such reagent-containing sample liquid and contacted with additional reagents before and / or after preparation of the liquid segment. In such cases, the concentration of the additional reagents in the liquid segment should be known with greater precision, as disclosed herein.

[0052] After separating the liquid segment 55 from the remaining portion 57 of the sample liquid 50, the reader can actuate oscillations in the pressure of the gas 54 in the gas chamber 20 and / or oscillations in the pressure of the gas 56 in the gas chamber 26. The oscillations may be performed as disclosed in the '325 and '858 applications, including, for example, through the use of piezoelectric actuators. 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 may be performed using respective piezoelectric actuators having actuation feet for separately compressing, decompressing, and / or oscillating, for example, synchronously, the top wall above each spaced apart location 22a, 22b, 22c and / or the gas chamber 26. Oscillating the top wall at each spaced apart location oscillates the volume occupied by the gas 54 in the gas chamber 20 (and therefore the pressure of the gas 54) by oscillating the spacing between the opposing inner walls at each spaced apart location. Alternatively, or in combination with the oscillation of the pressure of gas 54, the pressure of gas 56 may be oscillated, for example, synchronously at the same frequency, at the same frequency but out of phase, or at a frequency different from the frequency of oscillation of gas 54.

[0053] Oscillating the pressure of the distal gas 54 induces bulk movement of liquid within the liquid segment 55, thereby distributing the reagent 60 throughout the liquid segment and establishing a uniform concentration distribution therein. The bulk movement of liquid mixes the liquid and reagent in the liquid segment 55 more quickly than can be achieved by diffusion alone. The proximal gas-liquid interface 58 and the distal liquid-gas interface 52 of the liquid segment 55 prevent the reagent from leaving the liquid segment during oscillatory mixing. Thus, mixing within the liquid segment 55 provides a mixture with a known, uniform concentration of reagent 60. The volume of the liquid segment 55 is determined by (i) the volume of the microchannel between the intersection of the gas input opening 30′ of the separation gas channel 30 with the separation channel 28 and (ii) the capillary stop, e.g., the vent 32. The aforementioned volume of the microchannel is determined by the internal dimensions, e.g., cross-sectional area, of the microchannel and the distance along the microchannel between (i) the intersection of the gas entry opening 30′ of the separation gas channel 30 with the separation channel 28 and (ii) the vent 32 and / or hydrophobic layer, if present. Typically, the dimensions and distances are selected to provide a liquid segment having a volume, e.g., between about 0.2 μL and about 2.5 μL, e.g., between about 0.2 μL and about 0.750 μL, e.g., about 0.35 μL.

[0054] The liquid segment 55 may move distally along the microchannel, for example, within the analysis channel 18, by reducing the pressure of the gas 54 acting on the distal liquid-gas interface 52. For example, the pressure of the gas 54 may be reduced by decreasing the compression at one or more of the spaced locations 22a, 22b, 22c within the gas chamber 20. The reduction in compression may be performed, for example, through the use of piezoelectric actuators in contact with the outer walls of the microfluidic device 10 above the spaced locations, as disclosed in the '325 and '858 applications. Reducing the compression increases the volume of the gas chamber 20 by increasing the spacing between the opposing interior walls at each spaced location 22a, 22b, 22c.

[0055] Distal movement of the liquid segment 55 may be performed, for example, to bring the liquid of the liquid segment 55 into contact with other reagents disposed in the analysis channel 18 and / or to bring the liquid segment into a detection zone 18'' therein.

[0056] After the liquid segment 55 has been contacted and mixed with all of the reagents necessary to perform target determination (e.g., detection), the liquid segment is subjected to a detection step to perform such determination. Such a detection step may include, for example, fluorescent or other optical detection and / or electrochemical detection. Any of the detection steps disclosed in the '325 application may be performed. For example, the reagents may include magnetic particles, and the detection step may include exposing the liquid segment to a magnetic field to capture the magnetic particles. The detection step may then 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., releasing, the liquid of the liquid segment 55 in contact with the captured magnetic particles from the detection zone 18″ (e.g., separating the liquid of the liquid segment 55 from the captured magnetic particles) prior to detection. Alternatively, the detection step may then include depressurizing 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., drawing, the liquid of the liquid segment 55 in contact with the captured magnetic particles from the detection zone 18''. As another example, the detection step may then include depressurizing one or more, e.g., all, of the spaced apart locations 22a, 22b, 22c or the gas chamber 26 on the one hand, and compressing 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 segment 55 by a combination of releasing and drawing the gas pressure.

[0057] In embodiments, the microfluidic device 10 comprises two or more microfluidic networks 12, each of which may be configured to receive a sample liquid, mix it with one or more reagents, and separately perform an analysis on the sample liquid, e.g., to detect the presence or absence of a target in the sample liquid. In some embodiments, each microfluidic network comprises the same components as described herein for the microfluidic network 12, including, for example, a supply channel portion 16, a segmented separation channel portion 28, an analysis channel portion 18 including a reagent zone 18' and a detection zone 18", and a microfluidic channel having a gas chamber 20. In some embodiments, the supply channel portions for each microfluidic network branch off from a common channel extending from the application zone. Thus, two or more microfluidic networks are configured to simultaneously analyze the sample liquid.

[0058] Two or more microfluidic networks may be located adjacent to one another on a microfluidic device. In embodiments, at least some of the microfluidic networks have the same reagents to provide redundant analysis of the sample liquid. Such redundancy can serve as a check in case of any contamination or failure in a given microfluidic network and can also provide additional accuracy and tunability for any sensitivity with the sample liquid.

[0059] In some cases, at least some of the microfluidic networks have one or more different reagents, which allows the microfluidic device to detect one or more different targets in the sample liquid, and the detection of such different targets may occur simultaneously.

[0060] 5-9, another exemplary microfluidic network 112 of a microfluidic device (only a portion of the microfluidic network is shown) is depicted, which includes a tapered segment separation channel 128 for separating a liquid segment from a liquid input into the microfluidic network. The microfluidic network 112 includes a supply channel 16 extending from a sample application zone 14, a segment 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 a vent 132 and / or a hydrophobic strip 133 may be located on the inner surface of the analysis channel 118 and can act as a hydrophobic stop. The width w4 of the vent 132 along the analysis channel 118 is typically about 75 μm to about 300 μm, e.g., about 150 μm. The microfluidic network 112 also includes a separation gas channel 130 having an opening 130' connecting to the segment separation channel 128. The separation gas channel 130 leads to a separation gas bladder, which may be configured to operate as described for the separation gas bladder 26 of the microfluidic network 10. The distal portion of the analysis channel 118 includes a gas chamber opening 124 that connects to a gas chamber, which may be configured to operate as described for the separation gas bladder 26 of the microfluidic network 10. The microfluidic network 112 further includes electrical features (not shown) as described for the microfluidic device 10. The internal height of the microfluidic network 112 is typically between about 50 μm and about 200 μm, for example, about 110 μm.

[0061] The segment separation channel 128 has a length between a proximal separation origin 135 and a distal separation end 137. The length of the segment separation channel 128 is typically about 1.5 mm to about 4.5 mm, e.g., about 3 mm. The width of the segment 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 a width w2 at the proximal separation origin 135 to a narrower width w1 at the distal separation end w1. The width w2 is typically about 800 μm to 2200 μm, e.g., about 1650 μm. The width w1 is typically about 400 μm to 1200 μm, e.g., about 800 μm. The width ratio w2 / w1 is typically at least about 1.25, such as about 1.5 to 3.5, e.g., about 2. The width of the segment separation channel 128, going along it from the proximal separation origin 135 to the distal separation terminus 137, is typically about 12 mm. -1 ~approx. 25% mm -1 , for example, about 17.5% mm -1 The height of the segment separation channel 128 typically decreases at an average rate of . The height of the segment separation channel 128 is typically constant or substantially constant. As used herein, the term "substantially" may refer to a variation of no more than + / - 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 origin 135 to a smaller cross-sectional area A1 at the distal separation terminus 137. For example, the cross-sectional area A1 can be about 0.044 mm to about 0.13 mm, e.g., about 0.09 mm, and the second cross-sectional area A2 can be about 0.13 mm to about 0.24 mm, e.g., about 0.18 mm. The ratio of the cross-sectional areas (area A2 at proximal separation origin 135 / area A1 at distal separation end 137) can be the same as the ratio of the widths (w2 / w1), e.g., A2 approximately equals A1 × w2 / w1. The cross-sectional area of ​​the segment separation channel 128 can decrease at the same relative rate as its widths as it progresses from the proximal separation origin 135 to the distal separation end 137.

[0062] The separation opening 130' has a width w5 along the first tapered sidewall 128' of the segment separation channel 128. The width w5 is typically about 75 μm to 300 μm, e.g., about 150 μm. The distance d3 (FIGS. 6 and 8) between the center of the separation opening 130' and the distal separation end point 137 is about 300 μm to 1000 μm, e.g., about 750 μm. Proceeding distally along the segment separation channel 128 from the center of the separation opening 130' to the distal separation end point 137, the width between the tapered walls 128' and 128'' tapers as described above. Proceeding distally beyond the distal separation end point 137, the width of the microchannel widens from width w1 at the distal separation end point 137 to width w3 within the analysis channel 118. Width w3 is greater than width w1. The width w3 is usually 800 μm to about 3000 μm, for example, about 2000 μm. The width ratio w3 / w1 is usually about 1 to 3.5, for example, about 2.5.

[0063] The reagent zone 118' of the analysis channel 118 includes one or more reagents arranged and configured as described for the reagents 60 of the microfluidic device 10. For example, the reagents 60 may include reagents configured to prepare the sample for analysis. For example, the reagents may be a heparin reagent to inhibit clotting in a blood sample, or a lysis reagent configured to lyse cells and release their contents for analysis. In Figures 5-9, the microfluidic network 112 is shown with the sample liquid already contacted and solubilized with such reagents. The microfluidic network is used to prepare a liquid segment containing all of such contacted and solubilized reagents.

[0064] Prior to introducing a liquid sample into microfluidic network 112, ambient gas, e.g., air, surrounding the microfluidic device including network 112 can enter and exit microfluidic network 112 via application zone 14 and vent 132. There are no other paths by which gas can enter or exit microfluidic network 112. Thus, microfluidic network 112, e.g., its channels and chambers, is occupied by such ambient gas.

[0065] The method for forming a liquid segment using microfluidic network 112 proceeds similarly to the process described for microfluidic device 10. The method begins by inserting the microfluidic device including microfluidic network 112 into a reader (not shown), as disclosed in the '325 application. The reader compresses the gas chamber of microfluidic network 112 (e.g., distal to gas chamber channel 124) to an operative, fully compressed state, thereby releasing the gas therein through application zone 14 and vent 132. The separation gas chamber remains uncompressed. The method continues by introducing sample liquid 150 into microfluidic network 112 via application zone 114. Sample liquid 150 travels by capillary action along feed channel 16, through segment separation channel 128, and into analysis channel 118 until distal liquid-air interface 152 reaches vent 132 and / or hydrophobic strip 133, either of which can act as a capillary stop to prevent further movement of liquid sample 150. 5 , the sample liquid 150 then occupies the portion of the microfluidic network 112 extending from the air-liquid interface 152′ formed in the application zone 14 to the distal air-liquid interface 152 located at the vent 132. Within the analysis channel 118, the sample liquid 150 may 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.

[0066] The gas chamber of microfluidic network 112 and the portion of analysis channel 118 distal to distal liquid-gas interface 152 are occupied by gas 154, which has the same composition and properties as gas 54. The separation gas chamber and separation gas channel 130 of microfluidic network 112 are occupied by gas 156, which has the same composition and properties as gas 56. Gas 154 and gas 156 consist of the remaining ambient gas, e.g., air, that occupied microfluidic network 112 prior to the introduction of sample liquid 150. Because sample liquid 150 occupying microfluidic network 112 blocks vent 132 and application zone 14, gas 154 and gas 156 are isolated from each other and from the ambient gas surrounding the microfluidic device containing microfluidic network 112.

[0067] 6 , the pressure of gas 156 by the separation gas chamber of microfluidic network 112 is increased, for example, by compressing the separation gas chamber as described for separation gas chamber 56 of microfluidic device 10, thereby forcing gas 156 through separation gas channel 130 and into separation channel 128 via gas input opening 130′. The input of gas 156 into separation channel 128 forms initial gas bubble 141, which separates liquid segment 155 from the remaining liquid portion 157 in microfluidic network 112. Initial gas bubble 141 and liquid segment 155 form gas-liquid interface 158, and initial gas bubble 141 and remaining liquid 157 form gas-liquid interface 159.

[0068] The liquid in liquid segment 155 essentially consists of the amount of sample liquid 150 disposed within microfluidic network 112 between gas input opening 130' and distal air-liquid interface 152. The volume of liquid segment 155 is typically about 0.75 μL to about 4 μL, e.g., about 1.5 μL to about 3 μL, e.g., about 2 μL. A smaller amount of sample liquid, e.g., about 150 nL to about 5 nL, e.g., about 350 nL, can enter vent 132 and is not considered part of liquid segment 155. The liquid in remaining liquid portion 157 is the amount of sample liquid 150 disposed within microfluidic network 112 between gas input opening 130' and application zone 14.

[0069] 7-9, the pressure of gas 156 in the separation gas chamber of microfluidic network 112 further increases, increasing the volume of initial bubble 141 to final bubble 141′. Because walls 128′, 128″ taper distally from the location of gas-liquid interface 158 (e.g., the width of separation channel 128 between walls 128′, 128″ decreases distally along separation channel 128 from the location of gas-liquid interface 158), the radius of curvature r1 of gas-liquid interface 158 must decrease as gas-liquid interface 158 moves distally as additional gas is admitted from the separation gas chamber through opening 130′. Radius of curvature r1 is defined in a plane parallel to both the longitudinal axis of the separation channel and the greater of the width or height of the separation channel at the location of gas-liquid interface 158. 5-9, the radius of curvature r1 lies in a plane parallel to the longitudinal axis and width of the separation channel, since the width is greater than the height at the location of the gas-liquid interface 158. In embodiments, the radius of curvature of each interface is measured along a radial axis aligned with the longitudinal axis of the channel and perpendicular to the width of the channel at the location of the interface, and the radius of curvature of the interface may also be parallel to the plane of a substantially flat microfluidic device.

[0070] As liquid segment 155 is moved distally along the separation channel, the radius of curvature of gas-liquid interface 158 decreases, thereby increasing the surface tension and energy of this interface. Gas-liquid interface 158 therefore resists distal movement and remains essentially in the same position along segment separation channel 128 as additional separation gas is introduced. Because gas-liquid interface 158 remains essentially in the same position, liquid segment 155 also remains essentially in the same position, and essentially no additional liquid enters vent 132.

[0071] However, the gas-liquid interface 159 can move proximally within the segment separation channel 128 as the width of the channel 128 widens proximally, thereby increasing the radius of curvature of the interface 159, until the interface 159 forms an interface 159' having a radius of curvature r2 (r2 > r1). The radius of curvature r2 may 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 and 159' of the bubble 141'. For example, the ratio r2 / r1 may be approximately the same as the ratio w2 / w1 when the interface is at positions w2 and w1, as shown in FIG. 7. Increasing the radius of curvature of the interface 159' reduces the surface tension and energy of the interface 159' compared to the gas-liquid interface 159 of the initial bubble 141. Thus, in some cases, the required pressure or energy of the separation gas within segment separation channel 128 to move liquid segment 155 is greater than the remaining liquid 157 because the cross-sectional area of ​​gas-liquid interface 158 exposed to the separation gas pressure or energy is smaller compared to the larger cross-sectional area of ​​gas-liquid interface 159'. Thus, the incoming separation gas preferentially moves the remaining liquid 157 proximally rather than moving liquid segment 155 distally. The distance along the microchannel between gas-liquid interface 158 and gas input opening 130' is distance d1, the distance along the microchannel between gas-liquid interface 159' and gas input opening 130' is distance d2, and the ratio d2 / d1 is at least about 2.25, e.g., about 2.25 to 10, e.g., about 4.5. For example, d1 can be about 250 μm to 1000 μm, such as about 500 μm, and d2 can be about 1000 μm to about 2750 μm, such as about 2000 μm.

[0072] After separating the liquid segment 155 from the remainder 157 of the sample liquid 150 and forming the gas bubble 141′, the reader actuates an oscillation in the pressure of the gas 154 in the gas chamber of the microfluidic network 112 to facilitate mixing of the reagents in contact with the liquid segment 155. The oscillation may be performed as disclosed in the '325 and '858 applications for the device 10, for example, including the use of a piezoelectric actuator. The pressure of the gas 154 is then reduced, drawing the liquid segment 155 distally into contact with additional reagents 160 in the detection region 118″ of the analysis channel 118. The additional reagents 160 typically include one or more reagents configured to bind to a target in the sample. For example, the reagents 160 may include one or more different particles, including a binding agent, such as an antibody, for the target. The particles may include, for example, magnetic particles and fluorescent particles, and the particles may be configured to form a detectable sandwich with the target. The target may then be detected in the detection region 118″, for example, optically or electrochemically.

[0073] In embodiments, the microfluidic device 210 comprises two or more microfluidic networks 112, each of which may be configured to receive a sample liquid, mix it with one or more reagents, and separately perform an analysis on the sample liquid, e.g., to detect the presence or absence of a target in the sample liquid. In some embodiments, each microfluidic network comprises the same components as described herein for the microfluidic network 112, including, for example, the supply channel 16, the tapered-segment separation channel 128, the analysis channel 118 including the reagent zone 118′ and the detection zone 118″, the gas chamber, the separation gas chamber, the separation gas channel, the vent 132, and / or a hydrophobic layer in the form of a hydrophobic strip 133 that may be disposed on the inner surface of the analysis channel 118 and can act as a hydrophobic stop. In some embodiments, the supply channel portions for each microfluidic network branch off from a common channel extending from the application zone 14. Thus, two or more microfluidic networks are configured to simultaneously analyze the sample liquid.

[0074] Two or more microfluidic networks may be located adjacent to one another on a microfluidic device. In embodiments, at least some of the microfluidic networks have the same reagents to provide redundant analysis of the sample liquid. Such redundancy can serve as a check in case of any contamination or failure in a given microfluidic network and can also provide additional accuracy and tunability for any sensitivity with the sample liquid.

[0075] In some cases, at least some of the microfluidic networks have one or more different reagents, which allows the microfluidic device to detect one or more different targets in the sample liquid, and the detection of such different targets may occur simultaneously.

[0076] 10-16, 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, e.g., blood, and the other liquid is a diluent, such as a buffer solution. Other liquids may be used. Microfluidic device 210 includes a microfluidic network having a sample input zone 214, a primary sample input channel 216, a hematocrit input channel 216″, a hematocrit detection chamber 217, a secondary sample input channel 216′, a segment combining chamber 228, an analysis channel 218, a diluent application zone 219, and a diluent input channel 221.

[0077] First gas chamber 220 communicates with a distal portion of analysis channel 218. Gas chamber 220 may be configured as gas chamber 20 of device 10. Bonding gas chamber 226 communicates with segmented bonding chamber 228 via segmented gas channel 241 at segmented gas opening 241′, which may have the same dimensions as opening 130′ of microfluidic network 112.

[0078] Device 210 also includes electrical features for monitoring the presence of sample and diluent and for monitoring the compression state of gas chambers 220 and 226. Signal-emitting lead 229 includes signal-emitting electrodes 229', 229'', 229''', and 229'''' disposed within the microfluidic network so as to be in electrical communication with liquid present at each electrode's respective location. Signal-emitting lead 229 and corresponding electrodes are configured to emit time-varying signals as disclosed in the '325 application. Device 210 includes electrical sensing functionality including sensing electrode 251, sensing electrode 227, sensing electrode 223, sensing electrode 255, sensing electrode 231, sensing electrode 233, and sensing electrode 257. The aforementioned sensing electrodes cooperate with the emitting electrodes to detect the presence of liquid at each sensing electrode location as disclosed in the '325 application. Each of the emitting and sensing electrodes is disposed on an interior surface of the microchannel network of device 210. Additionally, microfluidic network 210 includes hydrophobic strips above or below sensing electrode 223 and above or below emitting electrode 229'''', which are obscured by the sensing or emitting electrodes in the figures. Each hydrophobic strip cooperates with sensing electrode 223 and emitting electrode 229'''' to form a hydrophobic barrier, e.g., a capillary stop, that prevents liquid from migrating distally or proximally beyond a location by capillary flow.

[0079] Operation of strip 210 proceeds as follows. Prior to operation, gas chamber 226 is compressed as described for gas chamber 26 of device 10. As seen in FIG. 10 , a blood sample is applied to application zone 214 and flows by capillary flow along primary sample input channel 216. A portion of the blood flows along hematocrit input channel 216″ and fills hematocrit detection chamber 217. Proper filling of chamber 217 with blood is confirmed by detecting a signal emitted by emitting electrode 229′ via sensing electrode 257. A second portion of the blood flows along secondary sample input channel 216 and forms blood segment 261 within the volume of segment coupling chamber 228, which is disposed between sensing electrode 223 (and corresponding hydrophobic strip) and emitting electrode 229′″ (and corresponding hydrophobic strip). The length along chamber 228 between sensing electrode 223 and emitting electrode 229'''' is about 500 μm to 2000 μm, e.g., about 1400 μm. The width of chamber 228 between sensing electrode 223 and emitting electrode 229'''' is about 500 μm to 1750 μm, e.g., about 1000 μm. The height within the chamber is about 50 μm to about 200 μm, e.g., about 110 μm. The volume of blood segment 261 is determined by the above dimensions and is typically about 75 nL to 500 nL, e.g., about 150 nL. Proper filling of blood is confirmed by detecting the signal emitted by emitting electrode 229'''' via sensing electrode 233.

[0080] 11 , for example, as described for gas chamber 20, the pressure of the gas in gas chamber 220 increases. The increase in gas pressure forces blood segment 261 proximally within chamber 228 past emitting electrode 229′″ (and corresponding hydrophobic strip) until proximal air-liquid interface 271 of blood segment 261 contacts sensing electrode 227, thereby placing sensing electrode 227 in electrical communication with emitting electrode 229′″. Upon detecting the presence of blood segment 261 at sensing electrode 227, compression of gas chamber 220 ceases.

[0081] 12a and 12b, diluent 267 (e.g., buffer solution) is introduced into diluent application zone 219. Diluent 267 flows along diluent introduction channel 221 by capillary action until the distal gas-liquid interface of diluent 267 migrates distally beyond vent 232. As best seen in FIG. 12b, where sensing electrode 227 is shown partially transparent for clarity, proximal interface 271 of blood segment 261 and distal interface 269 of diluent 267 are separated by a small amount of gas (e.g., air). The pressure of the gas in gas chamber 226 decreases, drawing the gas separating proximal interface 271 and interface 269, as well as the small amount of diluent 267, into channel 241 through opening 241′, thereby allowing blood segment 261 to contact and / or fuse with diluent 267. The hydrophobic strip and emitting electrode 229''' prevent the proximal interface 271 of the blood segment 261 from moving proximally when the pressure of the gas in the gas chamber 226 decreases, thereby maintaining the volume and position of the blood segment 261. Instead, the distal interface 269 of the diluent 267 moves distally along the segment joining chamber 228 until the interface 269 merges with the interface 271 of the blood segment 261.

[0082] Referring to FIG. 13 , the pressure of the gas in the gas chamber 220 decreases, drawing the blood segment 261 and diluent 267 distally along the analysis channel 218 until the distal gas-liquid interface 271′ establishes electrical communication between the emitting electrode 229′″ and the sensing electrode 255, at which point the presence of liquid is detected and operation of the gas chamber 220 ceases.

[0083] 14, the pressure of the gas in separation gas chamber 226 increases, forcing gas into chamber 228 through channel 241 and opening 241′, thereby dividing diluent 267 into diluent segment 275 and remainder portion 277. The division of diluent 267 into diluent segment 275 and remainder portion 277 occurs after fusing distal interface 269 of diluent 267 with interface 271 of blood segment 261.

[0084] 15, the pressure of the gas in gas chamber 220 is again reduced, drawing blood segment 261 and diluent segment 275 distally along analysis channel 218 until distal gas-liquid interface 271′ of blood segment 261 contacts sensing electrode 231, establishing electrical communication between sensing electrode 231 and emitting electrode 229′″, at which point the presence of liquid is detected and operation of gas chamber 220 is stopped. Analysis channel 218 may contain reagents to facilitate target determination, for example, reagents for determining the HbA1c content of blood segment 261. After stopping movement of blood segment 261 and diluent segment 275, the pressure of the gas in chamber 220 and / or separation gas chamber 226 is oscillated as described for device 10, thereby mixing blood segment 261, diluent segment 275, and such reagents to form sample segment 279. The oscillations may also be performed during at least a portion, e.g., most or all, of the time that blood segment 261 and diluent segment 275 travel distally. Exemplary suitable reagents and processes for determining HbA1c are disclosed in U.S. Patent Application No. 17 / 409,279, filed August 23, 2021, which is incorporated herein by reference in its entirety.

[0085] 16, the pressure of the gas in gas chamber 220 is again reduced, drawing sample segment 279 distally along analytical channel 218 until distal gas-liquid interface 271'' of sample segment 279 contacts sensing electrode 233, establishing electrical communication between sensing electrode 233 and emitting electrode 229''', at which point the presence of liquid is detected and the action of oscillating gas chamber 220 ceases. Analytical channel 218 may contain additional reagents disposed between sensing electrodes 231 and 233 that are contacted and mobilized by sample segment 279. The gas pressure in gas chamber 220 may be oscillated to enhance mixing of such reagents with sample segment 279. The presence of a target, e.g., HbA1c, is then detected.

[0086] 17-19, a microfluidic device 310, according to some embodiments, is configured to receive a particulate-containing liquid, separate at least a portion of the liquid from the particulates, form a segment containing the portion of the liquid separated from the particulates, and determine the presence or absence of at least one target within the liquid in the separated segment. For example, the particulate-containing liquid can be whole blood, the separated liquid can be plasma separated from red blood cells of the whole blood, and the at least one target can be a cardiac marker such as troponin I or troponin C.

[0087] The microfluidic device 310 includes a substantially planar substrate 311 defining a microfluidic network 312 therein. Beginning with a sample application zone 314 and proceeding from proximal to distal, the microfluidic network 312 may include a microfluidic channel having a supply channel portion 316, a segmented 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 overlies the sample application zone 314. The microfluidic device may further include i) electrical features such as electrodes for determining 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, such features are not shown in FIGS. 17 and 18. The dimensions, e.g., width, height, and length, as well as the function and operation, 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 device 10, 210 and the microfluidic network 112, for example.

[0088] 18, substrate 311 may include upper and lower layers 313 and 315 bonded to each other by intermediate layer 317. Upper and lower layers 313 and 315 may be formed from a polymer, such as polyester, and each may have a typical total thickness of about 80-130 μm, e.g., about 100 μm. Intermediate layer 317 may be formed from a central layer of polymer, such as polypropylene, with upper and lower layers of adhesive. Intermediate layer 317 may have a typical total thickness of about 80-150 μm, e.g., about 110 μm.

[0089] The lower interior surface of the microfluidic channel and application zone 314 may be defined by the upper surface 315' of the lower layer 315. The upper interior surface of the microfluidic network 312, including the microfluidic channel 316, may be defined by the lower surface 313' of the upper layer 313. The upper interior surface of the application zone 314 may be defined by the lower surface 319' of the porous membrane. The interior sidewall 314' of the application zone 314 and the opposing interior sidewalls 312', 312'' of the microfluidic channel 316 may be defined by the middle layer 317.

[0090] The upper layer 313 is defined by a sidewall 313'' and includes a first coating zone opening 321 having a maximum diagonal d1 (FIGS. 18 and 19) along an axis parallel to the plane of the substrate 311. The middle layer 317 defines an interior sidewall 314' and includes a second coating zone opening having a maximum diagonal d2 (FIGS. 18 and 19) along an axis parallel to the plane of the substrate 311. The first coating zone opening d1 in the upper layer 313 is larger than the second coating zone opening d2 in the middle layer 317, such that the first coating zone opening d1 exposes a peripheral portion 325 of the adhesive upper surface 317'. A peripheral portion of the lower surface 319' of the porous membrane is adhered to the peripheral portion 325 of the adhesive upper surface 317'. For example, porous membrane 319 may be secured to substrate 311 by positioning porous membrane 319 such that a peripheral portion of lower surface 319' of porous membrane 319 contacts and rests on peripheral portion 325 of adhesive upper surface 317'. Pressure and / or heat may be applied to secure porous membrane 319 and substrate 311 together. Peripheral portion 325 has a radial width d3 (FIG. 19) sufficient to bond porous membrane 319 to intermediate layer 317. Width d3 is typically between about 1 mm and 3 mm, e.g., about 2 mm.

[0091] The sample application zone 314 and the microfluidic channel 316 intersect at an intersection 327 from which the microfluidic channel 316 extends. A 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 portion 331 is beneath the porous membrane 319.

[0092] Intersection 327 defines a width w6 between first and second opposing interior sidewalls 312′, 312″ of microfluidic channel 316 at intersection 327. Width w6 is taken along a direction oriented approximately perpendicular to the longitudinal axis of microfluidic channel 316 at intersection 327 and parallel to a plane defined by microfluidic device 310. Width w6 may be, for example, about 1.5 mm. Microfluidic channel 316 defines a width w9 adjacent intersection 327. The width ratio w6 / w9 is typically about 1-3, e.g., about 2.

[0093] 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 typically less than about 1 mm, e.g., about 0.25 mm or about 0.5 mm. The protruding portion 331 has an overall length d4 taken from the sidewall 313'' of the first application zone opening 321 to the tip 361 of the protruding portion 331.

[0094] The width w7 of the protruding portion 331 at 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 about 20% to 75% of the width w6 of the intersection 327. The protruding portion 331 tapers from the width w7 of the intersection 327 to a smaller width w8 located within the sample application zone 314. The width ratio w8 / w7 is typically about 0.5 to 0.9, for example, about 0.75.

[0095] In embodiments, the protruding portions aid in the alignment of the porous membrane, which may help prevent or reduce the risk of the porous membrane blocking or at least partially blocking the opening to the microfluidic channel 316, for example.

[0096] The microfluidic device 310 includes a vent channel 350 extending from a vent intersection 351 between the sample application zone 314 and the vent channel 350. A distal portion 352 of the vent channel 350 is in gas communication with the ambient gas surrounding the substrate such that gas disposed within the sample application zone 314 can exit the sample application zone 314 through the vent channel 350 and / or ambient gas surrounding the microfluidic device 310 can enter the sample application zone 314 through the vent channel 350. A lower interior surface of the vent channel 350 is defined by an upper surface 315′ of the lower layer 315. An upper interior surface of the vent channel 350 is defined by a lower surface 313′ of the upper layer 313. Opposing interior sidewalls of the vent channel 350 are defined by the middle layer 317. A protruding portion 353 of the upper layer 313 that defines the upper inner surface of the vent channel 350 protrudes beyond the vent intersection 351 into the sample application zone 314 .

[0097] The microfluidic device 310 may operate as follows: A microparticle-containing liquid, e.g., blood, is applied to the upper surface of the porous membrane 319. The microparticles are retained on and / or within the porous membrane 319. The separated liquid, e.g., plasma, passes through the porous membrane 319 and into the sample application zone 314 below. The separated liquid passes through intersection 327 and into the microfluidic channel 316. Ambient gas enters and exits the sample application zone 314 via vent 352, thereby equalizing pressure therein as the separated liquid enters and exits the sample application zone 314. At least a portion of the separated liquid flows into the segment separation channel portion 328. A separated liquid segment is formed, for example, as disclosed for the embodiment of Figures 1-9. One or more targets are detected within the liquid in the liquid segment.

[0098] **** Numbered Embodiments Embodiment 1: A microfluidic device comprising a substantially planar substrate comprising a microfluidic network therein, the microfluidic network comprising: i) a distal gas chamber configured to adjust 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 input location, the separation gas chamber A microfluidic device configured to adjust the pressure of a separation gas so that sample liquid flows from an application zone through at least a portion of a microfluidic channel to a capillary stop located distal to a separation gas input location, such that when the separation gas is input into the microfluidic channel, a separation bubble is subsequently formed in the microfluidic channel, causing the microfluidic channel to separate the sample liquid therein into i) a liquid segment located distal to the separation bubble, and ii) a remaining amount of sample liquid located proximal to the separation bubble, and the liquid segment forms 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.

[0099] Embodiment 2: The microfluidic device of claim 1, wherein the microfluidic channel comprises a reagent zone distal to the separation gas input location and configured to contain one or more reagents.

[0100] Embodiment 3: The microfluidic device of claim 2, wherein the reagent is configured to solubilize with the sample liquid upon contact therewith.

[0101] Embodiment 4: The microfluidic device of claim 2 or 3, wherein the reagent is configured to allow detection of a target in the sample liquid upon contact therewith.

[0102] Embodiment 5: The microfluidic device of claim 4, wherein the microfluidic channel comprises a detection zone distal to the reagent zone and configured to detect the presence or absence of a target in the sample liquid.

[0103] Embodiment 6: The microfluidic device of 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 therebetween configured to be occupied by a distal gas.

[0104] Embodiment 7: The microfluidic device of claim 6, wherein the outer wall of the distal gas chamber is configured to contact a vibration member to vibrate and / or adjust the distal chamber spacing, thereby vibrating and / or adjusting the pressure of the distal gas.

[0105] Embodiment 8: The microfluidic device of claim 7, wherein adjusting the pressure of the distal gas facilitates movement of the liquid segment within the microfluidic channel.

[0106] Embodiment 9: The microfluidic device of claim 7, wherein the vibration member is configured to contact the outer wall of the distal gas chamber at a position spaced apart from the distal liquid-air interface by a distance along the microfluidic channel of at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

[0107] Embodiment 10: A microfluidic device according to any one of claims 1 to 9, wherein the separation gas chamber is configured to heat the separation gas therein to pressurize the separation gas and facilitate its introduction into the microfluidic channel to form a separation bubble.

[0108] Embodiment 11: A microfluidic device according to any one of claims 1 to 9, wherein the separation gas chamber comprises a third inner wall, a fourth inner wall, and a separation gas chamber spacing therebetween configured to be occupied by a separation gas.

[0109] Embodiment 12: The microfluidic device of claim 11, wherein the outer wall of the separation gas chamber is configured to contact a second vibration member to vibrate and / or adjust the separation gas chamber spacing, thereby vibrating and / or adjusting the pressure of the separation gas.

[0110] Embodiment 13: A microfluidic device according to any one of claims 1 to 13, wherein the separation bubble defines a volume of separation gas disposed between the proximal gas-liquid interface and the gas-liquid interface between the remaining volume of sample liquid and the separation bubble (RVSL gas-liquid interface), and the separation bubble is disposed within a separation zone of the microfluidic channel.

[0111] Embodiment 14: The microfluidic device of claim 13, wherein the separation zone of the microfluidic channel tapers distally from a larger cross-sectional area to a smaller cross-sectional area.

[0112] Embodiment 15: The microfluidic device of claim 13 or 14, wherein the proximal gas-liquid interface occupies a portion of the microfluidic channel having a cross-sectional area A1, and the RVSL gas-liquid interface occupies a portion of the microchannel having a cross-sectional area A2, A2 being greater than A1, such that the separation bubble comprises an asymmetric shape.

[0113] Embodiment 16: The microfluidic device of claim 15, wherein when separation gas is introduced into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is smaller than the radius of curvature of the RVSL gas-liquid interface, such that when further separation gas is introduced, the RVSL gas-liquid interface moves proximally but the proximal gas-liquid interface remains at the same or substantially the same position within the microfluidic channel, such that the liquid segments i) remain at the same or substantially the same position within the microfluidic channel and / or ii) have the same or substantially the same amount of volume within the microfluidic channel.

[0114] Embodiment 17: The ratio of A2 to A1 ("R A 17. The microfluidic device of claim 15 or 16, wherein the ρ is at least about 1.25.

[0115] Embodiment 18: Cross-sectional area A1 is about 0.04 mm 2 ~about 0.13mm 2 18. The microfluidic device according to claim 15, wherein:

[0116] Embodiment 19: A microfluidic device according to any one of claims 15 to 18, wherein the distance along the longitudinal axis of the microfluidic channel between the RSVL and the proximal air-liquid interface is about 1 to 3 mm.

[0117] Embodiment 20: The cross-sectional area of ​​the microfluidic channel between the RSVL and the proximal air-liquid interface is about 12% mm -1 ~approx. 25% mm -1 20. The microfluidic device according to claim 14, wherein the average rate of the decrease is 0.01.

[0118] Embodiment 21: A microfluidic device according to any one of claims 14 to 20, wherein the distance along the microfluidic channel between the RSVL gas-liquid interface and the separation gas input location is distance d1, the distance along the microfluidic channel between the proximal gas-liquid interface and the separation gas input location is distance d2, and the ratio d2 / d1 is between about 2.25 and 10.

[0119] Embodiment 22: A microfluidic device described in any one of claims 14 to 21, wherein the proximal gas-liquid interface is located within the separation zone, and the sample liquid of the liquid segment is substantially located within a reagent zone or detection zone of the microfluidic channel located distal to the separation zone, and the reagent zone or detection zone has a cross-sectional area A3 larger than the cross-sectional area A1.

[0120] Embodiment 23: A 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 and lower layers.

[0121] Embodiment 24: The microfluidic device of claim 23, wherein the middle layer comprises an upper surface having an adhesive that adheres to the upper layer, a lower surface having an adhesive that adheres to the lower layer, or both.

[0122] Embodiment 25: A microfluidic device according to claim 23 or 24, wherein the upper surface of the underlying layer defines the lower inner surface of the microfluidic channel and application zone.

[0123] Embodiment 26: A 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.

[0124] Embodiment 27: The microfluidic device of any one of claims 23 to 26, wherein the upper layer, the lower layer, the middle layer, or any combination thereof, comprises an opening that defines the application zone.

[0125] Embodiment 28: The microfluidic device of any one of claims 23 to 27, further comprising a porous membrane configured to overlie the application zone.

[0126] Embodiment 29: The microfluidic device of claim 28, wherein the porous membrane is configured to separate the sample liquid from the one or more microparticles.

[0127] Embodiment 30: A microfluidic device according to any one of claims 23 to 29, further comprising protrusions extending from the upper layer and / or the middle layer into the application zone.

[0128] Embodiment 31: A microfluidic device according to any one of claims 1 to 30, wherein the microfluidic device further comprises one or more further microfluidic networks, each of the one or more further microfluidic networks configured to separately receive a portion of the sample liquid from the application zone and analyze the sample liquid to detect a target therein.

[0129] Embodiment 32: The microfluidic device of claim 31, wherein two or more of the microfluidic channel and the one or more further microfluidic channels are configured to analyze a sample liquid simultaneously or sequentially.

[0130] Embodiment 33: A microfluidic device as described in claim 31 or 32, wherein at least one of the one or more further microfluidic channels is configured in the same manner as the microfluidic network as described in any one of claims 1 to 30.

[0131] Embodiment 34: a) introducing a sample liquid into a microfluidic channel in a microfluidic device, the microfluidic channel including a distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal liquid-gas interface therebetween; and 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 a remaining amount of the sample liquid introduced into the microfluidic channel, the liquid segment comprising: (i) a 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; A method comprising: c) separating a proximal gas-liquid interface of a liquid segment; c) moving the liquid segment to a reagent zone of a microfluidic channel by reducing the pressure of a distal gas, the reagent zone comprising at least one reagent disposed therein; and d) mixing a portion of the sample liquid in 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 reducing the pressure of the distal gas, ii) simultaneously with reducing the pressure of the distal gas, and / or iii) after reducing the pressure of the distal gas.

[0132] Embodiment 35: A method for analyzing a sample liquid to detect at least one target material therein, the method comprising: a) introducing the sample liquid into a microfluidic channel in 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 a portion of the sample liquid contacts at least one reagent disposed in a reagent zone of the microfluidic channel; and c) injecting a separation gas into the microfluidic channel at a location occupied by the sample liquid. 1. A method comprising: introducing, into a microfluidic channel, and thereby separating a segment of sample liquid from a remaining amount of sample liquid introduced into the microfluidic channel, the liquid segment comprising: (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 a separation gas and the portion of the sample liquid, the separation gas separating the proximal gas-liquid interface of the liquid segment from the remaining amount of sample liquid; and d) mixing the portion of the sample liquid in 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.

[0133] Embodiment 36: The method of claim 35, wherein moving the sample liquid is via capillary action.

[0134] Embodiment 37: The method of claim 35, wherein moving the sample liquid is via reducing the pressure of the distal gas.

[0135] Embodiment 38: The method of claim 37, wherein oscillating the pressure of the distal gas and / or the separation gas is performed i) before reducing the pressure of the distal gas, ii) simultaneously with reducing the pressure of the distal gas, and / or iii) after reducing the pressure of the distal gas.

[0136] Embodiment 39: The method of any one of claims 34 to 38, wherein oscillating the pressure of the distal gas and / or 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.

[0137] Embodiment 40: The step of vibrating includes oscillating the pressure of the distal gas, and during the step of vibrating, the distal liquid-gas interface 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 40. The method of claim 34, wherein the microfluidic channel occupies a position having a cross-sectional area of

[0138] Embodiment 41: The step of vibrating includes oscillating the pressure of the distal gas, and during the step of vibrating, the distal liquid-gas interface is adjusted to about 0.15 mm 2 Below, approximately 0.125mm 2 Below, approximately 0.1mm 2 Below, approximately 0.09mm 2 Less than or equal to 0.08 mm 2 41. The method according to any one of claims 34 to 40, wherein the position of a channel having a cross-sectional area of:

[0139] Embodiment 42: The method of any one of claims 34 to 41, wherein during the vibration step, the liquid segment has a volume of 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.

[0140] Embodiment 43: The method of any one of claims 34 to 42, wherein during the vibration 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.

[0141] Embodiment 44: The method of any one of claims 34 to 43, wherein introducing the sample liquid comprises moving the sample liquid by capillary action along the microfluidic channel until the distal sample liquid-gas interface contacts a capillary stop in the microfluidic channel.

[0142] Embodiment 45: The method of claim 44, wherein introducing the sample liquid comprises moving the sample liquid by capillary action along the microfluidic channel until the distal liquid-gas interface reaches and moves beyond the position where the separation gas is introduced.

[0143] Embodiment 46: The method of claim 43 or 44, wherein the capillary stop comprises one or more vents that provide gas communication between the microfluidic channel and a volume of gas disposed outside the microfluidic channel.

[0144] Embodiment 47: The method of claim 46, wherein the volume of gas comprises ambient air surrounding the microfluidic device.

[0145] Embodiment 48: A method according to any one of claims 34 to 47, wherein after the sample liquid is introduced into the microfluidic channel, a distal gas occupies a chamber of the microfluidic device that is isolated with respect to the ambient gas surrounding the microfluidic device.

[0146] Embodiment 49: The method of claim 34-45 or 48, wherein prior to introducing the liquid sample into the microfluidic channel, the microfluidic channel provides a sole pathway for gas communication between a distal gas and the exterior of the microfluidic device.

[0147] Embodiment 50: The method of any of claims 48 or 49, wherein after the liquid sample is introduced into the microfluidic channel, a distal gas occupies a chamber of the microfluidic device that is isolated relative to the ambient gas surrounding the microfluidic device.

[0148] Embodiment 51: The method of claim 46, wherein prior to loading the liquid sample into the microfluidic channel, the microfluidic channel and one or more vents provide the only path for gas communication between the distal gas and the exterior of the microfluidic device.

[0149] Embodiment 52: The method of any one of claims 48 to 51, wherein the ambient gas surrounding the microfluidic device is ambient air surrounding the microfluidic device.

[0150] Embodiment 53: The method of any one of claims 34 to 52, wherein oscillating the pressure of the distal gas comprises oscillating an 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.

[0151] Embodiment 54: The method of claim 53, wherein the region is a distal gas chamber of a microfluidic device and the first and second interior walls are interior walls of the chamber.

[0152] Embodiment 55: The method of claim 54, wherein vibrating the internal spacing between the first wall and the second wall of the distal gas chamber comprises vibrating the internal spacing at a location of the distal gas chamber spaced along the microfluidic channel from the distal liquid-gas interface.

[0153] Embodiment 56: The method of claim 55, wherein vibrating comprises contacting an outer wall of the distal gas chamber with a vibrating member.

[0154] Embodiment 57: The method of claim 56, wherein the vibration member contacts the outer wall of the distal gas chamber at a position spaced apart from the distal liquid-gas interface by a distance along the microfluidic channel of at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

[0155] Embodiment 58: The method of any one of claims 34 to 57, wherein after the liquid sample is introduced into the microfluidic channel, a separation gas occupies a separation gas chamber of the microfluidic device that is sealed against the ambient gas surrounding the microfluidic device.

[0156] Embodiment 59: The method of claim 58, wherein the ambient gas is air. Embodiment 60: The method of claim 58 or 59, wherein introducing the separation gas comprises heating the separation gas in a separation gas chamber.

[0157] Embodiment 61: The method of any one of claims 34 to 60, wherein the step of introducing a separation gas comprises increasing the pressure of the separation gas in a separation gas chamber.

[0158] Embodiment 62: The method of claim 61, wherein the method further comprises 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.

[0159] Embodiment 63: The method of any one of claims 34 to 62, wherein the method further comprises operating the microfluidic device using an instrument, and the method is performed without introducing any gas into the microfluidic channels from a gas source of the instrument.

[0160] Embodiment 64: A method according to any one of claims 34 to 63, wherein the only gas present in the microfluidic device before the sample liquid is introduced into the microfluidic channel is ambient air, and during the performance of the method the distal gas and the separation gas consist of the ambient air that was present in the microfluidic device before the sample liquid was introduced therein.

[0161] Embodiment 65: The method of any one of claims 34 to 64, wherein the microfluidic channel is a sample microfluidic channel and the step of introducing a separation gas comprises introducing the separation gas through a separation gas microfluidic channel that intersects with the sample microfluidic channel at a position occupied by the sample liquid.

[0162] Embodiment 66: The method of any one of claims 34 to 65, wherein the step of vibrating comprises simultaneously vibrating the pressure of the distal gas and the separation gas.

[0163] Embodiment 67: The method of claim 66, wherein the step of vibrating includes vibrating the pressure of the distal gas and the separation gas in phase with each other.

[0164] Embodiment 68: The method of claim 66, wherein the step of vibrating includes vibrating the pressures of the distal gas and the separation gas out of phase with each other.

[0165] Embodiment 69: A method according to any of claims 66 to 68, wherein the step of vibrating comprises oscillating the pressure of the distal gas and the separation gas at the same frequency and / or at different frequencies during at least a portion of the vibration.

[0166] Embodiment 70: The method of any one of claims 34 to 69, wherein the step of introducing a separation gas into the microchannel displaces at least a portion of the liquid sample from a separation zone in the microfluidic channel and forms a separation bubble therein, the separation bubble being positioned between the liquid segment and the remaining amount of sample liquid.

[0167] Embodiment 71: The method of claim 70, wherein the volume of the separation bubbles is at least about 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.

[0168] Embodiment 72: The method of claim 70, wherein the amount of separation bubbles 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.

[0169] Embodiment 73: The method of claim 71 or 72, wherein the amount of separation bubble defines the amount of separation gas that separates the proximal gas-liquid interface of the liquid segment from the gas-liquid interface between the remaining amount of sample liquid and the separation gas (the "RSVL gas-liquid interface").

[0170] Embodiment 74: The method of 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.

[0171] Embodiment 75: The method of any one of claims 70 to 74, wherein the proximal gas-liquid interface occupies a portion of a microfluidic channel having a cross-sectional area A1, and the RVSL gas-liquid interface occupies a portion of a microchannel having a cross-sectional area A2, A2 being greater than A1, such that the separation bubble comprises an asymmetric shape.

[0172] Embodiment 76: The method of claim 75, wherein when separation gas is introduced into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is smaller than the radius of curvature of the RVSL gas-liquid interface, such that when further separation gas is introduced, the RVSL gas-liquid interface moves proximally but the proximal gas-liquid interface remains in the same or substantially the same position, such that the liquid segments i) remain in the same or substantially the same position within the microfluidic channel and / or ii) have the same or substantially the same amount of volume within the microfluidic channel.

[0173] Embodiment 77: The method of claim 76, wherein mixing a portion of the sample liquid with at least one reagent comprises mixing the same or substantially the same amount of volume with the at least one reagent, thereby providing a limited reagent concentration within the portion of the sample liquid.

[0174] Embodiment 78: The ratio of A2 to A1 ("R A 78. The method of any one of claims 75 to 77, wherein the σ is at least about 1.25.

[0175] Embodiment 79: Cross-sectional area A1 is about 0.04 mm 2 ~about 0.13mm 2 The method according to any one of claims 75 to 78, wherein

[0176] Embodiment 80: The method of any one of claims 75 to 79, wherein the distance along the longitudinal axis of the microfluidic channel between the RSVL and the proximal air-liquid interface is about 1 to 3 mm.

[0177] Embodiment 81: The cross-sectional area of ​​the microfluidic channel between the RSVL and the proximal air-liquid interface is about 12% mm -1 ~approx. 25% mm -1 The method according to any one of claims 74 to 80, wherein the decrease is at an average rate of

[0178] Embodiment 82: The method of any one of claims 74 to 81, wherein the distance along the microfluidic channel between the RSVL gas-liquid interface and the position where the separation gas is injected into the microfluidic channel is distance d1, the distance along the microfluidic channel between the proximal gas-liquid interface and the position where the separation gas is injected into the microfluidic channel is distance d2, and the ratio d2 / d1 is about 2.25 to 10.

[0179] Embodiment 83: A method according to any one of claims 74 to 82, wherein the proximal gas-liquid interface is located within a separation zone, a portion of the sample liquid of the liquid segment is located substantially within an analysis zone of a microfluidic channel located distal to the separation zone, and the analysis zone has a cross-sectional area A3 greater than the cross-sectional area A1.

[0180] Embodiment 84: The method of any one of claims 34 to 83, further comprising analyzing a portion of the sample liquid to detect the presence or absence of the target therein.

[0181] Embodiment 85: The method of any one of claims 34 to 84, wherein at least one reagent comprises a binding reagent capable of specifically binding to a target in a portion of the sample liquid, and mixing the portion of the sample liquid with the at least one reagent allows binding of the binding reagent to the target to be detected and / or determined, making it possible to detect and / or determine the amount of binding reagent bound to the target.

[0182] Embodiment 86: The method of any one of claims 34 to 85, wherein at least one reagent is placed in a dry state in the microfluidic channel before the sample liquid is introduced into the microfluidic channel.

[0183] Embodiment 87: The method of claim 86, wherein the at least one reagent is configured to solubilize with the sample liquid upon contact therewith.

[0184] Embodiment 88: A method according to any one of claims 34 to 87, wherein the microfluidic device further comprises one or more further microfluidic channels, each of which is configured to carry out a method according to any one of claims 35 to 87 to individually analyze a sample liquid.

[0185] Embodiment 89: The method of claim 88, wherein two or more of the microfluidic channel and the one or more further microfluidic channels are configured to analyze the sample liquid simultaneously or sequentially.

[0186] Embodiment 90: The method of claim 88 or 89, wherein at least one of the one or more further microfluidic channels comprises a respective separation gas chamber configured to i) introduce a respective separation gas into the respective further microfluidic channel of the one or more further microfluidic channels, and ii) oscillate the respective pressure of the respective separation gas.

[0187] Embodiment 91: A method according to any one of claims 88 to 90, wherein at least one of the one or more further microfluidic channels comprises a respective distal gas chamber configured to: i) reduce the pressure of a respective distal gas to move a respective liquid segment distally along the respective further microfluidic channel of the one or more further microfluidic channels; and ii) oscillate the respective pressure of the respective distal gas.

[0188] Embodiment 92: a) introducing a quantity of sample liquid into a microfluidic channel in a microfluidic device, the microfluidic channel including a distal gas therein such that the sample liquid contacts the distal gas, thereby forming a distal sample liquid-gas interface therebetween; and b) introducing a separation gas into the microfluidic channel at a location occupied by the introduced quantity of sample liquid, thereby separating a segment of the sample liquid from a remaining quantity of the introduced quantity of sample liquid, the liquid segment comprising (i) a distal liquid-gas interface, and (ii) a proximal gas-liquid interface, the gas at the proximal gas-liquid interface being a separation gas, and the separation gas separating the liquid segment from the liquid-gas interface of the remaining quantity of the introduced quantity of 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 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 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 reducing the pressure of the distal gas, ii) simultaneously with reducing the pressure of the distal gas, and / or iii) after reducing the pressure of the distal gas.

[0189] Embodiment 93: A method for detecting at least one target material in a sample liquid, the method comprising: a) introducing a quantity of sample liquid into a microfluidic channel in a microfluidic device, the microfluidic channel comprising 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 a portion of the sample liquid contacts at least one reagent disposed in a reagent zone of the microfluidic channel; and c) introducing a separation gas into the microfluidic channel at a position occupied by the quantity of sample liquid, thereby separating a segment of the sample liquid from the remainder of the introduced quantity of sample liquid, a liquid segment having (i) a distal liquid-gas interface, (ii) a sample liquid in contact with at least one reagent, and (iii) a proximal gas-liquid interface, wherein the gas at the proximal gas-liquid interface is a separation gas, and the separation gas separates the proximal gas-liquid interface of the liquid segment from the liquid-gas interface of a remaining amount of the inputted quantity of sample liquid; and d) mixing 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 reducing the pressure of the distal gas, ii) simultaneously with reducing the pressure of the distal gas, and / or iii) after reducing the pressure of the distal gas.

[0190] Embodiment 94: A method according to any one of embodiments 92 to 93, wherein the oscillating pressure of the distal gas and / or 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.

[0191] Embodiment 95: The step of vibrating includes oscillating the pressure of the distal gas, and during the step of vibrating, the distal liquid-gas interface 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 95. The method of any one of embodiments 92 to 94, wherein the pores occupy positions in a channel having a cross-sectional area of

[0192] Embodiment 96: The step of vibrating includes oscillating the pressure of the distal gas, and during the step of vibrating, the distal liquid-gas interface is adjusted to about 0.15 mm 2 Below, approximately 0.125mm 2 Below, approximately 0.1mm 2 Below, approximately 0.09mm 2 Less than or equal to 0.08 mm 2 96. The method of any one of embodiments 92 to 95, wherein the location of the channel having the following cross-sectional area is occupied:

[0193] Embodiment 97: The method of any one of embodiments 92 to 96, wherein during the vibration step, the liquid segment has a volume of 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.

[0194] Embodiment 98: The method of any one of embodiments 92 to 97, wherein during the vibration 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.

[0195] Embodiment 99: A method according to any one of embodiments 92 to 98, wherein the step of introducing a certain amount 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 in the microfluidic channel.

[0196] Embodiment 100: A method according to any one of embodiments 92 to 99, wherein the step of introducing the sample liquid comprises moving the sample liquid by capillary action along the microfluidic channel until the distal sample liquid-gas interface reaches and moves beyond the position where the separation gas is introduced.

[0197] Embodiment 101: A method according to any one of embodiments 92 to 100, wherein the capillary stop comprises one or more vents that provide gas communication between the microfluidic channel and a certain amount of gas disposed outside the microfluidic channel.

[0198] Embodiment 102: The method of any one of embodiments 92 to 101, wherein the amount of gas is ambient air surrounding the microfluidic device.

[0199] Embodiment 103: A method according to any one of embodiments 92 to 102, wherein after a certain amount of liquid sample is introduced into the microfluidic channel, a distal gas occupies a chamber of the microfluidic device that is isolated from the ambient gas surrounding the microfluidic device.

[0200] Embodiment 104: A method according to any one of embodiments 92 to 103, wherein before a volume of liquid sample is introduced into the microfluidic channel, the microfluidic channel provides the only path for gas communication between the distal gas and the outside of the microfluidic device.

[0201] Embodiment 105: A method according to any one of embodiments 92 to 104, wherein after a certain amount of liquid sample is introduced into the microfluidic channel, a distal gas occupies a chamber of the microfluidic device that is isolated from the ambient gas surrounding the microfluidic device.

[0202] Embodiment 106: A method according to any one of embodiments 92 to 105, wherein before a volume of liquid sample is introduced into the microfluidic channel, the microfluidic channel and one or more vents provide the only path for gas communication between the distal gas and the outside of the microfluidic device.

[0203] Embodiment 107: The method of any one of embodiments 92 to 106, wherein the ambient gas surrounding the microfluidic device is ambient air surrounding the microfluidic device.

[0204] Embodiment 108: A method according to any one of embodiments 92 to 107, wherein oscillating the pressure of the distal gas comprises oscillating an internal spacing between a first internal wall of the region of the microchannel occupied by the distal gas and a second internal wall of the region.

[0205] Embodiment 109: The method of any one of embodiments 92 to 108, wherein the region is a chamber of a microfluidic device and the first and second interior walls are interior walls of the chamber.

[0206] Embodiment 110: A method described in any one of embodiments 92 to 109, wherein vibrating the internal gap between the first wall and the second wall of the chamber comprises vibrating the internal gap at a position of the chamber spaced along the microchannel from the distal liquid-gas interface.

[0207] Embodiment 111: The method of any one of embodiments 92 to 110, wherein vibrating comprises contacting the outer wall of the chamber with a vibrating member.

[0208] Embodiment 112: A method according to any one of embodiments 92 to 111, wherein the vibration member contacts the outer wall of the chamber at a position spaced apart from the distal liquid-air interface by a distance along the microchannel of at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 15 mm.

[0209] Embodiment 113: A method according to any one of embodiments 92 to 112, wherein after a certain amount of liquid sample is introduced into the microfluidic channel, a separation gas occupies a chamber of the microfluidic device that is sealed against the ambient gas surrounding the microfluidic device.

[0210] Embodiment 114: The method of any one of embodiments 92 to 113, wherein the ambient gas is air.

[0211] Embodiment 115: The method of any one of embodiments 92 to 116, wherein introducing the separation gas comprises heating the separation gas in a chamber occupied by the separation gas.

[0212] Embodiment 116: The method of any one of embodiments 92 to 115, wherein the step of introducing the separation gas comprises increasing the pressure of the separation gas, and the method comprises decreasing the pressure of the distal gas during at least a portion of the step of increasing the pressure of the separation gas.

[0213] Embodiment 117: A method according to any one of embodiments 92 to 116, wherein the method comprises operating a microfluidic device using an instrument, and the method is carried out without introducing any gas into the microfluidic channels from a gas source of the instrument.

[0214] Embodiment 118: A method according to any one of embodiments 92 to 117, wherein the only gas present in the microfluidic device before the sample liquid is introduced into the microfluidic channel is ambient air, and during the execution of the method, the distal gas and the separation gas consist of the ambient air that was present in the microfluidic device before the sample liquid was introduced.

[0215] Embodiment 119: The method of any one of embodiments 92 to 118, wherein the microfluidic channel is a sample microfluidic channel and the step of introducing a separation gas comprises introducing the separation gas through a separation gas microfluidic channel that intersects with the sample microfluidic channel at a position occupied by the sample liquid.

[0216] Embodiment 120: The method of any one of embodiments 92 to 119, wherein the step of vibrating comprises simultaneously vibrating the pressure of the distal gas and the separation gas.

[0217] Embodiment 121: The method of any one of embodiments 92 to 120, wherein the step of vibrating comprises vibrating the pressure of the distal gas and the separation gas in phase with each other.

[0218] Embodiment 122: The method of any one of embodiments 92 to 121, wherein the step of vibrating comprises vibrating the pressure of the distal gas and the separation gas out of phase with each other.

[0219] Embodiment 123: A method according to any one of embodiments 92 to 122, wherein the step of vibrating includes oscillating the pressure of the distal gas and the separation gas at the same frequency and / or at different frequencies during at least a portion of the vibration.

[0220] Embodiment 124: A method according to any one of embodiments 92 to 123, wherein the step of injecting a separation gas into the microchannel displaces at least a portion of the liquid sample from the separation zone within the microchannel, and the amount of separation gas occupying the microchannel after injection is at least about 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.

[0221] Embodiment 125: A method according to any one of embodiments 92 to 124, wherein the step of injecting a separation gas into the microchannel displaces at least a portion of the liquid sample from the separation zone in the microchannel, and the amount of separation gas occupying the microchannel after injection is less than about 5 μL, less than about 3.5 μL, less than about 2.75 μL, less than about 1.75 μL, less than about 1.5 μL, less than about 1.25 μL, less than about 1 μL, less than about 0.75 μL, or less than about 0.5 μL.

[0222] Embodiment 126: A method according to any one of embodiments 92 to 125, wherein the amount of separation gas occupying the microchannel after introduction defines the amount of separation gas separating the liquid-gas interface of the remaining amount of sample liquid introduced from the proximal gas-liquid interface of the liquid segment.

[0223] Embodiment 127: The method of any one of embodiments 92 to 126, wherein at least one reagent comprises a binding reagent capable of specifically binding to the target, and the method further comprises binding the binding reagent to the target and detecting the amount of binding reagent bound to the target.

[0224] Embodiment 128: The method according to any one of embodiments 92 to 127, wherein at least one reagent is placed in the microchannel in a dry state before the amount of sample liquid is introduced.

[0225] Embodiment 129: A method comprising: preparing a liquid segment in contact with and / or containing at least one reagent in 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 a liquid-gas interface of a quantity of liquid disposed in the microchannel proximal to the liquid segment; and (iii) the gas at the distal liquid-gas interface is a distal gas disposed in the microfluidic channel distal to 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, thereby forming a first mixture.

[0226] Embodiment 130: The method described in embodiment 129, wherein the amount of liquid placed in the microchannel proximal to the liquid segment is a residual amount of liquid remaining from a first amount of liquid, and the method includes, prior to the mixing step, introducing the first amount of liquid into the microchannel and separating the liquid segment from the residual amount of liquid.

[0227] Embodiment 131: A method according to any one of embodiments 129 to 130, wherein the step of separating the liquid segment from the remaining amount of liquid comprises injecting a separation gas into the microchannel through a separation gas microfluidic channel that intersects the microfluidic channel at the position occupied by the first amount of liquid.

[0228] Embodiment 132: A method according to any one of embodiments 129 to 131, wherein the step of introducing the sample liquid comprises moving the first quantity of liquid by capillary action along the microfluidic channel until at least a portion of the first quantity of liquid reaches and moves beyond the position where the separation gas microfluidic channel intersects with the microfluidic channel.

[0229] Embodiment 133: A method according to any one of embodiments 129 to 132, wherein the step of introducing a first amount of liquid into a microchannel comprises moving the first amount of liquid along the microfluidic channel by capillary action until the distal liquid-gas interface of the first amount of liquid contacts a capillary stop in the microfluidic channel.

[0230] Embodiment 134: A method according to any one of embodiments 129 to 133, wherein the capillary stop comprises one or more vents that provide gas communication between the microfluidic channel and a certain amount of gas disposed outside the microfluidic channel.

[0231] Embodiment 135: The method of any one of embodiments 129 to 134, wherein the amount of gas placed outside the microfluidic channel is the ambient air surrounding the microfluidic device.

[0232] Embodiment 136: A method according to any one of embodiments 129 to 135, wherein before a volume of liquid sample is introduced into the microfluidic channel, the microfluidic channel provides the only path for gas communication between the distal gas and the outside of the microfluidic device.

[0233] Embodiment 137: A method according to any one of embodiments 129 to 136, wherein after a certain amount of liquid sample is introduced into the microfluidic channel, a distal gas occupies a chamber of the microfluidic device that is isolated from the ambient gas surrounding the microfluidic device.

[0234] Embodiment 138: A method according to any one of embodiments 129 to 137, wherein before a volume of liquid sample is introduced into the microfluidic channel, the microfluidic channel and one or more vents provide the only path for gas communication between the distal gas and the outside of the microfluidic device.

[0235] Embodiment 139: A method of forming a liquid in a microchannel of a microfluidic device, comprising: disposing a liquid in a microchannel; and forming a gas bubble in 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 gas 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 gas bubble and the second portion of the liquid occupies a portion of the microchannel having a cross-sectional area A2, and wherein the ratio A2 / A1=R A is at least about 1.25, such as about 1.5 to 3.5, for example about 2.

[0236] Embodiment 140: The cross-sectional area A1 is about 0.04 mm to about 0.13 mm, for example about 0.09 mm, and the second cross-sectional area A2 is R A The method of embodiment 139, wherein the value is equal to ×A1.

[0237] Embodiment 141: The method according to any one of embodiments 139 to 140, wherein the distance along the longitudinal axis of the microchannel between the first gas-liquid interface and the second gas-liquid interface is about 1 to 3 mm, for example about 2 mm.

[0238] Embodiment 142: The method described in any one of embodiments 139 to 141, wherein the amount of the air bubble placed in the microchannel between the first air-liquid interface and the second air-liquid interface is from about 200 nL to about 750 nL, for example about 350 nL.

[0239] Embodiment 143: The method of any one of embodiments 139 to 142, wherein the cross-sectional area of ​​the first microchannel tapers from a first area to a second, smaller area as one proceeds along the longitudinal axis of the microchannel from the location of the first gas-liquid interface to the location of the second gas-liquid interface.

[0240] Embodiment 144: The cross-sectional area of ​​the microchannel between the first gas-liquid interface and the second gas-liquid interface is about 12% mm -1 ~approx. 25% mm -1 , for example, about 17.5% mm -1 The method of any one of embodiments 139 to 143, wherein the decrease is at an average rate of

[0241] Embodiment 145: The method of any one of embodiments 139 to 144, wherein the microchannel has an internal height h1 and an internal width w1 at the location of the microchannel occupied by the first gas-liquid interface, where w1>h1.

[0242] Embodiment 146: The method of any one of embodiments 139 to 145, wherein the microchannel has an internal height h2 and an internal width w2 at the location occupied by the second gas-liquid interface, where w2>w1>h2.

[0243] Embodiment 147: The method of any one of embodiments 139 to 146, wherein the ratio w2 / w1 is approximately the same as the ratio RA of the second cross-sectional area to the first cross-sectional area.

[0244] Embodiment 148: The method of any one of embodiments 139 to 147, wherein h1 and h2 are approximately the same, for example essentially identical, for example, h1 and h2 can each be about 50 μm to about 200 μm, for example about 110 μm.

[0245] Embodiment 149: The method of any one of embodiments 139 to 148, wherein w1 is about 400 to 1200 μm, for example about 800 μm.

[0246] Embodiment 150: The method of any one of embodiments 139 to 149, wherein w2 is about RA × w1 μm.

[0247] Embodiment 151: The method of any one of embodiments 139 to 150, wherein w1 is about 800 μm and w2 is about 1600 μm.

[0248] Embodiment 152: The method according to any one of embodiments 139 to 151, wherein the first portion of the liquid comprises a distal gas-liquid interface disposed in the microchannel distal to the first gas-liquid interface.

[0249] Embodiment 153: The method of any one of embodiments 139 to 152, wherein the gas at the distal air-liquid interface is confined within the distal portion of the microchannel of the microfluidic device.

[0250] Embodiment 154: The method of any one of embodiments 139 to 153, wherein the second portion of the liquid comprises a proximal gas-liquid interface, and the gas at the gas-liquid interface is a gas of the ambient atmosphere surrounding the microfluidic device, for example air.

[0251] Embodiment 155: A method according to any one of embodiments 139 to 154, wherein the microchannel is a first microchannel and the step of forming bubbles comprises injecting gas for the bubbles from a second microchannel that intersects with the first microchannel at a gas injection opening occupied by the liquid.

[0252] Embodiment 156: A method according to any one of embodiments 139 to 155, wherein the distance along the microchannel between the first gas-liquid interface and the gas input opening is distance d1, the distance along the microchannel between the second gas-liquid interface and the gas input position is distance d2, and the ratio d2 / d1 is at least about 2.25, for example about 2.25 to 10, for example about 4.5.

[0253] Embodiment 157: A method according to any one of embodiments 139 to 156, wherein placing a liquid in a microchannel comprises, before forming a bubble, injecting the liquid into an application zone of the microchannel and allowing the liquid to flow by capillary action along the microchannel until the distal gas-liquid interface of the liquid reaches a capillary stop located distal to the gas injection opening in the microchannel.

[0254] Embodiment 158: A method according to any one of embodiments 139 to 157, wherein flowing the liquid by capillary action comprises flowing a distal gas-liquid interface of the liquid along a distal portion of a microchannel located distal to the gas input opening, the distal portion of the microchannel having a width w3 greater than width w1.

[0255] Embodiment 159: The method according to any one of embodiments 139 to 158, wherein the ratio w3 / w1 is from about 2 to about 6, for example about 3.

[0256] Embodiment 160: The method according to any one of embodiments 139 to 159, wherein the distal portion of the microchannel has a cross-sectional area A3, and the ratio A3 / A1 is from about 2 to about 6, for example about 3.

[0257] Embodiment 161: A method according to any one of embodiments 139 to 160, wherein the distal portion of the microchannel has a height h3, and h3, h2, and h1 are approximately the same, e.g., essentially identical, e.g., h3, h2, and h1 can each be about 50 μm to about 200 μm, e.g., about 110 μm.

[0258] Embodiment 162: A method according to any one of embodiments 139 to 161, wherein the step of forming a bubble comprises injecting an initial amount of gas for the bubble into a microchannel through a gas injection 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 injection opening is distance d1', the distance along the microchannel between the second gas-liquid interface of the initial bubble and the gas injection position is distance d2', and the ratio d2' / d1' is about 1.

[0259] Embodiment 163: A method according to any one of embodiments 139 to 162, wherein the step of forming a bubble includes injecting additional gas into the initial bubble through a gas input opening, so that the distance along the microchannel between the second gas-liquid interface of the bubble and the gas input opening increases relative to the distance along the microchannel between the first gas-liquid interface of the bubble and the gas input opening until the distance along the microchannel between the second gas-liquid interface of the bubble and the gas input opening becomes d2 and the distance along the microchannel between the first gas-liquid interface of the bubble and the gas input opening becomes d1.

[0260] Embodiment 164: In any one of embodiments 1 to 163, the liquid comprises a sample liquid obtained from a mammal, such as a human.

[0261] Embodiment 165: In any one of embodiments 1 to 164, the sample liquid comprises blood, serum, plasma, saliva, urine, sputum, or material obtained from a swab, such as a nasopharyngeal swab.

[0262] Embodiment 166: In any one of embodiments 1 to 165, the liquid is a mixture comprising a sample liquid and a further liquid, such as a buffer.

[0263] Embodiment 167: In any one of embodiments 1 to 166, the liquid in the first portion of the liquid comprises a reagent configured to facilitate the determination of one or more targets present in the first portion of the liquid.

[0264] Embodiment 168: In any one of embodiments 1 to 167, the method comprises contacting the liquid of the first portion of the liquid with a reagent after the step of forming a gas bubble.

[0265] Embodiment 169: In any one of embodiments 1 to 168, the method comprises contacting the liquid of the first portion of the liquid with a reagent before the step of forming the bubbles.

[0266] Embodiment 170: In any one of embodiments 1 to 169, the method comprises contacting the liquid of the first portion of the liquid with a further, different reagent after the step of forming the bubbles.

[0267] Embodiment 171: In any one of embodiments 1 to 170, the reagent contacted prior to the step of forming the bubbles is a reagent configured to inhibit clotting of the blood sample, such as a heparin-containing reagent.

[0268] Embodiment 172: In any one of embodiments 1 to 171, the further different reagent comprises one or more reagents configured to bind to a target.

[0269] Embodiment 173: In any one of embodiments 1 to 172, the further different reagent comprises one or more different particles comprising a binding agent for the target, such as an antibody.

[0270] Embodiment 174: In any one of embodiments 1 to 173, the particles comprise magnetic particles and fluorescent particles, and the particles are configured to form a detectable sandwich with the target.

Claims

1. a. A microfluidic device comprising a substantially planar substrate having a microfluidic network therein, the microfluidic network comprising: i. a distal gas chamber configured to regulate a 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; iii. a separation gas chamber communicating with the microfluidic channel via a separation gas channel intersecting the microfluidic channel at a separation gas input location; the separation gas chamber configured to adjust the pressure of a separation gas within at least a portion of the microfluidic network; 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 input location, such that when the separation gas is input into the microfluidic channel, a separation bubble is subsequently formed within the microfluidic channel, causing the microfluidic channel to separate the sample liquid therein into i) a liquid segment located distal to the separation bubble, and ii) a remaining amount of sample liquid located 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. Microfluidic devices.

2. The microfluidic device of claim 1 , wherein the microfluidic channel comprises a reagent zone distal to the separation gas input location and configured to contain one or more reagents.

3. The microfluidic device of claim 2 , wherein the reagent is configured to solubilize with the sample liquid upon contact therewith.

4. 4. The microfluidic device of claim 2 or 3, wherein the reagent is configured to allow detection of a target in the sample liquid upon contact therewith.

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

6. 6. The microfluidic device of claim 1, wherein the distal gas chamber comprises a first interior wall, a second interior wall, and a distal chamber spacing therebetween configured to be occupied by the distal gas.

7. The microfluidic device of claim 6, wherein the outer wall of the distal gas chamber is configured to contact a vibration member to vibrate and / or adjust the distal chamber spacing, thereby vibrating and / or adjusting the pressure of the distal gas.

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

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

10. 10. The microfluidic device of claim 1, wherein the separation gas chamber is configured to heat the separation gas therein to pressurize the separation gas and facilitate its introduction into the microfluidic channel to form the separation bubble.

11. 10. The microfluidic device of claim 1, wherein the separation gas chamber comprises a third inner wall, a fourth inner wall, and a separation gas chamber spacing therebetween configured to be occupied by the separation gas.

12. The microfluidic device of claim 11, wherein the outer wall of the separation gas chamber is configured to contact a second vibration member to vibrate and / or adjust the separation gas chamber spacing, thereby vibrating and / or adjusting the pressure of the separation gas.

13. 14. The microfluidic device of claim 1, wherein the separation bubble defines a volume of separation gas disposed between the proximal gas-liquid interface and a gas-liquid interface between the remaining volume of the sample liquid and the separation bubble (RVSL gas-liquid interface), and the separation bubble is disposed within a separation zone of the microfluidic channel.

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

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

16. 16. The microfluidic device of claim 15, wherein when the separation gas is introduced into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is smaller than the radius of curvature of the RVSL gas-liquid interface, such that when the separation gas is further introduced, the RVSL gas-liquid interface moves proximally but the proximal gas-liquid interface remains at the same or substantially the same position within the microfluidic channel, such that the liquid segments i) remain at the same or substantially the same position within the microfluidic channel and / or ii) have the same or substantially the same amount of volume within the microfluidic channel.

17. The ratio of A2 to A1 ("R A 17. The microfluidic device of claim 15 or 16, wherein the ρ is at least about 1.

25.

18. Cross-sectional area A1 is approximately 0.04 mm ２ ~Approx. 0.13mm ２ The microfluidic device according to any one of claims 15 to 17, wherein

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

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

21. 21. The microfluidic device of claim 14, wherein the distance along the microfluidic channel between the RSVL gas-liquid interface and the separation gas input location is distance d1, the distance along the microfluidic channel between the proximal gas-liquid interface and the separation gas input location is distance d2, and the ratio d2 / d1 is between about 2.25 and 10.

22. 22. The microfluidic device of claim 14, wherein the proximal gas-liquid interface is located within the separation zone, the sample liquid of the liquid segment is located substantially within a reagent zone or detection zone of the microfluidic channel located distal to the separation zone, and the reagent zone or detection zone has a cross-sectional area A3 that is larger than the cross-sectional area A1.

23. The microfluidic device of 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. 24. The microfluidic device of claim 23, wherein the middle layer comprises a top surface having an adhesive that adheres to the upper layer, a bottom surface having an adhesive that adheres to the lower layer, or both.

25. 25. The microfluidic device of claim 23 or 24, wherein the upper surface of the underlying layer defines the lower interior surfaces of the microfluidic channel and the application zone.

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

27. The microfluidic device of any one of claims 23 to 26, wherein the upper layer, the lower layer, the middle layer, or any combination thereof comprises an opening that defines the application zone.

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

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

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

31. 31. The microfluidic device of any one of claims 1 to 30, further comprising one or more further microfluidic networks, each of said one or more further microfluidic networks configured to separately receive a portion of said sample liquid from said application zone and analyze said sample liquid to detect a target therein.

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

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

34. a) introducing a sample liquid into a microfluidic channel in 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) injecting a separation gas into the microfluidic channel at the location occupied by the sample liquid, thereby separating a segment of the sample liquid from a remaining amount 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 liquid-gas interface between the separation gas and the portion of the sample liquid, the separation gas separating the proximal liquid-gas interface of the liquid segment from the remaining amount 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 including at least one reagent disposed therein; d) mixing the portion of the sample liquid in 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; Including, oscillating the pressure of the distal gas and / or separation gas is performed i) before reducing the pressure of the distal gas, ii) simultaneously with reducing the pressure of the distal gas, and / or iii) after reducing the pressure of the distal gas. method.

35. 1. A method for analyzing a sample liquid to detect at least one target material therein, said method comprising: a) introducing the sample liquid into a microfluidic channel in 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 a portion of the sample liquid contacts at least one reagent disposed in a reagent zone of the microfluidic channel; c) injecting a separation gas into the microfluidic channel at the location occupied by the sample liquid, thereby separating a segment of the sample liquid from a remaining amount of the sample liquid introduced into the microfluidic channel, the liquid segment comprising (i) the distal liquid-air interface, (ii) a portion of the sample liquid in contact with the at least one reagent, and (iii) a proximal liquid-air interface disposed between the separation gas and the portion of the sample liquid, the separation gas separating the proximal liquid-air interface of the liquid segment from the remaining amount of the sample liquid; d) mixing the portion of the sample liquid in 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; A method comprising:

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

37. 36. The method of claim 35, wherein displacing the sample liquid is via reducing the pressure of the distal gas.

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

39. 39. The method of any one of claims 34 to 38, wherein oscillating the pressure of the distal gas and / or 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 step of vibrating includes oscillating the pressure of the distal gas, and during the step of vibrating, the distal liquid-gas interface 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 40. The method of any one of claims 34 to 39, wherein the microfluidic channel occupies a position having a cross-sectional area of

41. the step of vibrating includes oscillating the pressure of the distal gas, and during the step of vibrating, the distal liquid-gas interface is ２ Below, approximately 0.125mm ２ Below, approximately 0.1mm ２ Below, approximately 0.09mm ２ Less than or equal to 0.08 mm ２ 41. The method of any one of claims 34 to 40, wherein the position of the channel having a cross-sectional area of:

42. 42. The method of any one of claims 34-41, wherein during the step of vibrating, the liquid segment has a volume of 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.

43. 43. The method of any one of claims 34-42, wherein during the step of vibrating, 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. 44. The method of any one of claims 34 to 43, wherein introducing the sample liquid comprises moving the sample liquid by capillary action along the microfluidic channel until the distal sample liquid-gas interface contacts a capillary stop in the microfluidic channel.

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

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

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

48. 48. The method of any of claims 34 to 47, wherein after the sample liquid is introduced into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device that is isolated with respect to an ambient gas surrounding the microfluidic device.

49. 49. The method of claim 34, 45, or 48, wherein prior to introducing the liquid sample into the microfluidic channel, the microfluidic channel provides a sole pathway for gas communication between the distal gas and the exterior of the microfluidic device.

50. 50. The method of claim 48 or 49, wherein after the liquid sample is introduced into the microfluidic channel, the distal gas occupies a chamber of the microfluidic device that is isolated from an ambient gas surrounding the microfluidic device.

51. 47. The method of claim 46, wherein prior to loading the liquid sample into the microfluidic channel, the microfluidic channel and the one or more vents provide the only path for gas communication between the distal gas and the exterior of the microfluidic device.

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

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

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

55. 55. The method of 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 location of the distal gas chamber spaced along the microfluidic channel from the distal liquid-air interface.

56. 56. The method of claim 55, wherein said vibrating comprises contacting an outer wall of the distal gas chamber with a vibrating member.

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

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

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

60. 60. The method of claim 58 or 59, wherein introducing the separation gas comprises heating the separation gas in the separation gas chamber.

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

62. 62. The method of claim 61, wherein the method further comprises 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. 63. The method of any one of claims 34 to 62, further comprising operating the microfluidic device with an instrument, wherein the method is performed without introducing any gas into the microfluidic channels from a gas source of the instrument.

64. 64. The method of any one of claims 34 to 63, wherein the only gas present in the microfluidic device prior to introducing the sample liquid into the microfluidic channel is ambient air, and wherein during the performance of the method, the distal gas and the separation gas consist of the ambient air that was present in the microfluidic device prior to introducing the sample liquid therein.

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

66. 66. The method of any one of claims 34 to 65, wherein the step of vibrating comprises simultaneously vibrating the pressure of the distal gas and the separation gas.

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

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

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

70. 70. The method of any one of claims 34 to 69, wherein the step of introducing the separation gas into the microchannel displaces at least a portion 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 amount of the sample liquid.

71. 71. The method of claim 70, wherein the volume of the separation bubble is at least about 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.

72. 71. The method of 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 of claim 71 or 72, wherein the amount of separation bubbles defines an amount of separation gas that separates the proximal gas-liquid interface of the liquid segment from the gas-liquid interface between the remaining amount of the sample liquid and the separation gas ("RSVL gas-liquid interface").

74. 74. The method of 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. 75. The method of 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 and the RVSL gas-liquid interface occupies a portion of the microchannel having a cross-sectional area A2, A2 being greater than A1, such that the separation bubble comprises an asymmetric shape.

76. 76. The method of claim 75, wherein, upon introduction of the separation gas into the microfluidic channel, the radius of curvature of the proximal gas-liquid interface is smaller 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, but the proximal gas-liquid interface remains in the same or substantially the same position, such that the liquid segments i) remain in the same or substantially the same position within the microfluidic channel, and / or ii) have the same or substantially the same amount of volume within the microfluidic channel.

77. 77. The method of claim 76, wherein mixing the portion of the sample liquid with the at least one reagent comprises mixing the same or substantially the same amount of volume with the at least one reagent, thereby providing a defined reagent concentration within the portion of the sample liquid.

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

25.

79. Cross-sectional area A1 is approximately 0.04 mm ２ ~Approx. 0.13mm ２ The method according to any one of claims 75 to 78, wherein

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

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

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

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

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

85. 85. The method of any one of claims 34 to 84, wherein the at least one reagent comprises a binding reagent capable of specifically binding to the target in the portion of the sample liquid, and wherein mixing the portion of the sample liquid with the at least one reagent detects binding of the binding reagent to the target, allowing the amount of binding reagent bound to the target to be detected and / or determined.

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

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

88. 88. The method of any one of claims 34 to 87, wherein the microfluidic device further comprises one or more further microfluidic channels, each of the one or more further microfluidic channels configured to carry out a method of any one of claims 35 to 87, for individually analysing the sample liquid.

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

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

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

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