Thermally actuated microfluidic valve

Abstract

A microfluidic device includes (i) a microfluidic liquid network including a microfluidic liquid channel and (ii) a microfluidic temperature responsive material (TRM) network including a TRM chamber and a microfluidic TRM channel extending from the TRM chamber to a TRM channel opening in a wall of the microfluidic liquid channel A bolus of TRM is disposed within the TRM chamber. During operation of the microfluidic device, the TRM of the microfluidic TRM network may be actuated to move at least some of the TRM out of the TRM chamber, along the TRM channel, and into the microfluidic liquid channel, thereby sealing the microfluidic liquid channel.

Description

[0001] THERMALLY ACTUATED MICROFLUIDIC VALVE

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a microfluidic device comprising a thermally actuated valve useful for sealing one or more portions of a microfluidic network.

[0004] INCORPORATION BY REFERENCE

[0005] International Application Nos. PCT / US2021 / 013325, filed 13 January 2021 (the “’325 Application”) and PCT / GB2023 / 050189, filed 27 January 2023 (the ‘“189 Application”) are incorporated herein by reference in their entireties.

[0006] BACKGROUND OF THE INVENTION

[0007] Microfluidic devices are used to manipulate and / or process small volumes of fluids, e.g., liquids or gases. For example, microfluidic devices may be used in assays for determining the presence of one or more targets in a liquid sample. Such devices typically include a network of microfluidic channels and chambers in which a fluid is moved and processed. Elements such as valves, passive features (e.g., hydrophobic patches), and pumps may be used to control and direct such fluids.

[0008] SUMMARY OF THE INVENTION

[0009] A microfluidic device of the present invention includes a microfluidic temperature responsive material (TRM) network (TRM network) including an amount, e.g., a bolus, of TRM disposed therein and a TRM channel. Optionally, the microfluidic device includes a microfluidic liquid network and the TRM channel includes a TRM channel opening into the microfluidic liquid network. At least some, e.g., substantially all or all, of the TRM channel is disposed between the amount of TRM and the TRM channel opening. The TRM channel opening may be an opening in a wall, e.g., a side wall, of the microfluidic liquid network. For example, the microfluidic liquid network may include a microfluidic liquid channel and the TRM channel opening may be an opening in a wall, e.g., a side wall, of the microfluidic liquid channel. Alternatively, or in combination with embodiments including a microfluidic liquid network, the microfluidic device includes a vent network having a vent junction disposed along the TRM channel. At least some of the TRM channel is disposed between the amount of TRM and the vent junction. The vent network permits gas to enter or escape the microfluidic device.A TRM is a material having a mobility, e.g., a flowability, that depends on temperature. The TRM is relatively immobile, e.g., relatively non-flowable or immovable, when below a transition temperature and relatively mobile, e.g., relatively flowable or movable, when above the transition temperature. The transition temperature is typically higher than ambient temperatures to which the microfluidic device is subjected when not in use such that, e.g., the TRM remains relatively immobile during storage and shipping. For example, the transition temperature may be at least about 40 °C, e.g., at least about 50 °C.

[0010] Examples of TRM’s suitable for use in any of the disclosed microfluidic devices and methods using TRM’s include waxes, olefins, alkanes, e.g., straight chain alkanes, aliphatic alcohols, e.g., long chain aliphatic alcohols, polymers, plastics, and combinations thereof. Suitable alkanes include, e.g., docosane, (C22H46; transition temperature about 43.8°C), tricosane, (C23H48; transition temperature about 47.4°C), tetracosane, (C24H50; transition temperature about 54°C), and combinations thereof. Suitable aliphatic alcohols include, e.g., 1 -pentadecanol, (C15H32O; transition temperature about 45.5°C), 1-hexadecanol, (C16H34O; transition temperature about 49.3°C), 1 -heptadecanol, (C17H36O; transition temperature about 54°C), and combinations thereof. In exemplary embodiments, the TRM of the microfluidic device or methods using a TRM, the TRM is tricosane, 1-hexadecanol, or a combination thereof.

[0011] In any of the embodiments of the microfluidic device, a volume of the amount, e.g., bolus, of TRM may be at least about 0.4 pL, e.g., at least about 0.6 pL, at least about 0.8 pL or at least about 1.0 pL. The volume of the bolus of TRM is typically about 5 pL or less, e.g., about 4 pL or less, about 3 pL or less, or about 2 pL or less. For example, the volume of TRM may be between about 0.7 pLand 1.2 pL, e.g., about 1.0 pL.

[0012] During use of the microfluidic device, the TRM of the TRM network may be actuated, e.g., by heating the TRM to a temperature at least as high as the transition temperature of the TRM. The heating may be performed using, e.g., one or more heat sources in thermal communication with, e.g., underlying, at least some, e.g., most or all, of the TRM network of the microfluidic device. For example, such heat source(s) is typically in thermal communication with at least some, e.g., substantially all or all of the amount of TRM and at least some, e.g., substantially all or all, of the TRM channel. In embodiments including a microfluidic liquid network, the heat source(s) may be in thermal communication with, e.g., underlie, at least a portion of the microfluidic liquid network. For example, the heat source(s) may be in thermal communication with, e.g., underlie, a regionof a microfluidic liquid channel adjacent the TRM channel opening. The length of such region may be, e.g., at least about 5 mm or at least about 7.5 mm extending from the TRM channel opening in at least one, e.g., two directions along the microfluidic liquid channel. The heat source(s) is configured to heat the TRM to a temperature at least as high as, or higher than, the transition temperature.

[0013] Upon actuation, at least some of the TRM may be moved along the TRM channel, e.g., by a gas pressure differential as disclosed herein. In embodiments including a microfluidic liquid network, at least some of the TRM is moved along the TRM channel and into the microfluidic liquid network, e.g., into a microfluidic liquid channel thereof, via the TRM channel opening. The TRM obstructs the portion of the microfluidic liquid network at the location occupied by the TRM by preventing fluids from passing thereby. In embodiments alternatively or also including a vent network, at least some of the TRM is moved along the TRM channel at least to the location of the vent junction, thereby obstructing the vent junction to prevent gas from entering or escaping the microfluidic device via the vent network. In any embodiments, the TRM may be cooled or allowed to cool following actuation so that the TRM again becomes relatively immobile in the obstructing position within the microfluidic liquid network, e.g., within the microfluidic liquid channel, and / or within the TRM channel at the location of the vent junction. Accordingly, the TRM functions as a thermally actuated valve for selectively sealing one or more portions of the microfluidic liquid network and / or of the vent network.

[0014] In any embodiments of the microfluidic device, a heat source may be integrated with the microfluidic device, e.g., adjacent to or within an upper or lower layer of the microfluidic device. Such integrated heat source may be operated by a reader used to operate the microfluidic device. Alternatively, or in combination, the microfluidic device may be used with a separate heat source disposed adjacent to, e.g., in contact with, an external surface, e.g., a lower or upper surface, of the microfluidic device. For example, such separate heat source may be disposed within and operated by a reader used to operate the microfluidic device. In embodiments of the microfluidic device including a microfluidic liquid network, a heat source(s) configured to actuate the TRM may also be configured to heat a portion of the microfluidic liquid network, e.g., a reaction / detection chamber thereof. Alternatively, a microfluidic device may include a separate integral heat source or be used with a separate external heat source configured to heat such portion of the microfluidic liquid network. Examples of suitable heat sources include heat sources that generate heatthrough joule heating. Other examples, include infrared heat sources and chemical heat sources that generate heat through exothermic reactions.

[0015] In any embodiments of the microfluidic device, the TRM network may include a TRM chamber and at least some, e.g., substantially all or all of the TRM is initially disposed within the TRM chamber. The TRM chamber and TRM channel are in communication via a distal TRM chamber opening of the TRM chamber. In embodiments including a microfluidic liquid network, the TRM channel is disposed between the TRM chamber and the distal TRM channel opening. In embodiments including a vent network, at least some, e.g., at least 25%, at least 35%, at least 50%, or substantially all, or all, of the TRM channel is disposed between the TRM chamber and the vent junction. In embodiments including the TRM chamber, a heat source used to actuate the TRM is in thermal communication with the TRM chamber. The heat source may also be in thermal communication with at least some, e.g., most or all of the TRM channel.

[0016] In any of the embodiments of the foregoing microfluidic device including a TRM chamber, a volume of the TRM chamber may be at least about 0.4 pL, e.g., at least about 0.6 pL, at least about 0.8 pL or at least about 1.0 pL. The volume of the TRM chamber is typically about 5 pL or less, e.g., about 4 pL or less, about 3 pL or less, or about 2 pL or less. For example, the TRM chamber may have a length of between about 500 and about 1500 pm, e.g., about 1000 pm, a width of between about 300 pm and about 1000 pm, e.g., about 700 pm, and a height of between about 100 pm and about 200 pm, e.g., about 140 pm. In such embodiments, substantially all, e.g., all of the TRM within the TRM network may be initially disposed within the TRM chamber.

[0017] In the foregoing embodiments of the microfluidic device including a TRM chamber, a portion of the microfluidic TRM network disposed proximally to the TRM chamber opening may be sealed with respect to sources of fluid disposed external to the microfluidic device. For example, the microfluidic TRM network may include a compressible gas chamber disposed proximally to the TRM chamber. When the TRM is in a mobile state, e.g., upon heating, compressing an external surface of the gas chamber compresses the gas therein creating a gas pressure differential acting on the TRM and capable of moving the at least some of the TRM out of the TRM chamber through the TRM chamber opening and along the TRM channel. In embodiments of the microfluidic device including a microfluidic network, such TRM may be moved out of the TRM channel opening and into the microfluidic liquid network, e.g., into a microfluidic liquid channel thereof to thereby obstruct the microfluidic liquid network / channel. In embodiments of the microfluidic deviceincluding a vent network, such TRM may obstruct the vent junction disposed along the TRM channel.

[0018] In the foregoing embodiments of the microfluidic device including a microfluidic liquid network, a length of the TRM channel between the amount of TRM in its initial location (e.g. in the TRM chamber) and the TRM channel opening may be at least about 5 mm, e.g., at least about 6.5 mm, at least about 7.5 mm, at least about 8.5 mm, at least about 10 mm, or at least about 12.5 mm. Such length is typically about 15 mm or less, e.g., about 12.5 mm or less, or about 10 mm or less. In embodiments including a TRM chamber, such length is the length of the TRM channel between the TRM chamber opening and the TRM channel opening.

[0019] In any of the foregoing embodiments of the microfluidic device, at least a portion of the microfluidic TRM channel may have an average transverse cross-sectional area of about 0.06 mm2or less, e.g., about 0.05 mm2or less, about 0.04 mm2or less, or about 0.03 mm2or less. The average transverse cross-sectional area of the portion of the microfluidic TRM channel may be at least about 0.015 mm2, e.g., at least about 0.020 mm2, at least about 0.025 mm2. In embodiments of the microfluidic device including a microfluidic liquid network, at least about 30%, at least about 50%, at least about 70%, at least about 90%, or essentially all of the total length of the microfluidic TRM channel between the TRM and / or the TRM chamber opening and the TRM channel opening may have such transverse cross-sectional area. The transverse cross-sectional area the TRM channel opening in such embodiments may be about the same as the cross-sectional area of such portion of the TRM channel. In embodiments of the microfluidic device including a vent network, at least about 30%, at least about 50%, at least about 70%, at least about 90%, or essentially all of the total length of the microfluidic TRM channel between the TRM and / or the TRM chamber opening and the vent junction may have such average cross-sectional area.

[0020] In any of the embodiments of the microfluidic device including a TRM chamber, most, essentially all, e.g., all, of the TRM associated with the TRM network may be located within the TRM chamber prior to use of the microfluidic device. For example, the TRM channel may be essentially free of, e.g., free of, TRM prior to use. Typically, the TRM only enters the TRM channel upon actuation, e.g., by heating the TRM and applying a gas pressure differential thereto, during the use of the microfluidic device. In such embodiments, the TRM channel may be essentially fully occupied, e.g., fully occupied by a gas such as gas (e.g., air) from the ambient gas (e.g., air) surrounding the device prior to actuation.A transverse cross-sectional area of a portion of a microfluidic liquid network, e.g., a microfluidic liquid channel or reaction / detection chamber thereof, TRM network, e.g., a TRM channel orTRM chamber thereof, or vent network, e.g., a vent channel thereof, means the cross-sectional area of such feature, chamber or channel, taken along an axis that is perpendicular to a longitudinal axis of such feature at the location the area is defined. Typically, the microfluidic device is generally planar, the longitudinal axis of such portion is parallel to the plane, and the transverse cross-sectional area of the network / channel is perpendicular to the plane.

[0021] In any of the foregoing embodiments of the microfluidic device including a microfluidic liquid network, the cross-sectional area of the TRM channel opening may be about 0.06 mm2or less, e.g., about 0.05 mm2or less, about 0.04 mm2or less, or about 0.03 mm2or less. The cross-sectional area of the TRM channel opening may be at least about 0.015 mm2, e.g., at least about 0.020 mm2, at least about 0.025 mm2. Such a TRM channel opening advantageously limits or prevents liquid present within the microfluidic liquid network from entering the TRM channel and passing therealong, e.g., by capillary action.

[0022] In any embodiments of the foregoing microfluidic devices including a microfluidic liquid network, a transverse cross-sectional area of the microfluidic liquid network, e.g., of the microfluidic liquid channel, adjacent the TRM channel opening may be larger than a transverse cross-sectional area of the TRM channel opening. For example, a ratio of the transverse cross-sectional area of the microfluidic liquid network / channel adjacent the TRM channel opening to the transverse cross-sectional area of the TRM channel opening may be at least about 2, e.g., at least about 3, or at least about 4. Such ratio may be about 10 or less, e.g., about 8 or less, about 6 or less, or about 5 or less.

[0023] In any embodiments of the foregoing microfluidic device including a microfluidic liquid network, the transverse cross-sectional area of the microfluidic liquid network / channel adjacent the TRM channel opening may be at least about 0.06 mm2, e.g., at least about 0.07 mm2, at least about 0.08 mm2, at least about 0.09 mm2, or at least about 0.1 mm2. The aforementioned transverse cross-sectional area of the microfluidic liquid network / channel is typically about 1.5 mm2or less, e.g., about 1.25 mm2or less, or about 1 mm2or less. In such embodiments, the average transverse cross-sectional area of the microfluidic liquid network / channel in a portion of the microfluidic network / channel that includes the TRM channel opening may be about the same asthe aforementioned transverse cross-sectional area. For example, the portion of the microfluidic liquid network / channel having such average transverse cross-sectional area may be at least about 3 mm, e.g., at least about 5 mm, at least about 7.5 mm, at least about 10 mm, or at least about 12.5 mm long along a longitudinal axis of the microfluidic liquid network / channel.

[0024] In any of the foregoing embodiments of the microfluid device including a microfluidic liquid network, optionally, none of the dimensions (e.g. height or width) of the transverse cross-section of the TRM channel opening are greater than any of the corresponding dimensions (e.g. height or width) of the transverse cross-section of the microfluidic liquid network / channel adjacent the TRM channel opening. In other words, in such optional embodiments, each of the dimensions (e.g. height or width) of the transverse cross-section of the microfluidic liquid network / channel adjacent the TRM channel opening are at least as large and optionally greater than each of the corresponding dimensions (e.g. height or width) of the transverse cross-section of the TRM channel opening.

[0025] In any of the foregoing embodiments of the microfluid device, the TRM channel may include at least one, e.g., 2, 3, 4 or more, sharp angle(s) along the longitudinal axis thereof. A sharp angle along the longitudinal axis of TRM channel 46 is an angle of at least about 25°, at least about 35°, at least about 45°, at least about 60°, or at least 75°. Typically, such sharp angle is about 180° or less, about 150° or less, or about 115° or less. For example, such sharp angle(s) may be between about 75° and about 105°, e.g., about 90°. Such sharp angle(s) may be formed along a TRM channel having a longitudinal axis that lies entirely within a single plane or may be formed along a TRM channel that includes one or more portions that depart from a single plane. For example, a TRM channel may include a first portion having a longitudinal axis parallel to a first plane, e.g., a plane generally defined by the microfluidic device, and a second portion having a longitudinal axis parallel to a second, different plane that intersects the first plane at an angle thereto. In embodiments of the microfluidic device including a microfluidic liquid network, the sharp angle(s) is / are disposed between the TRM and the TRM channel opening. In such embodiments including a TRM chamber, the sharp angle(s) is are disposed between the TRM chamber opening and the TRM channel opening. In embodiments of the microfluidic device including a vent network, the sharp angle(s) is / are typically disposed between the TRM and the vent junction. In such embodiments including a TRM chamber, the sharp angle(s) is / are disposed between the TRM chamber opening and the vent junction. A sharp angle along the longitudinal axis of the TRMchannel advantageously limits or prevents liquid that may have entered the TRM channel from passing therealong beyond the sharp angle(s), e.g., by capillary action.

[0026] In any of the foregoing embodiments of the microfluidic device, the microfluidic TRM channel may include at least one step (e.g., a step up). The step(s) typically has a height of at least about 100 pm, e.g., at least about 125 pm, along an axis oriented perpendicular to a plane defined by the microfluidic device and / or to the transverse longitudinal axis of the portion of the TRM channel immediately preceding the step. The height of the step is typically less than about 500 pm, e.g., less than about 250 pm, along the perpendicular axis. For example, the height of the step may be about 140 pm. Each step may form at least one, e.g., 2, sharp angles along the TRM channel. The step is typically spaced apart from the amount of TRM by a proximal portion of the TRM channel. The proximal portion of the microfluidic TRM channel disposed between the TRM and the step, if present, typically has a longitudinal axis disposed within a plane of the microfluidic device. The length of such proximal portion of the microfluidic TRM channel is typically at least about 1 mm, e.g., at least about 2.5 mm, at least about 3 mm, at least about 4 mm, or at least about 5 mm. The length is typically about 10 mm or less, e.g., about 7.5 mm or less, or about 5 mm or less. In embodiments of the microfluidic device including a step, the lower internal surface of such proximal portion of the microfluidic TRM channel is typically disposed a first distance along an axis perpendicular to the plane of the microfluidic device from a lower external surface of the microfluidic device and / or from a heat source used to heat the TRM and the lower internal surface of the portion of the TRM channel at the height of the step is disposed a second, greater distance along the perpendicular axis from the lower external surface of the microfluidic device, e.g., from the heat source. The difference between the first and second distances is typically the same as the height of the step.

[0027] In any of the foregoing embodiments of the microfluidic device, the TRM channel may include a drop (e.g., a step down), e.g., in embodiments including a step. The drop is typically disposed at a location disposed between the TRM chamber and the TRM channel opening, e.g., between the step, if present, and the TRM channel opening. Alternatively, the drop may be disposed at a location disposed between the step, if present, and the TRM chamber. The drop typically has a height of at least about 100 pm, e.g., at least about 125 pm, along an axis oriented perpendicular to the microfluidic device and / or the transverse longitudinal axis of the portion of the TRM channel immediately preceding the drop. The height of the drop is typically less than about 500 pm, e.g., less than about 250 pm, along the perpendicular axis. For example, the height of the drop maybe about 140 pm. The heights of the step and the drop, if both present, may be about the same, e.g., the same. Each drop may form at least one, e.g., 2, sharp angles along the TRM channel. In any of the embodiments including a step and a drop, a central portion typically has a longitudinal axis disposed within a plane of the microfluidic device. The length of such central portion of the microfluidic TRM channel disposed between the step and the drop is typically at least about 1 mm, e.g., at least about 2.5 mm, at least about 3 mm, at least about 4 mm, or at least about 5 mm. The length of such central portion of the microfluidic TRM channel disposed between the step and the drop is typically about 10 mm or less, e.g., about 7.5 mm or less, about 5 mm or less, or about 4 mm or less. The lower internal surface of such central portion of the microfluidic TRM channel disposed between the step and the drop typically has a height of about the same as the step as compared to the lower internal surface of a proximal portion of the TRM channel between the TRM chamber and the step and / or the lower internal surface of a distal portion of the TRM channel between the drop and the TRM channel opening. The distal portion of the microfluidic TRM channel disposed between the drop and the TRM channel opening typically has a longitudinal axis disposed within a plane of the microfluidic device. The length of the distal portion of the microfluidic TRM channel disposed between the drop and the TRM channel opening is typically at least about 1 mm, e.g., at least about 2.5 mm, at least about 3 mm, at least about 4 mm, or at least about 5 mm. The length is typically about 10 mm or less, e.g., about 7.5 mm or less, or about 5 mm or less. In embodiments of the microfluidic device including a step and a drop and used with a heat source, the lower internal surface of the central portion of the TRM channel disposed between the step and the drop, is typically disposed at the second, greater, distance from the heat source and the portion of the distal TRM channel disposed between the drop and the TRM channel opening is typically disposed at the first, smaller, distance from the heat source.

[0028] In any of the foregoing embodiments of the microfluidic device, the microfluidic liquid network may include a liquid application zone, e.g., a sample amplification zone, in fluidic communication with the microfluidic liquid network, e.g., at a proximal portion or proximal terminus thereof. For example, the liquid application zone may be in fluidic communication with a microfluidic liquid channel of the microfluidic device, e.g., at a proximal portion or proximal terminus thereof. The microfluidic network may include a chamber such as a reaction chamber and / or a detection chamber in fluidic communication with the microfluidic liquid channel, e.g., at a distal portion or distal terminus thereof disposed downstream of the sample application zone. In any of such embodiments, the chamber may be a single chamber operable to perform or be used for bothreaction and detection functions. Such a reaction / detection chamber may include reagents configured to facilitate a sample processing and / or detection function, e.g., cell lysis, labeling reaction, or polynucleotide amplification.

[0029] In any of the foregoing embodiments of the microfluidic device including a microfluidic liquid channel, the microfluidic liquid channel may be a first microfluidic liquid channel and the microfluidic liquid network may include a second microfluidic liquid channel in fluidic communication with the first microfluidic liquid channel, e.g., at a junction disposed in a distal portion or distal terminus thereof. In embodiments with a liquid application zone, the junction between the first and second microfluidic channels is disposed downstream of the liquid application zone. In embodiments with a reaction and / or detection chamber(s), such second microfluidic liquid channel may fluidly connect the first microfluidic liquid channel and the reaction and / or detection chamber(s), which may also be refered to as reaction and / or detection zone(s).

[0030] In any of the foregoing embodiments of the microfluidic device including a vent network, the vent network may include a vent opening and a vent channel that places the vent opening in gaseous communication with a vent junction. The vent opening is typically exposed to an ambient gas surrounding the microfluidic device. The average transverse cross sectional area of the vent channel may be about the same as or smaller than the average transverse cross sectional area of the TRM channel. In some embodiments, a lower internal surface of at least a portion of the TRM channel is disposed a first distance relative to a heat source used to heat the TRM and the vent junction and optionally a lower internal surface of at least a portion of the vent channel is / are disposed at a second, greater distance from the heat source. In embodiments of the microfluidic device including a step, the difference between the first and second distances is typically the same as the height of the step. Disposing the vent junction and optionally a portion of the vent channel a greater distance from a heat source facilitates cooling of the TRM at such locations so that the TRM solidifies enhancing the ability of the TRM to obstruct the vent network.

[0031] In embodiments of the microfluidic device, the device includes at least two layers: a lower layer and an upper layer secured together, e.g., by thermal bonding or an adhesive. One of the layers, e.g., the lower layer, typically defines side walls and one of the upper or lower internal walls of at least a portion, e.g., most or all, of the TRM network, the microfluidic liquid network (if present) and / or vent network (if present). Features defining such walls may be formed by, e.g., a mechanical process, e.g., stamping, an optical cutting process, e.g., laser ablation, a chemicalprocess, e.g., lithography such as photolithography, a molding process, e.g., injection molding or a combination thereof. The other layer, e.g., the upper layer, defines the other of the lower or upper internal walls of at least a portion, e.g., most or all, of the of the TRM network, the microfluidic liquid network (if present) and / or vent network (if present). In such embodiments, the thickness of the adhesive (if used) to adhere the lower and upper layers typically does not substantially contribute, e.g., essentially does not contribute to the thickness of the device or the TRM network, microfluidic network (if present), or vent network (if present) as compared to the thickness of the lower and upper layers. The TRM network, microfluidic network (if present), or vent network (if present) of such device may include any of the features of such elements disclosed herein.

[0032] In embodiments of the microfluidic device, the device includes at least three layers: a lower layer and an upper layer secured together by a central layer, e.g., an adhesive layer. Typically, at least a portion, e.g., substantially all or all, upper and lower internal surfaces of the TRM network, microfluidic network (if present), or vent network (if present) are respectively defined by a lower (internal) surface of the upper layer and an upper (internal) surface of the lower layer. Internal side walls of at least a portion, e.g., substantially all or all, of the TRM network, microfluidic network (if present), or vent network (if present) may be defined by the central layer. For example, the central layer may define at least some, e.g., most or all, of the side walls of the TRM channel, e.g., including one or more sharp angles therealong. Features defining such side walls may be formed by, e.g., a mechanical process, e.g., stamping, an optical cutting process, e.g., laser ablation, a chemical process, e.g., lithography such as photolithography, a molding process, e.g., injection molding or a combination thereof.

[0033] In embodiments of the microfluidic device, e.g., in embodiments including a step and / or a drop along the TRM channel, at least one of the lower or upper layers may define a slot extending at least partially therealong. At least one portion, e.g., at least two portions, of the slot overlies (if in the upper layer) or underlies (if in the base layer) at least a portion of the TRM channel defined in the central layer, e.g., in the adhesive layer. Remaining portions of the slot typically overlie (if in the upper layer) or underlie (if in the base layer) the intact portions of the central layer, e.g., portions of the central layer that have not been removed to define side walls of the TRM network. A fourth layer overlies, if the slot is in the upper layer, or underlies, if the slot is in the base layer, and seals the slot. In embodiments in which the slot is defined in the upper layer and having a step and a drop along the TRM channel, the portion of the TRM channel defined therebetweenhas a lower wall defined by the upper surface of the central layer, side walls defined by the slot along the upper layer, and an upper wall defined by a lower internal surface of the fourth layer. In embodiments in which the slot is defined in the lower layer and having a step and a drop along the TRM channel, the portion of the TRM channel defined therebetween has a lower wall defined by the upper internal surface of the fourth layer, side walls defined by the slot along the lower layer, and an upper wall defined by a lower internal surface of the central layer. In such embodiments with a step and a drop along the TRM channel, the central layer typically defines the height of the step and the drop.

[0034] In embodiments of the microfluidic device having a fourth layer, a step and a drop along the TRM channel, and a microfluidic liquid network, a proximal portion of the TRM channel extending from the TRM chamber may be defined by a proximal slot in the central layer that defines the side walls of such portion of the TRM channel. A distal portion of the TRM channel that includes the TRM channel opening with the microfluidic liquid network may be defined by a distal slot in the central layer that defines the side walls of such portion of the TRM channel. The slot in the upper (or base layer) overlies (or underlies) and connects the proximal and distal slots in the central layer. The proximal slot in the central layer may be a dead end slot that does not directly connect to any other feature of the microfluidic network or TRM network in the absence of the slot in the upper or lower layer. The distal slot in the central layer may be a dead end slot that connects directly only with the microfluidic liquid network and not with any other portion of the TRM network in the absence of the slot in the upper or base layer. The longitudinal axes of the proximal and distal slots in the central layer may be generally aligned or at a diverging or converging angle to one another. The longitudinal axis of the slot in the upper (or lower layer) is typically opposed to, i.e., not parallel with, both the proximal and distal slots in the central layer. Because the slot in the upper (or lower layer) opposes the proximal and distal slots in the central layer, the slot in the upper (or lower layer) will still connect the proximal and distal slots in the central layer even if the upper or base layer and central layer are not perfectly aligned, e.g., transversely or longitudinally, during manufacturing.

[0035] In embodiments of the microfluidic device having a fourth layer, a vent junction, and vent channel, at least a first (proximal) portion of the vent channel may be defined by the slot in the upper (or lower layer). For example, for embodiments in which the slot is defined in the upper layer, a lower internal surface of a first portion of the vent channel is defined by an upper internal surface of the central layer, side walls of the first portion of the vent channel are defined by side walls of the slotalong the upper layer, and upper internal walls of the first portion of the vent channel are defined by a lower internal surface of the fourth layer. The first (proximal) portion of the vent channel may extend beyond the fourth layer to a vent opening in the upper layer. Alternatively, the vent channel may include a drop. A second (distal) portion of the vent channel extends from the drop to a vent opening in the upper layer. A lower internal surface of such second (distal) portion of the vent channel is defined by an upper internal surface of the lower layer, side walls of the second (distal) portion of the vent channel are defined by side walls of the central layer, and an upper internal wall of the second (distal) portion of the vent channel is defined by a lower internal surface of the upper layer. The height of the drop in the vent channel is defined by the thickness of the central layer.

[0036] Methods of using foregoing embodiments of the microfluidic device, include the following. TRM present in a TRM chamber of a microfluidic device is actuated, e.g., by using a heat source(s) to heat the TRM to a temperature at least as high as the transition temperature of the TRM. At least some of the resulting mobile TRM is moved out of the TRM chamber and along the TRM channel. The heat source(s) may be disposed and arranged according to any of the heat source embodiments disclosed herein.

[0037] When performed using a microfluidic device having a vent network, the step of moving the at least some TRM along the TRM channel may include obstructing a vent junction disposed along the TRM channel thereby preventing gas and / or liquid from passing through the vent junction and out of a vent opening in gaseous communication with the vent junction. The vent junction and vent opening may be gaseously connected by a vent channel and may be disposed and arranged pursuant to the foregoing embodiments of the microfluidic device. When performed using a microfluidic device having a microfluidic liquid network, alternatively or in combination with a vent network, the step of moving the TRM along the TRM channel may include moving at least some of the TRM out of a TRM channel opening and into a microfluidic liquid network, e.g., into a microfluidic liquid channel thereof, of the microfluidic device thereby obstructing the microfluidic liquid network, e.g., the microfluidic liquid channel thereof. In any of the foregoing methods, the step of moving the at least some TRM along the TRM channel may include moving the at least some TRM through one or more sharp angles along the TRM channel and / or up a step and / or down a drop each disposed and arranged along the TRM channel pursuant to any of the embodiments of such sharp angle(s), step(s), and / or drop(s) disclosed herein. The step of moving the at least some TRM up the step may include moving the at least some TRM a greater distancefrom a lower external surface of the microfluidic device and / or from a heat source used to heat the TRM. The step of moving the at least some TRM down the drop may include moving the at least some TRM to a smaller distance to a lower external surface of the microfluidic device and / or to a heat source used to heat the TRM. Alternatively, depending on the arrangement of the heat source and the microfluidic device, the step of moving the at least some TRM up the step may include moving the at least some TRM to a smaller distance to the external surface of the microfluidic device and / or to the heat source used to heat the TRM and the step of moving the at least some TRM down the drop may include moving the at least some TRM a greater distance from the external surface of the microfluidic device and / or to the heat source used to heat the TRM.

[0038] In the foregoing method, the at least some TRM may be moved out of the TRM chamber and along the TRM channel by, e.g., applying a gas pressure differential to the TRM. A gas pressure differential may be achieved, e.g., by increasing the gas pressure proximally to the TRM within or adjacent to the TRM chamber and / or decreasing the gas pressure disposed distally of the TRM within the TRM channel. The gas pressure increase or decrease may be obtained by respectively compressing a gas bladder in gaseous communication with a proximal portion of the TRM chamber and / or decompressing a gas bladder in gaseous communication with a distal portion of the TRM chamber. Subsequent to obstructing the vent junction and / or the microfluidic liquid network / channel, the TRM may be cooled or allowed to cool to a temperature below the transition temperature such that the TRM within the vent junction and / or microfluidic liquid network, e.g., within the microfluidic liquid channel, again becomes relatively immobile, e.g., non-flowable.

[0039] In embodiments of the method performed using a microfluidic device having a microfluidic liquid network including a reaction chamber, detection chamber, and / or combination thereof, the step of obstructing the microfluidic liquid network / channel may include sealing such chamber(s) to prevent material, e.g., reactants and / or products, from exiting the chamber(s) and / or from the microfluidic device. The method may include, prior to the step of obstructing the microfluidic liquid network / channel, introducing a liquid sample to such chamber. The method may further include combining and / or reacting such liquid sample and reagents within such chamber. The method may further include detecting one or more targets present in the liquid sample. For example, the method may include amplifying one or more target polynucleotides within the chamber(s) and detecting the amplified target polynucleotides. The step of obstructing the microfluidic liquid network / channel is typically performed prior to completion of the reacting step.In embodiments of the foregoing microfluidic method, the TRM channel includes a third portion having a longitudinal axis located at a third distance from the heat source, wherein the second distance is greater than the third distance. The first and third distances may be the same. The method includes moving at least some of the heated TRM along the first and second portions of the TRM channel and then moving at least some of the heated TRM along the third portion of the TRM channel. The method may include, following the step of moving the at least some heated TRM along the third portion of the TRM channel, moving at least some of the heated TRM into a microfluidic liquid channel of a microfluidic liquid network of the microfluidic device. The method may include, after moving the at least some heated TRM into the microfluidic liquid channel, allowing the TRM to cool to a temperature below the transition temperature of the TRM thereby obstructing the microfluidic liquid channel.

[0040] In embodiments, a microfluidic method includes heating, using a heat source disposed in thermal communication with a microfluidic device, a bolus of TRM disposed in a microfluidic TRM network of the microfluidic device, e.g., within a TRM chamber of the TRM network. The step of heating includes heating at least some, e.g., all, of the bolus of TRM to a temperature at least as high as a transition temperature of the TRM. The method further includes moving at least some of the heated TRM along a TRM channel of the TRM network. The TRM channel optionally includes a vent junction with a vent channel. In such embodiments, the step of moving at least some of the heated TRM along the TRM channel includes obstructing the junction with the vent channel thereby preventing fluids, e.g., gases and / or liquids, from exiting the microfluidic device via the vent channel. Alternatively, or in combination, the method includes moving at least some of the heated TRM along the TRM channel, e.g., beyond the vent junction if present, and into a portion of a microfluidic liquid network of the microfluidic device, e.g., into a microfluidic liquid channel of the microfluidic liquid network. The method may include, after obstructing the junction with the vent channel if present, cooling and / or allowing, the TRM obstructing the vent junction to cool to a temperature below the transition temperature of the TRM thereby rendering the TRM obstructing the vent junction immobile. The method may include, after moving the at least some heated TRM into the microfluidic liquid channel if present, cooling and / or allowing the TRM within the microfluidic liquid network, e.g., microfluidic liquid channel, to cool to a temperature below the transition temperature of the TRM thereby obstructing the portion of the microfluidic liquid network, e.g., obstructing the microfluidic liquid channel. The method may include, prior to the step of moving the at least some TRM along the TRM channel, introducing a liquid, e.g., a liquidsample, a liquid reagent such as a buffer, or combination thereof, into the microfluidic liquid channel. The liquid may be moved along the microfluidic liquid channel. The step of introducing the liquid and / or the step of moving the liquid along the microfluidic liquid channel may include expelling gas from the microfluidic liquid channel, along the TRM channel, and out of the microfluidic device through the vent channel if present. The step of moving the at least some TRM along the TRM channel may include, prior to or simultaneously with the step of obstructing the vent junction if present, increasing a distance between the at least some TRM in the TRM channel and the heat source. For example, in any of the embodiments, the TRM channel may include a step and the step of moving the at least some TRM along the TRM channel may include moving the at least some TRM up the step and further from the heat source, e.g., prior to or simultaneously with the step of obstructing the vent junction if present. The TRM network, microfluidic network, and vent network of such methods may have any of the features, arrangement, and dimensions of any of such elements disclosed herein.

[0041] In embodiments, a method of manufacturing any of the foregoing microfluidic devices includes forming a structure defining lower internal walls and side walls of at least a portion of a TRM network and of at least portions of one or both of a microfluidic liquid network and a vent network. An amount of TRM is deposited within a portion of the structure corresponding to the TRM network, e.g., within a location corresponding to a TRM chamber thereof. An upper layer is secured, e.g., adhered, to the structure to define at least some, e.g., most or all, upper internal walls of the TRM network including at the location of the structure in which the TRM is disposed. The upper layer may also define upper internal walls of at least portions of the at least one of the microfluidic liquid network and vent network. The step of depositing the TRM is typically performed with the TRM at a temperature above the transition temperature and the structure at a temperature below the transition temperature. Typically, neither the structure nor the completed microfluidic device is heated to a temperature above the transition temperature until the microfluidic device is operated to perform a diagnostic assay as disclosed herein.

[0042] In some embodiments of the method of manufacturing the microfluidic device, the TRM network of the microfluidic device includes a TRM channel and, after depositing the TRM within the TRM network but prior to operating the device to perform a diagnostic assay, the TRM channel remains free of TRM. For example, in embodiments of the manufactured microfluidic device including a microfluidic liquid network, the TRM is spaced apart from the microfluidic liquid network, e.g., froma TRM channel opening to a microfluidic liquid channel thereof, by at least most, substantially all, or all of the TRM channel.

[0043] Examples of liquids that can be used with the foregoing microfluidic devices and methods include liquid samples containing or suspected of containing one or more targets to be detected, liquid reagents, and combinations thereof. Exemplary liquid samples include whole blood, e.g., venous blood or fingerstick blood, blood based liquids, e.g., serum or plasma, nasopharyngeal samples, saliva, sputum, and urine. Exemplary liquid reagents include diluents, buffers, e.g., extraction buffers or lysing buffer, and combinations thereof.

[0044] Any of the foregoing microfluidic devices may include one or more non-liquid reagents, e.g., dried or lyophilized reagents, deposited within one or more of the microfluidic liquid channel(s) and / or detection / reaction chamber(s) thereof. Such reagents are mobilized by the liquid(s), e.g., liquid samples and / or liquid reagents and typically facilitate the preparation of a liquid sample and / or detection of one or more targets within a sample liquid. Exemplary reagents include, e.g., one or more of detection or assay label(s), (e.g., magnetic particle(s) and optical label(s), e.g., fluorophores), lysing reagent(s) (e.g., for lysing red blood cells or other cells of the sample), buffering reagent(s) (e.g., for controlling a pH of a liquid), agglutination reagent(s) (e.g., for separating plasma from red blood cells of a blood-based sample), DNA / RNA amplification reagent(s) (e.g., for amplifying a target polynucleotide), or other reagents useful in performing an assay for a target(s). Exemplary targets include, e.g., targets indicative of a disease state or physiological condition, e.g., cardiometabolic or infectious disease states or conditions and targets indicative of the presence of pathogens, e.g., viruses or bacteria. Such targets include, e.g., biomolecules such as proteins, polynucleotides, lipids, antigens, antibodies, and combinations thereof.

[0045] The TRM channel of the present microfluidic devices and methods advantageously reduces or prevents liquid disposed within the microfluidic liquid network from contacting the TRM prior to actuation thereof. In embodiments of the microfluidic device in which the TRM channel includes a step and / or a drop, the step and / or drop may enhance the capability of the TRM channel to prevent such liquid from contacting the TRM prior to actuation. Embodiments of the microfluidic device and methods including a vent junction disposed along the TRM channel advantageously permit a single bolus of TRM to obstruct both a vent and a portion of the microfluidic network thereby preventing liquid from exiting the microfluidic device. Embodiments of the microfluidicdevice in which a vent junction and / or portion of a vent channel are disposed at a greater distance from a heat source than portions of the TRM channel advantageously permit the TRM to cool more rapidly below the transition temperature thereby reducing or preventing the TRM from exiting the device via the vent.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 illustrates a diagnostic system including a diagnostic reader and a microfluidic device of the invention.

[0048] FIG. 2 is a top planar view of the microfluidic device of FIG. 1 ;

[0049] FIG. 3 is a top planar view of the microfluidic device of FIG. 1 with a rib and a label having been removed to reveal underlying features of the device;

[0050] FIG. 4a — 4d collectively, depict an exploded perspective view of the microfluidic device of FIG. 2 with electrical features used for detection of liquids within the microfluidic device and for detection of the mechanical actuation status of gas bladders of the microfluidic device, the label and an amount of temperature responsive material having been removed for clarity and with FIG. 4a depicting a rib, FIG. 4b depicting an upper layer (substrate), FIG. 4c depicting a central adhesive layer, and FIG. 4d depicting a lower layer (substrate) of the microfluidic device;

[0051] FIG. 5a is a magnified top planar view of a portion of the microfluidic device as shown in FIG. 2, illustrating the microfluidic device prior to actuation of a TRM disposed therein;

[0052] FIG. 5b illustrates the microfluidic device of FIG. 5a subsequent to actuation of the TRM disposed therein; and

[0053] FIG. 6 is a side cross-sectional view of the microfluidic device as shown in FIG. 2, also showing a microfluidic device support and heater of the diagnostic reader; illustrating the microfluidic device prior to actuation of a TRM disposed therein.

[0054] DESCRIPTIONWith reference to FIG. 1, a diagnostic system 201 includes a diagnostic reader 211 and a microfluidic device (strip) 10. Reader 211 includes an input port 213, which receives microfluidic device 10, and a touchscreen 215 by which a user can enter and receive information relevant to the operation of reader 211. Reader 211 operates the received microfluidic device 10 to determine the presence and / or amount of at least one target (e.g., a biomolecule such as at least one protein, at least one polynucleotide, or combinations thereof) present in a sample liquid applied to the microfluidic device 10. A temperature responsive material (TRM) disposed within the microfluidic device 10 can be actuated to obstruct or seal at least one portion of the microfluidic device 10, e.g., to prevent fluid, e.g., liquid and / or gas, from passing along a portion of a microfluidic network within the microfluidic device 10 and / or to prevent fluid from exiting, e.g., leaking from a vent opening of the microfluidic device 10. Reader 211 includes features for operating the microfluidic device 10 including one or more of a magnetic field generator, an optical detection system, one or more flow controllers, and contacts to receive and / or supply electrical signals to electrical components, e.g., liquid and / or mechanical sensing electrodes, of the microfluidic device 10 as disclosed in the ‘325 Application. Reader 211 may also include one or more heating system(s) or heating system controller(s) configured to actuate the TRM of the microfluidic device 10 and / or to heat a reaction chamber and / or detection chamber thereof. Alternatively, or in combination with such heating system of reader 211, the microfluidic device 10 may include a heating system operable by a heating system controller of reader 211 to perform such actuation.

[0055] With reference to FIGS. 2 and 3, the microfluidic device 10 includes a microfluidic liquid network 12 in gaseous communication with a microfluidic liquid network gas bladder 14 (a gas bladder may also be referred to as a gas chamber), a microfluidic temperature responsive material (TRM) network (TRM network) 16, in gaseous communication with a microfluidic TRM network gas bladder 18, and a microfluidic vent network (vent network) 20. Microfluidic device 10 is generally planar and has a major (longitudinal) axis ai and a minor (transverse) axis a2. A perpendicular axis a3extends perpendicularly to the plane generally defined by microfluidic device 10 (FIG. 6).

[0056] Microfluidic device 10 is composed of an upper substrate 22, a lower substrate 24, a central adhesive layer 26, a rib 28, and a label 30 (see, e.g., FIGS. 4a — 4d and 6). The term substrate is used herein interchangeably with the term layer. A substrate (layer) may be formed of a single layer or of two or more integrated lamina. Upper and lower substrates 22, 24 are each composed of a polyester film, about 100 pm thick. Upper substrate 22 has a lower (inner) surface 22a and an upper (outer) surface 22b. Lower substrate 24 has a lower (outer) surface 24a and an upper(inner) surface 24b. Upper substrate 22 defines a longitudinally extending slot 70, a transverse slot 70’ intersecting a proximal terminus 71’ of slot 70, and a vent slot 70”. Each of slots 70, 70’, 70” extend through upper layer 22. Slot 70 has a width of about 200 pm, a height of about 100 pm, and a length of between about 2 cm and about 4 cm between the proximal terminus 71’ and a distal terminus 71 thereof. Each of slots 70’, 70” has a width of about 200 pm, a height of about 100 pm, and a length of between about 2 mm and 5 mm, e.g., about 3 mm. Slots 70, 70’, 70” may be formed by, e.g., a mechanical process, e.g., stamping, an optical cutting process, e.g., laser ablation, a chemical process, e.g., lithography such as photolithography, a molding process, e.g., injection molding ora combination thereof.

[0057] Central adhesive layer 26 is about 140 pm thick. Central adhesive layer 26 has a lower (inner) adhesive surface 26a and an upper (inner) adhesive surface 26b. Upper (inner) surface 24b of lower substrate 24 and lower (inner) surface 22a of upper substrate 22 are adhered in opposition by surfaces 26a / 26b of adhesive layer 26. A lower (inner) surface 28a of rib 28 is adhered to upper (outer) surface 22b of upper substrate 22 via, e.g., a thin layer of adhesive or by thermal or pressure bonding. Rib 28 covers the entirety of each of the longitudinal slot 70 and the transverse slot 70’. Rib 28 is composed of a polymer, such as polyester, and typically includes an adhesive backing to adhere to upper (outer) surface 22b of upper substrate 22. Rib 28 is about 5 mm wide, 32.5 mm long, and 0.4 mm thick.

[0058] Microfluidic liquid channel network 12 includes, a sample application zone 32, and proceeding proximally to distally from the sample application zone 32, a first microfluidic liquid channel 34, a second microfluidic liquid channel 36, and a reaction / detection chamber 38. First and second liquid channels 34, 36 are in fluidic communication via a channel junction 37 therebetween. A distal portion 40 of the reaction / detection chamber 38 is in gaseous communication with liquid network gas bladder 14. Central adhesive layer 26 occupies less than all of the area of surfaces 22a, 24b between upper and lower substrates 22, 24 to define side walls 42 of the microfluidic liquid channel network 12 and side walls 42’ of the liquid network gas bladder 14. Features defining such side walls may be formed by, e.g., any of the processes used to form the slots 70, 70’, 70”. Within the microfluidic liquid channel network 12, upper internal walls (surfaces) are defined by portions of the lower surface 22a of the upper substrate 22 unoccupied by adhesive layer 26, e.g., overlying absent portions of adhesive layer 26, and lower internal walls (surfaces) are defined by those portions of the upper surface 24b of the lower substrate 24 unoccupied by central adhesive layer 26, e.g., underlying the absent portions of adhesive layer 26.Dimensions of the microfluidic liquid channel network 12 include the following. First and second microfluidic liquid channels 34, 36 are each about 800 pm wide and about 140 pm high. First microfluidic liquid channel 34 has a length of between about 20 and 30 mm between the sample application zone 32 and a distal terminus 49 of the first microfluidic liquid channel 34 (FIG. 5a). Second microfluidic liquid channel 36 has a length of between about 3 and 5 mm between the junction 37 with the first microfluidic liquid channel 34 and an opening 43 to the reaction / detection chamber 38.

[0059] Microfluidic TRM network 16 includes, a microfluidic TRM chamber (TRM chamber) 44, and proceeding proximally to distally from the TRM chamber 44, a microfluidic TRM channel (TRM channel) 46, and a microfluidic TRM channel opening (TRM channel opening) 48 in sidewall 42 of the first microfluidic liquid channel 34. A proximal portion 51 of the TRM chamber 44 is in gaseous communication with the TRM network gas bladder 18. An amount of, e.g., bolus, 45 of TRM is initially disposed within TRM chamber 44. A TRM is a material having a mobility, e.g., a flowability, that depends on temperature. The TRM is relatively immobile, e.g., relatively non-flowable or immovable, when below a transition temperature of the TRM and relatively mobile, e.g., relatively flowable or movable, when above the transition temperature. The TRM of the microfluidic device 10 is an aliphatic alcohol such as tricosane C23H48, transition temperature about 47.4°C, 1 -hexadecanol, (C16H34O), transition temperature about 49.3°C, or a combination thereof.

[0060] A shown in FIG. 5a, TRM channel 46 includes a proximal portion 50, a central portion 52, and a distal portion 54. TRM chamber 44, proximal portion 50, and distal portion 54 have side walls 56 defined by adhesive layer 26 (FIGS. 4c, 6). TRM network gas bladder 18 has side walls 56’ defined by adhesive layer 26 (FIG. 4c). TRM network gas bladder 18, TRM chamber 44, proximal portion 50, and distal portion 54 have upper and lower walls respectively defined by lower surface 22a of upper substrate 22 and upper surface 24b of lower substrate 24 as for the microfluidic liquid network 12. More particularly, the side walls 56 of the proximal portion 50 of the TRM channel 46 are defined by a proximal dead end slot 58 in the central adhesive layer 26 that extends from a TRM chamber opening 60 of the TRM chamber 44 to a distal terminus 62 (FIG.

[0061] 4c). Side walls 56 of the distal portion 54 of the TRM channel 46 are defined by a distal dead end slot 64 in adhesive layer 26 that extends from a proximal origin 66 to the TRM channel opening 48.Central TRM channel portion 52 (FIG. 5a) has side walls 68 (FIG. 4b) defined by longitudinal slot 70 within upper layer 22, a lower (internal) wall 72 defined by upper surface 26b of adhesive layer 26, and an upper internal wall 73 defined by lower surface 28a of rib 28. Referring to FIG. 6, sidewall 56 of proximal TRM channel portion 50 defines a step 86 between proximal portion 50 and central portion 52 of TRM channel 46. Sidewall 56 of distal TRM channel portion 54 defines a drop 88 between central portion 52 and distal channel portion 54 of TRM channel 46. Each of step 86 and drop 88 forms a sharp angle Z of about 90° along the longitudinal axis of TRM channel 46. A sharp angle along the longitudinal axis of TRM channel 46 is an angle of at least about 30°, at least about 45°, at least about 60°, or at least 75°. Typically, such sharp angle is about 180° or less, about 150° or less, or about 120° or less. Such sharp angle in a TRM channel may be formed without, or in combination with, a step and / or drop. For example, a sharp angle along a TRM channel may be formed within a TRM channel that occupies a single plane of a microfluidic device rather than with a step and / or a drop which depart from a single plane of the microfluidic device. A sharp angle may be formed, as seen in FIG. 6, by the intersection of two walls or surfaces that are linear, at least within a distance of at least about 1 mm, 2 mm, 3 mm, or 4 mm of the intersection. A sharp angle within a TRM channel, e.g., formed without or by a step and / or a drop, contributes to limiting and / or preventing sample liquid from passing along TRM channel 46 beyond the sharp angle, e.g., by capillary action, and contacting TRM bolus 45 and / or exiting microfluidic device 10 via vent network 20. Limiting and / or preventing sample liquid from entering TRM channel 46 and / or contacting the TRM bolus 45 within the TRM chamber 44 and / or TRM channel 46 prior to actuation thereof advantageously contributes to the ability of the TRM to obstruct first microfluidic liquid channel 34 and / or vent network 20 upon actuation thereof. Limiting and / or preventing liquid from exiting vent network 20 advantageously permits vent network to vent gas from microfluidic liquid network 12 thereby facilitating the movement of liquid therein while also preventing such liquid from exiting, e.g., leaking, from microfluidic device 10 via vent network 20.

[0062] Dimensions of TRM network 16 include the following. TRM chamber 44 has a length of about 7 mm, a width of about 1 mm, and a height of about 140 pm. Proximal and distal TRM channel portions 50, 54 each have a length of about 4 mm, a width of about 200 pm, and a height of about 140 pm. TRM channel opening 48 has a height and width about the same as distal TRM channel portion 54. Thus, a transverse cross-sectional area of TRM channel opening 48 (about 0.028 mm2) is smaller than a transverse cross-sectional area of first microfluidic liquid channel 34 at alocation adjacent such opening (about 800 pm x 140 pm « 0.11 mm2). A ratio of the transverse cross-sectional area of the first microfluidic liquid channel 34 adjacent the TRM channel opening 48 to the cross-sectional area of TRM channel opening 48 is typically at least about 2, e.g., at least about 3, or at least about 4. Such ratio is typically about 10 or less, e.g., about 8 or less, about 6 or less, or about 5 or less. Because of the smaller transverse cross-sectional area of TRM channel opening 48, capillary forces (e.g., surface tension) advantageously limit or prevent sample liquid present in first microfluidic liquid channel 34 from entering TRM channel 46, e.g., by capillary action, and contacting bolus 45 of TRM within TRM chamber 44 and / or from exiting the microfluidic device 10 via the vent network 20.

[0063] Central TRM channel portion 52 has a length between proximal and distal TRM channel portions 50, 54 of about 1.25 mm, a width of about 200 pm, and a height of about 100 pm. A transverse cross-section of central TRM channel portion 52 (about 0.02 mm2) is smaller than a transverse cross-section of one or both of proximal and distal TRM channel portions 50, 54 (about 0.028 mm2). A ratio of the transverse cross-section of central TRM channel portion 52 to the transverse cross-section of one or both of proximal and distal TRM channel portions 50, 54 is typically between about 0.6 and 1.0, e.g., between about 0.65 and 0.85, e.g., about 0.7. A TRM channel 46 having such a portion of smaller transverse cross-sectional area than a preceding and / or following portion of the TRM channel 46 advantageously limits or prevents liquid that might have entered the TRM channel 46 from passing into and beyond such smaller portion, e.g., by capillary action, and contacting bolus 45 of TRM within TRM chamber 44 and / or exiting the microfluidic device 10 via the vent network 20. Step 86 and drop 88 each have a height of about 140 pm along perpendicular axis a3(as shown in FIG. 6). Thus, the lower wall within central portion 52 of TRM channel 46 (defined by upper surface 26b of central adhesive layer 26, is disposed a greater distance from a heater element 92 than the lower wall within proximal and distal channel portions 50, 54 (which lower wall is defined by upper internal surface 24b of lower substrate 24 (FIG. 6).

[0064] With reference to FIGS. 4a, 4b, 5, 6, slot 70 in upper layer 22 fluidically connects proximal and distal TRM channel portions 50, 54, which, having side walls formed by dead-end slots 58, 64 in central adhesive layer 26 (FIG.4c), would otherwise not be connected along TRM channel 46. (The side cross-sectional view shown in Fig. 6 is taken through and along slot 70.) Slot 70 extends beyond proximal and distal TRM channel portions 50, 54 parallel to major axis ai of microfluidic device 10. Proximal and distal TRM channel portions 50, 54 extend beyond slot 70 generally along minor axis a2of microfluidic device 10. That is, slot 70 fluidically connects proximal anddistal TRM channel portions 50, 54 between a first location 74 of proximal portion 50 disposed intermediate TRM chamber opening 60 and distal terminus 62 and a second location 76 of distal portion 54 disposed intermediate proximal origin 66 and TRM channel opening 48 (FIG. 5a). A distance between first location 74 and distal terminus 62 and between second location 76 and proximal origin 66 is typically between about 0.5 mm and about 2.5 mm, e.g., between about 0.75 mm and about 1.5 mm. This configuration reduces manufacturing tolerances required when assembling the various substrates and layers of the microfluidic device 10 because slot 70 will still place proximal and distal TRM channel portions 50, 54 in fluidic communication via central TRM channel portion 52 even if the upper layer 22 with slot 70 is mispositioned along major axis a1 and / or minor axis a2 of microfluidic device 10 with respect to dead end slots 58, 64.

[0065] Vent network 20 includes, a vent junction 78 with distal TRM channel portion 54, and proceeding proximally to distally from the vent junction 78, a microfluidic vent channel (vent channel) 80 and a microfluidic vent opening (vent opening) 82. In the absence of TRM within TRM channel 46 and / or first microfluidic channel 34, vent opening 82 is in gaseous communication with microfluidic network 12 via TRM channel opening 48, distal TRM channel portion 54, vent junction 78 and vent channel 80. Thus, gas may be expelled from microfluidic liquid network 12 via distal portion 54 of TRM channel 46 and vent network 20 thereby preventing gas pressure buildup from hindering the movement of liquids therein.

[0066] Vent channel 80 includes a proximal portion 80’ and a distal portion 80”. Proximal portion 80’ of vent channel 80 has side walls defined by slot 70 within upper layer 22, a lower wall defined by upper surface 26b of adhesive layer 26, and an upper wall defined by lower surface 28a of rib 28 (FIG. 6). Distal portion 80” of vent channel 80 has side walls defined by adhesive layer 26 and upper and lower walls respectively defined by lower surface 22a of upper substrate 22 and upper surface 24b of lower substrate 24 as disclosed for the microfluidic liquid network 12. A drop 84 between proximal and distal vent channel portions 80’, 80” defines the intersection therebetween.

[0067] Microfluidic device 10 also includes electrical features disposed and configured to permit a reader to monitor the proper filling of microfluidic device 10 with sample liquid, the proper movement of sample liquid therein as well as monitoring the operation (e.g., the compression state) of each gas bladder 14, 18. The electrical features may be arranged and configured and operated by reader211 generally as disclosed in the ‘325 application and / orthe ‘858 application. For example, microfluidic device 10 includes electrodes configured to perform both a liquid sensing functionand a mechanical sensing function as disclosed in the ‘325 application and / or the ‘858 application. For example, as shown in FIG. 2, lead 125’ extends from a contact 127 through gas chamber 14 to a liquid sensing electrode 125. A lead 129 extends from a contact 131 into gas chamber 14. Gas chamber 14 also includes a bridging contact 133. When gas chamber 14 is fully compressed, bridging contact 133 brings lead 125’ and lead 129 into electrical communication, which is sensed by the reader via contacts 127,131. When leads 127,131 are not in electrical communication (i.e. , when gas chamber 14 is not in a fully compressed state), a reader operating microfluidic device 10 may also use liquid sensing electrode 125 to sense the presence of liquid in reaction / detection chamber 38. A supply electrode 121 connected via lead 121’ to a contact 123 emits a signal that is detected by liquid sensing electrode 125 as disclosed in the ‘325 application and / or the ‘858 application.

[0068] In use, microfluidic device 10 may be operated as follows to determine the presence and / or amount of one or more targets present in a liquid sample. A user inserts microfluidic device 10 into input port 213 of reader 211 (as shown in FIG.1). Lower (outer) surface 24a of lower substrate 24 contacts and is supported by a device support 90 within reader 211 (FIG. 6). A heater element 92 within device support 90 is placed in thermal communication with TRM chamber 44 and TRM channel 46. A separate heater (not shown) within reader 211 and within device support 90 is placed in thermal communication with reaction / detection chamber 38 of microfluidic device 10. Contacts within reader 211 engage contacts, including contacts 127, 131, 123 of microfluidic device 10 and flow controllers of reader 211 (e.g., piezo electric actuators) (not shown) engage and compress gas bladder liquid network gas bladder 14 of microfluidic device 10 as disclosed in the ‘325 Application. The user applies a liquid sample to application zone 32 of microfluidic network 12. The liquid sample flows by capillary action along first liquid channel 34 until a distal gas-liquid interface of the liquid sample reaches a distal terminus thereof. Liquid sample also proceeds at least partway along second microfluidic liquid channel 36. As the liquid sample flows distally along first liquid channel 34, gas disposed within microfluidic liquid network 12 exits microfluidic device 10 via distal portion 54 of TRM channel 46 and vent network 20. By allowing gas downstream of the liquid sample to exit microfluidic device 10, vent network 20 prevents a gas pressure increase that would otherwise hinder further distal movement of the liquid sample. Reader 211 detects the presence of the liquid sample by supply electrode 121 and liquid sensing electrode 125 as disclosed in the ‘325 application. Reader 211 then actuates the flowcontroller(s) to decompress liquid network gas bladder 14 to draw the liquid sample further along second liquid channel 36 and into reaction / detection chamber 38 as disclosed in the ‘325 application. The liquidsample mobilizes reagents, e.g., non-liquid reagents, deposited within microfluidic network 12, e.g., within one or more of first microfluidic channel 34, second microfluidic channel 36, or reaction / detection chamber 38. The reagents facilitate the preparation of a liquid sample and / or detection of at least one target in the liquid sample.

[0069] Reader 211 actuates the heater to increase the temperature of bolus 45 of TRM within TRM chamber 44 to at least the transition temperature thereof. Reader 211 actuates the flow controller of TRM gas bladder 18 to compress the gas bladder thereby increasing the pressure of the gas disposed proximally to TRM 45. At least some of the heated TRM is expelled from TRM chamber 44 through TRM chamber opening 60, moved along proximal TRM channel portion 50, up step 86, along central TRM channel portion 52, down drop 88, along distal TRM channel portion 54, out of TRM channel opening 48 and into first microfluidic liquid channel 34. The TRM moves along first microfluidic liquid channel 34 beyond channel junction 37 with second liquid channel 36. Portions of first microfluidic liquid channel 34 adjacent and proximal to junction 37 are spaced apart from, i.e. , are not directly over heater 92, and so are cooler than the TRM chamber 44 and TRM channel 46. Therefore, TRM within first microfluidic liquid channel 34 rapidly returns to an immobile state and obstructs fluid from passing therealong. For example, material, e.g., reactants and / or products such as amplicons, within reaction / detection chamber 38 and second liquid channel 36 cannot pass from the reaction / detection chamber 38, along first microfluidic liquid channel 34 and out of sample application zone 32. As TRM moves along proximal TRM channel portion 50 and central TRM channel portion 52, gas disposed between the TRM and TRM channel opening 48 is expelled through vent network 20 advantageously preventing the gas from displacing liquid sample disposed within microfluidic network 12.

[0070] In addition, at least some of the TRM within TRM channel 46 obstructs vent junction 78 and may enter proximal portion 80’ of vent channel 80. Because vent junction 78 and proximal portion 80’ of vent channel 80 are disposed a greater distance from underlying heater 92 than proximal and distal TRM channel portions 50, 54, the temperature of the TRM at vent junction 78 and within proximal portion 80’ of vent channel 80 falls more rapidly below the transition temperature than it would if at a smaller distance to heater 92. Therefore, the TRM at vent junction 78 and within proximal portion 80’ of vent channel 80 returns more rapidly to an immobile state and obstructs vent network 20 without passing out of vent opening 82 via vent channel 80. With vent junction 78 obstructed by TRM, neither gases nor liquids within microfluidic network 12 may exit microfluidic device via vent network 20.Reader 211 continues to operate microfluidic device 10 to determine the presence and / or amount of one or more targets present in the liquid sample within reaction / detection chamber 38. Such determination may include performing an amplification reaction of one or more target polynucleotides, a lysing step to release one or more targets present within cells of the liquid sample, immunological detection, e.g., via antibodies, or combination thereof. In some instances, the liquid sample may include one or more pathogens, e.g., viruses or bacteria. Advantageously, the TRM and TRM network of the microfluidic device prevents materials, e.g., amplicons and / or pathogenic materials, within the microfluidic network 12 and / or reaction / detection chamber 38 from exiting microfluidic device 10 and contaminating reader 211 , the workplace, and / or the user.

Claims

CLAIMSWhat is claimed, is:

1. A microfluidic device, comprising:(i) a microfluidic network comprising a microfluidic liquid channel;(ii) a microfluidic temperature responsive material (TRM) network comprising an amount of TRM disposed in a first position therein, a TRM channel opening in a wall of the microfluidic liquid channel, and a TRM channel disposed between the first position of the TRM and the TRM channel opening; and(iii) optionally, a vent network comprising a vent opening and a vent junction in a wall of the TRM channel, wherein the vent opening is in gaseous communication with the microfluidic liquid channel via at least the vent junction, at least a portion of the TRM channel, and the TRM channel opening.

2. The microfluidic device of claim 1, wherein the microfluidic liquid channel defines a transverse cross-sectional area at a location adjacent the TRM channel opening, the TRM channel opening defines a cross-sectional area, and a ratio of the cross-sectional area of the microfluidic liquid channel to the cross sectional area of the TRM channel opening has a (i) minimum of at least about 2, e.g., at least about 3, or at least about 4 and (ii) maximum of about 10 or less, e.g., about 8 or less, about 6 or less, or about 5 or less.

3. The microfluidic device of claim 2, wherein (a) the cross-sectional area of the TRM channel opening has a (i) maximum of about 0.06 mm2or less, about 0.05 mm2or less, about 0.04 mm2or less, or about 0.03 mm2or less and (ii) a minimum of at least about 0.015 mm2, at least about 0.020 mm2, or at least about 0.025 mm2and / or (b) the transverse cross- sectional area of the microfluidic liquid channel at the location adjacent the TRM channel opening has a (i) minimum of at least about 0.06 mm2, e.g., at least about 0.07 mm2, at least about 0.08 mm2, at least about 0.09 mm2, or at least about 0.1 mm2and (ii) a maximum of about 1.5 mm2or less, about 1.25 mm2or less, or about 1 mm2or less.

4. The microfluidic device of any of the foregoing claims, wherein the microfluidic liquid channel is a first microfluidic channel, the microfluidic network comprises a second microfluidic channel in fluidic communication with the first microfluidic channel via asecond channel opening disposed at a location along a wall of the first microfluidic channel, and a distance along a longitudinal axis of the first microfluidic channel between the TRM channel opening and the second channel opening has a (i) minimum of at least about 1.5 mm, at least about 2 mm, or at least about 2.5 mm and (ii) a maximum of about 10 mm or less, 7.5 mm or less, or about 5 mm or less.

5. The microfluidic device of claim 4, wherein the first microfluidic channel is in fluidic communication with a microfluidic chamber via the second channel opening and the second microfluidic channel and further wherein the microfluidic chamber is configured as a reaction chamber, a detection chamber, or a combination thereof.

6. The microfluidic device of any of the foregoing claims, wherein a length of the TRM channel along a longitudinal axis thereof between the first position of the TRM and the TRM channel opening has a (i) minimum of at least about 5 mm, at least about 6.5 mm, at least about 7.5 mm, at least about 8.5 mm, at least about 10 mm, or at least about 12.5 mm and (ii) a maximum of about 20 mm or less, about 15 mm or less, or about 13 mm or less.

7. The microfluidic device of any of the foregoing claims, wherein at least about 30%, at least about 50%, at least about 70%, at least about 90%, or essentially all of a total length of the TRM channel between the first position of the TRM and the TRM channel opening has an average transverse cross-sectional area having a (i) maximum of about 0.06 mm2or less, about 0.05 mm2or less, about 0.04 mm2or less, or about 0.03 mm2or less and (ii) a minimum of at least about 0.015 mm2, at least about 0.020 mm2, or at least about 0.025 mm2.

8. The microfluidic device of any of the foregoing claims, wherein the microfluidic device comprises the vent network and the vent network comprises a vent channel disposed between the vent opening and the vent junction and the vent opening is in gaseous communication with the microfluidic liquid channel via the vent channel, the vent junction, at least a portion of the TRM channel, and the TRM channel opening.

9. The microfluidic device of any of the foregoing claims, wherein the microfluidic device is generally planar, a lower internal surface of a first portion of the TRM channel occupies a first plane parallel to the plane of the microfluidic device, a lower internal surface of a second portion of the TRM channel occupies a second plane parallel to the plane of the microfluidic device, a lower internal surface of a third portion of the TRM channel occupiesthe first plane, the first and second planes are spaced apart from one another by a first distance along an axis perpendicular to the plane of the microfluidic device and the second portion of the TRM channel is disposed between the first and third portions thereof.

10. The microfluidic device of claim 9, wherein the TRM channel comprises a step disposed between the first and second portions thereof, a drop disposed between the second and third portions thereof, and the step and the drop each have a height equal to the first distance.

11. The microfluidic device of claim 9 or 10, wherein the microfluidic device comprises the vent network and at least a lower portion of the vent junction and, optionally, at least a portion of the vent channel, if present, occupies the second plane.

12. The microfluidic device of any of the foregoing claims, wherein the TRM network comprises a TRM chamber spaced apart by the TRM channel from the TRM channel opening and the amount of TRM is initially disposed in the TRM chamber.

13. The microfluidic device of claim 12, wherein (a) the TRM chamber comprises a TRM chamber opening to the TRM channel, a ratio of a transverse cross-sectional area of the TRM chamber at a location adjacent the TRM chamber opening to a cross-sectional area of the TRM chamber opening has (i) a minimum of at least about 2, e.g., at least about 3, or at least about 4 and (ii) a maximum of about 10 or less, e.g., about 8 or less, about 6 or less, or about 5 or less and / or (b) the TRM chamber opening has a cross-sectional area having (i) a maximum of about 0.06 mm2or less, about 0.05 mm2or less, about 0.04 mm2or less, or about 0.03 mm2or less and (ii) a minimum of at least about 0.015 mm2, at least about 0.020 mm2, or at least about 0.025 mm2.

14. The microfluidic device of any of the foregoing claims, wherein the TRM comprises an alkane such as tricosane, an aliphatic alcohol such as 1 -hexadecanol, or a combination thereof.

15. The microfluidic device of any of the foregoing claims, wherein the microfluidic device comprises a heat source integrated therewith and configured to heat the TRM within the TRM network to a temperature at least as high as the transition temperature thereof.

16. A system, comprising a microfluidic device of any of claims 1 — 15 and a reader, wherein the reader includes a heat source configured to heat the TRM within the TRM network to a temperature at least as high as the transition temperature thereof when the microfluidicdevice is disposed in an operable position therewith.

17. f method of using any of the microfluidic devices of claims 1 — 15 or the system of claim 16, comprising: heating the TRM to at least the transition temperature thereof, moving the TRM along the TRM channel, out of the TRM channel opening and into the microfluidic channel, wherein the TRM obstructs (i) the microfluidic channel at the location of the TRM therein and (ii) if present, the vent junction of the vent network.

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