Mechanical microfluidic manipulation

Abstract

Methods and devices for mechanically controlling microfluidic movement using force applicators and elastically deformable sheets are described herein. These devices may include mechanical microfluidic devices and cartridges. Microfluidic droplets may be moved or displaced within an air gap of a cartridge by applying a compressive force locally to selectively reduce the gap width of the air gap near the microfluidic droplets and move the microfluidic droplets toward the reduced gap. Compressive forces may also be used to split, join, mix, or perform other operations on the microfluidic droplets.

Description

[Technical field]

[0001] Claiming priority This application is a continuation of U.S. Patent Application No. 18 / 062,007, filed on December 5, 2022, entitled "SEQUENCING BY SYNTHESIS USING MECHANICAL COMPRESSION," U.S. Patent Application No. 18 / 062,011, filed on December 5, 2022, entitled "METHODS OF MECHANICAL MICROFLUIDIC MANIPULATION," U.S. Provisional Patent Application No. 63 / 298,973, filed on January 12, 2022, entitled "MICROFLUIDIC TWO-DIMENSIONAL CAPILLARY MANIPULATION DEVICES AND METHODS," and U.S. Provisional Patent Application No. 63 / 393,815, filed on July 29, 2022, entitled "MECHANICAL MICROFLUIDIC MANIPULATION DEVICES AND METHODS." No. 63 / 417,302, entitled "MECHANICAL MICROFLUIDIC MANIPULATION DEVICES AND METHODS," filed on October 18, 2022, and U.S. Provisional Patent Application No. 63 / 418,028, entitled "MECHANICAL MICROFLUIDIC MANIPULATION DEVICES AND METHODS," filed on October 20, 2022, each of which is incorporated herein by reference in its entirety.

[0002] INCORPORATION BY REFERENCE All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] background Microfluidics can achieve up to 10 times the volume of a liter. 15Microfluidic devices deal with very small amounts of fluid, down to femtoliters (fL), which is a fraction of a milliliter. Fluids at the micrometer scale behave very differently than everyday ones: these unique characteristics are key to new scientific experiments and innovations. Microfluidic devices may involve ultra-small devices that contain microchannels and chambers and tunnels through which fluids flow or are contained.

[0004] For example, digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale fluid droplets. DMF has rapidly gained popularity for chemical, biological, and medical applications because it can directly control multiple reagents (no pumps, valves, or tubing required); it can easily handle both solids and liquids (no channels to clog); and the hydrophobic surfaces (typically Teflon-coated) in contact with the fluid droplets are chemically inert and therefore compatible with difficult reagents (e.g., organic solvents, corrosive chemicals). However, conventional DMF devices use relatively large electric fields selectively applied to electrode arrays to manipulate the droplets. The generation and control of these electric fields requires specialized and complex circuitry capable of withstanding the relatively high voltages. Summary of the Invention

[0005] Summary of the Disclosure Methods and apparatus (e.g., devices and systems, including cartridges) for preparing, manipulating, and / or analyzing fluid droplets, such as microfluidic droplets, are described herein. For example, microfluidic devices are described herein that may be particularly useful for handling and analyzing clinical, laboratory, biological, or chemical samples. These devices may generally operate by applying a mechanical force, such as a compressive force, to an elastically deformable sheet that at least partially covers the air gap over a sub-region of the air gap to reduce the height of the air gap in a region proximate to the droplet and move the droplet toward this region of reduced height. Controlling the relative height of the air gap near the droplet (e.g., by controlling the application of a force to deform the elastically deformable sheet) can efficiently and rapidly move the droplet around the air gap, allowing for droplet processing, including droplet merging, droplet splitting, droplet mixing, droplet cooling / heating (e.g., thermal cycling of the droplet), and the use of magnetic particles within the droplet (e.g., to bind / remove material from the droplet).

[0006] Described herein are apparatus (systems, devices, etc.) for controlling microfluidic movement, e.g., droplet movement, on a surface by mechanical means. These apparatus may be referred to herein as mechanical microfluidic actuation devices ("mechanical microfluidic actuators") and may include a force applicator for applying a force to an elastically deformable sheet that at least partially encapsulates an air gap in which one or more droplets reside. The elastically deformable sheet may be part of a mechanical microfluidic actuator device (referred to herein as a "mechanical microfluidic actuator" or "mechanical microfluidic device") or may be part of a separate or integrated cartridge that is operated by a mechanical microfluidic device. In some examples, the cartridge may include a first (e.g., upper) elastically deformable sheet and a second (e.g., lower) sheet that are held apart (e.g., by a frame) to form an air gap in which one or more droplets can be manipulated by a force applicator (e.g., a stylus, etc.). The mechanical microfluidic device may include a force applicator, a force applicator driver subassembly (e.g., a force applicator subassembly), and a thermal subassembly for controlling the temperature of one or more regions of the air gap. Any of these mechanical microfluidic devices may also include a magnetic control subassembly for controllably applying a magnetic field within the air gap. In some examples, the device may include a cartridge holder for securing a cartridge to a cartridge seat or mounting area of ​​the mechanical microfluidic actuator. Optionally, the device may include a vacuum / suction subassembly for securing a cartridge to a mounting area of ​​the mechanical microfluidic actuator. In some examples, the mechanical microfluidic device may include a fluid handling (e.g., pipetting) subassembly for adding and / or removing fluids from the air gap.Other subassemblies forming part of the mechanical microfluidic device may include an imaging subassembly (e.g., for imaging a droplet in the air gap) and / or a sensing subassembly (e.g., for sensing a droplet or sensing other inputs from the air gap and mechanical microfluidic actuator). The mechanical microfluidic devices described herein may also include one or more control inputs (e.g., keyboard, touch screen, buttons, switches, etc.) and / or one or more outputs (e.g., displays, LEDs, wireless communication outputs / inputs, etc.) and hardware, software, and / or firmware for controlling the same. In some cases, the same features may be used for the control inputs and outputs. In general, the mechanical microfluidic actuators described herein may include one or more controllers for controlling and coordinating the operation of the various subassemblies.

[0007] For example, a microfluidic device is described herein that includes: a cartridge having a first sheet and a second sheet fixed generally parallel facing each other at a predetermined distance with an air gap therebetween; and a controller configured to selectively reduce the predetermined distance in one or more regions within the air gap proximate to a fluid droplet positioned within the air gap to move the fluid droplet within the air gap of the cartridge. The controller and / or cartridge may be part of a mechanical microfluidic actuator; in some examples, the cartridge may be separate from the mechanical microfluidic device and may be removable from the mechanical microfluidic actuator. The cartridge may be single-use or reusable (e.g., washable, sterilizable, etc.). In some cases, the cartridge may be integrated within the mechanical microfluidic actuator.

[0008] The second sheet may be elastically deformable or non-deformable. In some examples, the second sheet is made of the same material (e.g., elastic material) as the first sheet. The second sheet may be configured to be secured (by suction) to a system that holds the cartridge so that the cartridge makes consistent thermal contact with the second sheet so that the temperature can be changed quickly and efficiently by heating / cooling a localized area (thermal control area) of the second sheet to heat / cool the droplet in the air gap in an area above the thermal control area. In some examples, the droplet may be moved and / or pinned by deforming the lower sheet to change the height of a localized area of ​​the air gap; alternatively or additionally, the droplet may be moved and / or pinned by deforming the upper sheet to change the height of a localized area of ​​the air gap. When the force deforming the sheets (either or both of the first and second sheets) is removed, the sheets may return to a neutral, non-deformed state such that the air gap returns to approximately the same predetermined distance.

[0009] In any of these examples, the controller may be configured to selectively reduce the predetermined distance in the one or more regions within the air gap without reducing the predetermined distance in one or more adjacent regions. The sheet may have a first surface facing the air gap. Generally, either or both surfaces of the air gap may be hydrophobic and oleophobic; for example, the first surface may be hydrophobic and oleophobic and / or the plate may include a hydrophobic and oleophobic surface facing the air gap. Generally, the surfaces (or materials forming the sheet) described herein may be oleophobic in addition to being hydrophobic.

[0010] In any of these examples, the air gap may include at least one input / output port. For example, the sheet and / or plate may be configured to introduce a first fluid droplet into the air gap by including one or more input and / or output ports. In some examples, a fluid droplet may be inserted into (or removed from) the air gap. The inlet / outlet port may be a cut-out area of ​​the first sheet. The cut-out (opening) may be 1 mm to 10 cm long and 1 mm to 10 cm wide (e.g., 5 mm to 7 cm long, 5 mm to 4 cm wide, etc.). The opening through the first sheet may be offset from the edge of the sheet, for example, by 5 mm or more (e.g., 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 1 cm or more, 1.5 cm or more, 2 cm or more, etc.). The inlet / outlet port may be configured such that tension is maintained on the sheet in the area around the inlet / outlet. Hence, the inlet / outlet edges may be placed in tension.

[0011] The air gap may generally be of any suitable height ("thickness") when no force is applied to the sheets. For example, the air gap may have a height of about 0.4 mm to about 7 mm (e.g., about 0.5 mm to about 6 mm, about 0.5 mm to about 5 mm, about 0.5 mm to about 4 mm, etc.). Generally, the devices (including, but not limited to, cartridges) described herein may include an air gap formed between a first (e.g., upper) sheet and a second (e.g., lower) sheet. The controller may be configured to apply a compressive force to the sheets to selectively reduce the predetermined distance.

[0012] For example, described herein is a microfluidic device comprising: a cartridge comprising a first sheet and a second sheet fixed generally parallel facing each other at a predetermined distance with an air gap therebetween; and a controller configured to selectively reduce the predetermined distance in one or more regions within the air gap proximate to a fluid droplet positioned within the air gap to move a fluid droplet within the air gap of the cartridge.

[0013] The controller may generally be configured to selectively reduce the predetermined distance in one or more regions within the air gap without reducing the predetermined distance in one or more adjacent regions by applying a force (pressure) to elastically deform the first sheet (e.g., the upper sheet). The first sheet may have a hydrophobic first surface facing the air gap; the second sheet may have a second surface facing the air gap, and the second surface facing the air gap may also be hydrophobic and oleophobic. The cartridge may further include at least one input port on the first sheet and / or the second sheet configured to introduce a first fluid droplet into the air gap.

[0014] The controller may generally be configured to apply (and move) the compressive force across the sheet to selectively reduce a predetermined distance, whereby as the compressive force moves across the sheet, the width of the air gap dynamically changes to draw in droplets to follow the reduced height of the air gap, which movement may be continuous or periodic.

[0015] Generally, the first (and / or second) sheet is configured to elastically deform in response to a compressive force.

[0016] Generally, the first (and / or second) sheet is configured to elastically deform in response to a compressive force. In any of these methods and devices, the first sheet and / or the second sheet may be partially or completely clear (e.g., optically transparent) or opaque, depending. In some cases, both the first and second sheets are clear. In some instances, only the second sheet is clear. In some instances, only the first sheet is clear.

[0017] A sheet, as used herein, includes at least one surface that extends in a plane (e.g., in the x, y directions). In some examples, the sheet may be relatively thin, as in the first sheet that is elastically deformable. In some examples, the sheet may include regions of different thicknesses. The second sheet may not be elastically deformable. In some cases, the second sheet is rigid. In some cases, the second sheet may include regions of different thicknesses. In some examples, the second sheet may be elastically deformable. In some examples, the second sheet may be referred to as a layer, a plate, a base, or the like.

[0018] Any of these devices may include a force applicator (e.g., a stylus in some examples) positioned to apply a force to the outer surface of the first sheet to reduce the height of the air gap in a localized region, where the controller may be configured to direct movement of the force applicator relative to the outer surface (e.g., such that the force applicator moves across the outer surface). The force applicator may be a mechanical force applicator (e.g., a stylus, roller, ball point, etc.) and / or a pressure applicator (e.g., a jet applicator, etc.). The force applicator may be contact or non-contact. In some examples, the force applicator may include a source of pressurized fluid.

[0019] As mentioned, at least one of the first or second sheets may be elastically deformable. In some examples, the first sheet may be referred to as an upper sheet or an upper sheet, and the second sheet may be referred to as a lower sheet or a lower sheet. The second sheet may also be referred to as a plate in some examples.

[0020] In devices that include a cartridge, the cartridge may include a frame to which the first sheet (and / or the second sheet) is tensioned to maintain the first sheet generally parallel to the second sheet facing the second sheet. The frame may include a tensioner. In some examples, tension may be applied during fabrication, and the sheet may be attached (e.g., mechanically and / or chemically, e.g., by adhesive) to the frame under tension. The frame may surround the periphery of the cartridge. In some examples, the cartridge may be divided into regions, such as lanes, chambers, etc., that can be separated by one or more walls (e.g., partitions, etc.). The partitions may be affixed (e.g., glued, welded, etc.) to the upper and / or lower sheets. In some examples, the frame and spacer are combined into a single component, which is referred to herein as a spacer frame.

[0021] The second sheet may be of the same material as the first sheet (e.g., an elastic material) or may be of a different material. In some examples, the second sheet is formed of a non-elastic material. In some examples, the second sheet is formed of a material that is more rigid or stiff than the first sheet. The second sheet may be referred to as a plate in some examples. In any of the devices and methods described herein, the second sheet may be relatively thermally conductive to allow heating therethrough. In some examples, both the first sheet and the second sheet comprise an elastic material that is held taut in the frame of the cartridge. The first and second sheets may be of any suitable thickness, and may be the same thickness or different thicknesses. For example, the first and / or second sheets may be 0.05 mm to 5 mm thick (e.g., about 0.075 mm to 2 mm, about 0.1 mm to 5 mm, about 0.1 mm to 4 mm, about 0.2 mm to 4 mm, about 0.1 mm to 3.5 mm, about 0.2 mm to 3.5 mm, etc.).

[0022] The first and second sheets may be formed of any suitable material. In any of these devices and methods, the first and second sheets are formed of an elastomeric material. In some examples, the first sheet is a polyester material (e.g., TPE, TPU, etc.).

[0023] Any of these devices may include a controller configured to operate the force applicator to apply a force (e.g., a "compressive force") against the first or second sheet to reduce the height of the portion of the air gap proximate to the droplet in order to move the droplet (e.g., by temporary capillary action) to a region of reduced height. The controller generally controls the force applicator, including, for example, controlling the z-height based on the height of the air gap and / or controlling the x and / or y movement of the force applicator along the outer surface of the first (or in some instances second) sheet. The force applicator may drag the droplet as it moves along the upper sheet; the droplet may follow the region of reduced height air gap formed by the moving force applicator.

[0024] In general, the devices and methods described herein may operate by temporarily and dynamically reducing the height (or, in some instances, width) of an air gap formed between two surfaces in front of the droplet, such that the droplet is pulled toward the region of reduced height. The act of pulling the droplet by reducing the height of the air gap is sometimes referred to herein as the application of a temporary and / or dynamic capillary action force.

[0025] The controller may receive input from one or more sensors (e.g., optical sensors, electrical sensors, force sensors, pressure sensors, etc.), including a sensor that identifies the position of the droplet. In some examples, the force applicator includes one or more sensors. The controller may be configured to execute preprogrammed and / or dynamic steps, including moving the force applicator in a pattern to accomplish one or more fluid handling operations, such as splitting or dividing droplets, combining droplets, mixing droplets, washing the droplets (or materials within the droplets, such as magnetic materials, magnetic beads, etc.). For example, the controller may be configured to apply a pinning compressive force to divide a first fluidic droplet, and to apply an actuation compressive force near the pinning compressive force to elongate and form a second fluidic droplet from the first fluidic droplet, where the pinning compressive force is greater than the actuation compressive force. The controller may be configured to apply a pinning compressive force to split the first fluidic droplet, and to apply an actuation compressive force near the pinning compressive force to elongate and form a second fluidic droplet from the first fluidic droplet, where the pinning compressive force is greater than the actuation compressive force.

[0026] In some examples, the controller is configured to apply a compressive force to the sheet between two or more separate fluidic droplets and release the compressive force to combine the two or more separate fluidic droplets into a single fluidic droplet. In some examples, the controller is configured to alternately apply a first compressive force and a second compressive force different from the first compressive force to the sheet to mix the two or more separate fluidic droplets together. Thus, the controller may be configured to mix the two or more fluidic droplets together by repeatedly applying and releasing a compressive force to the sheet in proximity to the two or more fluidic droplets.

[0027] The controller may also be configured to coordinate one or more processes in addition to controlling the movement of the force applicator. For example, a coordinator may control and / or coordinate the addition of fluid into the air gap (e.g., dispensing of fluid, which may be performed manually, automatically, or semi-automatically). The controller may also control or coordinate the activity of one or more heaters, heat spreaders, and magnets. For example, in some examples, the controller may be configured to control a magnet to attract ferrous particles suspended in the first fluid droplet. The controller may further be configured to resuspend one or more ferrous particles in the fluid droplet by applying and releasing a compressive force to the fluid droplet and disabling the magnet. The first sheet may further comprise two or more pinning posts disposed on the surface and extending into the gap configured to restrict the movement of the first fluid droplet. Any of these devices may include a heater and / or heat spreader disposed under a second surface opposite the two or more pinning posts. Alternatively, the devices described herein may be used without the posts.

[0028] Any of the devices described herein may include a well into which a droplet may be moved. The well may be part of a cartridge and / or part of a mechanical microfluidic actuation device ("mechanical microfluidic actuator"). In some examples, the well is formed on a base or footprint of the mechanical microfluidic actuator. For example, the cartridge may fit into the footprint of the mechanical microfluidic device and may conform to the shape of the footprint that includes one or more wells. In some examples, the well is part of the cartridge, e.g., the well is configured to confine the movement of a fluid droplet.

[0029] Any of these devices (e.g., mechanical microfluidic actuator devices) may include a base having a cartridge pedestal configured to secure the cartridge, whereby the second sheet is held against (in contact with) the cartridge pedestal with at least one region of the cartridge in communication with a heating element disposed below the pedestal and configured to heat the fluid droplets in the air gap. In some examples, the mechanical microfluidic device may include a securement, such as a clamp, to secure the cartridge to and / or within the pedestal. In some examples, the pedestal may include a number of suction ports to apply negative pressure to hold the cartridge (e.g., the second or lower sheet of the cartridge) to the pedestal. The use of suction may be particularly beneficial to secure the cartridge snugly in place so that good thermal contact is maintained. The use of suction may also allow for the transfer of three-dimensional structures (e.g., wells, ridges, shelves, etc.) to the cartridge. (e.g., the lower sheet of the cartridge may conform to the shape of the pedestal).

[0030] In examples having a well, a heater, heat spreader, magnets, etc. may be configured to effect the well. In some examples, the heater and / or magnets may be arranged to effect droplets in the air gap in areas that are not part of the well, including rail areas. In general, the heater and / or magnets may be part of the cartridge or part of the actuation device (e.g., drive unit).

[0031] Methods of manipulating one or more microfluidic droplets using mechanical actuation of the droplets are also described herein. For example, methods are described herein that include the steps of: introducing a first fluid droplet into an air gap formed between a first sheet having a hydrophobic and oleophobic first surface and a second sheet having a hydrophobic and oleophobic second surface, the first sheet and the second sheet being fixed generally parallel facing each other at a predetermined distance with an air gap between them; and applying a force (e.g., a mechanical force) to elastically deform the first sheet to reduce the distance of the air gap between the first sheet and the second sheet in a region proximate to the fluid droplet in the air gap to move the fluid droplet in the air gap. Applying the force may include moving the force along an outer surface of the sheet to selectively reduce the distance between the first sheet and the second sheet such that the droplet follows the applied force. In any of these examples, applying the force may include moving a stylus relative to the first sheet. The applying of the force may include driving movement of a pressure applicator to apply a compressive force to the outer surface of the first sheet, where the pressure applicator is controlled by the controller. Any of these methods may include forming a second fluidic droplet from the fluidic droplet by applying a pinning compressive force to the first sheet to split the fluidic droplet, and applying an actuation compressive force to the first sheet near the pinning compressive force to elongate and form a second fluidic droplet, where the pinning compressive force is greater than the actuation compressive force.

[0032] For example, a method of microfluidically manipulating a droplet may include the steps of: introducing a droplet into an air gap formed between an elastically deformable first sheet and a second sheet, the first sheet facing the second sheet at a distance to form an air gap having a gap width of a predetermined distance in a neutral state; applying a compressive force to the first sheet using a mechanical force applicator to form a region of locally reduced gap width in the air gap adjacent to the droplet, thereby drawing the droplet toward the region of locally reduced air gap; and moving the droplet in the air gap by translating the mechanical force applicator along an outer surface of the first sheet to translate the region of locally reduced gap width in the air gap such that the droplet follows the mechanical force applicator.

[0033] In any of these methods, the step of moving the droplet may include moving the droplet along a rail region of the air gap, where the rail region has a gap width that is smaller than a gap width of a region surrounding the rail region in the air gap.

[0034] For example, a method of microfluidically manipulating a droplet may include the steps of: introducing a droplet into an air gap formed between an elastically deformable first sheet and a second sheet, the first sheet facing the second sheet at a distance to form an air gap having a gap width of a predetermined distance in a neutral state, the air gap being open to atmospheric pressure and unpressurized, and the droplet being positioned within a rail region of the air gap having a gap width smaller than the gap width of a region surrounding the rail region; applying a compressive force to the first sheet using a mechanical force applicator to form a region of locally reduced gap width in the air gap proximate to the droplet, thereby drawing the droplet by capillary action (e.g., temporary and / or dynamic capillary action) toward the region of locally reduced gap in the air gap; and moving the droplet along the rail region of the air gap by translating the mechanical force applicator along an outer surface of the first sheet to translate the region of locally reduced gap width in the air gap, thereby pulling the droplet within the air gap.

[0035] Any of these methods may include moving the droplet into a well formed by the second sheet. The well may be used to modify the droplet. For example, the well may be used to heat / cool the droplet and / or react a substance in the droplet. The methods described herein may include controlling the temperature of the well. For example, a method of microfluidically manipulating a droplet may include the following steps: introducing the droplet into an air gap formed between a first sheet that is hydrophobic, oleophobic, and elastically deformable and a second sheet that is hydrophobic and oleophobic, the first sheet facing the second sheet at a distance to form an air gap having a gap width of a predetermined distance in a neutral state, the air gap being open to atmospheric pressure and unpressurized; forming an area of ​​locally reduced gap width in the air gap adjacent to the droplet, thereby drawing the droplet by temporary capillary action toward the area of ​​locally reduced air gap. applying a compressive force to the first sheet using a mechanical force applicator to compress the droplet; moving the droplet into a well formed by the second sheet by translating the mechanical force applicator along an outer surface of the first sheet to translate a region of locally reduced gap width in the air gap, thereby pulling the droplet into the well within the air gap; modifying the droplet within the well; and moving the droplet out of the well by translating the mechanical force applicator along the outer surface of the first sheet to translate a region of locally reduced gap width in the air gap away from the well, thereby pulling the droplet out of the well. Any of these methods may include moving the droplet out of the well by translating the mechanical force applicator along the outer surface of the first sheet, where the region of locally reduced gap width in the air gap is translated away from the well, thereby pulling the droplet out of the well.

[0036] Thus, in general, any of these methods may include modifying the droplet in the air gap, which may include one or more of reacting one or more substances in the droplet, heating the droplet, adding a substance to the droplet, and applying energy to the droplet.

[0037] As mentioned, the first sheet may have a first hydrophobic and oleophobic surface positioned opposite a second hydrophobic and oleophobic surface of the second sheet.

[0038] Generally, the methods and apparatus described herein may manipulate droplets in an air gap by reducing the gap width (e.g., the distance between the upper and lower sheets); reducing the gap width may pull the droplet into the area of ​​reduced height by temporary capillary action. It may be particularly beneficial to have an air gap that is open to atmospheric pressure and may not be pressurized. This is in contrast to systems that drive the droplet by pressure (e.g., squeezing a fluidic material between the sheets), either trying to push the droplet or alternatively sucking the droplet by negative pressure.

[0039] Any of these methods may include applying a compressive force to the first sheet using a mechanical force applicator to draw the droplets towards the area where the air gap is locally reduced by temporary capillary action.

[0040] Generally, these methods may be part of any one of the following methods: nucleic acid extraction, library preparation, sequencing, and protein synthesis. For example, the introducing, applying, and transferring steps may be part of one or more of the following methods: nucleic acid extraction, library preparation, sequencing, and protein synthesis.

[0041] Generally, any suitable mechanical force applicator may be used. For example, the tip of the mechanical force applicator may have a rounded profile, a circular profile, an elliptical profile, a rectangular profile, or a square profile. In some examples, the tip of the mechanical force applicator includes a roller (e.g., has a wheel).

[0042] Any of these methods and devices may be configured to detect light transmitted through or reflected from the droplet. Any of these methods or devices may be configured to apply a voltage to the droplet from a mechanical force applicator or from a region below the second sheet. Any of these methods may include attracting magnetic particles suspended within the droplet by a magnet in the mechanical force applicator or in the region below the second sheet.

[0043] In general, these methods may include mixing the droplets in the air gap. The mixing may be chaotic or gentle. For example, chaotic mixing may be performed on the droplets by moving a mechanical force applicator (or any force applicator) in the z-axis across the first sheet and repeatedly applying and removing a compressive force by the mechanical force applicator. Alternatively or additionally, mixing of the droplets may be performed by moving a mechanical force applicator relative to (in contact with) the first sheet in the plane of the first sheet, e.g., in the direction of the x-axis and / or y-axis.

[0044] The methods and apparatus described herein may be configured to separate a droplet by applying a pinning compressive force to the first sheet; and applying an actuation compressive force to the first sheet near the pinning compressive force to elongate and split the droplet, where the pinning compressive force is greater than the actuation compressive force. Any of these methods may include removing all or a portion of the droplet from the air gap through an opening in the first sheet. Any of these methods may include introducing the droplet from a mechanical force applicator by passing the droplet through an opening in the first sheet.

[0045] As mentioned, methods of combining droplets using mechanical actuation through an elastically deformable sheet are also described herein. For example, any of these methods may include applying a compressive force to a first sheet to deform the first sheet between two or more separate fluid droplets; and releasing the compressive force to combine the two or more separate fluid droplets into a single fluid droplet.

[0046] Also described herein are methods of separating droplets using mechanical actuation through an elastically deformable sheet. For example, the method may include alternately applying a first compressive force and a second compressive force different from the first compressive force to a first sheet to mix two or more separate fluid droplets together in an air gap.

[0047] Also described herein are methods of mixing droplets using mechanical actuation through an elastically deformable sheet. For example, any of these methods may include repeatedly applying and releasing a compressive force to the outer surface of the first sheet in an area of ​​the first sheet that is in close proximity to two or more fluid droplets in the air gap to mix the two or more fluid droplets together. Any of these methods and devices may perform mixing by moving the mechanical force applicator in the y-axis or x-axis (e.g., in the plane of the sheet) relative to the first sheet in addition to or instead of mixing by moving the mechanical force applicator in the z-axis, which may result in gentler mixing than the chaotic mixing that results from moving the mechanical force applicator in the z-direction. Gentle mixing may be particularly preferred when mixing long polynucleotides to avoid shear.

[0048] These methods may also be used with magnetic beads or particles, which may be concentrated (e.g., with a magnet as described herein), washed, and resuspended. The method may include any of the steps of moving, mixing, splitting, and combining droplets. A controller may be configured to perform and control any of these steps. Also described herein is a method of attracting ferrous particles suspended within a fluid droplet with a magnet outside the air gap. Any of these methods may also include resuspending one or more ferrous particles in the fluid droplet by applying a compressive force to an outer surface of the first sheet on or in proximity to the fluid droplet in the air gap; and disabling the controllable magnet.

[0049] In some examples, the methods described herein may include constraining movement of the first fluidic droplet by two or more pinning posts disposed within the air gap. Alternatively or additionally, the methods may include using a well to hold the droplet, particularly during temperature adjustment (e.g., thermal cycling). Holding the droplet in the well (and / or "pinning" the droplet with one or more pinning posts) may prevent unintended movement of the droplet during operation, including particularly during heating of the droplet. Any of the methods described herein may include heating the droplet confined by the well and / or pinning posts with a heating element. Any of these methods may include heating the fluidic droplet in the well, for example, with a heating element (of a mechanical microfluidic actuator).

[0050] In use, any of these methods and apparatus may be used with a coating substance, which may be referred to as a gloss coat (e.g., equivalently referred to as a drop gloss, gloss coating, or gloss substance). The gloss coat may be a substance with low surface tension (e.g., oil) and may be immiscible with the droplets. If the droplets are aqueous, the gloss coating substance may be a hydrophobic substance. The gloss coating may be applied before or after the droplets are applied into the air gap. The gloss coat may be removed, for example, by wicking into a substance that is absorbent for the gloss substance. The gloss coat may be particularly useful in preventing evaporation of the droplets within the air gap.

[0051] Methods that include electroporating cells or particles within a droplet are also described herein. For example, any of these methods may include applying energy to electrodes on a plate and / or a force applicator to create temporary pores in the cell membranes of cells within the fluid droplet.

[0052] In general, the methods and devices described herein may be particularly useful for processing fluid droplets having a variety of small and medium volumes. For example, the methods described herein may be used to process droplets of about 10 -15 ~10 -6 It may be useful for fluid droplets having a volume of liters.

[0053] Mechanical microfluidic devices are also described herein. These microfluidic devices may include a cartridge mounting surface ("seat"); a force applicator configured to contact and apply a compressive force to an elastically deformable outer surface of a cartridge when the cartridge is mounted on the cartridge mounting surface; a force applicator driver configured to move the force applicator across the deformable outer surface; and a controller coupled to control the force applicator driver to move the force applicator relative to the deformable outer surface of the cartridge to dynamically reduce the height of the air gap in the cartridge to move a fluid droplet in the air gap of the cartridge. The force applicator driver may include one or more motors to move the force applicator in x, y, and / or z. While most of the examples provided herein are configured to move the force applicator while keeping the cartridge fixed (e.g., moving the force applicator relative to the elastically deformable sheet and the air gap), in some examples the device may be configured to move the cartridge (e.g., the elastically deformable sheet and the air gap) while keeping the force applicator fixed; alternatively, both the force applicator and the cartridge (e.g., the elastically deformable sheet and the air gap) may move relative to each other.

[0054] A mechanical microfluidic device may include one or more force applicators. In some examples, multiple force applicators may be controlled independently (in parallel or in series). In some examples, a mechanical microfluidic device may switch between one type of force applicator with another type of force applicator. In some examples, a mechanical microfluidic device may be configured to perform multiple simultaneous actuations using multiple different force applicators.

[0055] The force applicator may have any suitable shape, particularly one that applies sufficient compression to the elastically deformable sheet to reduce the height of the air gap while preventing or limiting damage to the sheet. For example, the tip of the force applicator may be configured to have a profile that includes a circle, an oval, a rectangle, or a square. In some examples, the force applicator includes a wheel, a ball point, or a roller.

[0056] In some examples, the force applicator may be adapted to perform one or more additional functions in addition to applying a force to the elastically deformable sheet of the air gap to reduce the height of the air gap to drive movement of the droplet in the air gap (e.g., by 2D capillary action, also referred to herein as temporary or dynamic capillary action). In some examples, the tip of the force applicator includes a thermal output configured to control a temperature of the tip. In some examples, the tip of the force applicator includes a light source. In some examples, the tip of the force applicator includes a light source and the cartridge mounted source includes a light sensor configured to detect light transmitted or reflected through the fluid droplet.

[0057] In some examples, the force applicator includes an electrode configured to apply a voltage (e.g., to perform electroporation). In some examples, the force applicator includes a magnet. For example, the force applicator may be further configured to provide a variable magnetic field strength. In some examples, the force applicator includes a sonication probe configured to emit at least one of sound waves and ultrasound waves.

[0058] Methods of moving a droplet using mechanical actuation through an elastically deformable sheet are also described herein. For example, these methods may include introducing a fluid droplet into an air gap formed between a first elastically deformable sheet and a second sheet, the first sheet facing the second sheet generally parallel to each other and spaced apart by a predetermined distance to form an air gap; and applying a compressive force to the elastically deformable sheet using a force applicator to move the droplet in the air gap, thereby reducing the predetermined distance in at least one area proximate to the fluid droplet in the air gap.

[0059] Any of these methods may also include moving a force applicator across the elastically deformable sheet while applying a compressive force such that the droplet conforms to a region of reduced distance (e.g., height) in the gap formed by the force applicator. The tip of the force applicator may be configured to have a rounded, circular, elliptical, rectangular, or square profile. In some examples, the tip is a roller, ball point, or wheel.

[0060] Any of the methods described herein may also include controlling a temperature of the area below the second sheet to control the temperature of the fluid droplet in the air gap. In some examples, the method may include controlling a temperature of the force applicator to control the temperature of the fluid droplet. In some examples, the method may include detecting light transmitted or reflected through the fluid droplet. The light may be part of a sensor for detecting the presence of the droplet and / or one or more characteristics of the droplet. The light may be emitted by a light source on the force applicator.

[0061] Any of these methods may include applying a voltage from a force applicator or from a region below the second sheet. The first sheet and / or the second sheet may be a dielectric. Any of these methods may include attracting ferrous particles (e.g., magnetic beads) suspended within the fluid droplet with a magnet in the force applicator or in the region below the second sheet. In some examples, the method may include removing ferrous particles from the fluid droplet. The method may include mixing the liquid droplet by repeated application and removal of a compressive force by the force applicator. Any of these methods may include aspirating the fluid droplet through an opening in the sheet using a force applicator.

[0062] In some examples, the method may include delivering a fluid droplet into the air gap from a force applicator. Introducing the fluid droplet may include passing the droplet from the force applicator through an opening in the elastically deformable sheet. The method may include applying at least one of sonic and ultrasonic energy to the fluid droplet from the force applicator.

[0063] Thus, methods and apparatus (e.g., devices and systems, including cartridges) are described herein for preparing, manipulating, and / or analyzing fluid droplets, such as microfluidic droplets. For example, microfluidic devices are described herein that may be particularly useful for handling and analyzing clinical, laboratory, biological, or chemical samples. These devices may generally operate by applying a force, such as a compressive force, to an elastically deformable sheet that at least partially covers the air gap over a sub-region of the air gap to reduce the height of the air gap in a region proximate to the droplet and move the droplet toward this region of reduced height. By controlling the relative height of the air gap near the droplet (e.g., by controlling the application of a force to deform the elastically deformable sheet), the droplet can be efficiently and rapidly moved around the air gap, which allows for the manipulation of the droplets, including combining the droplets, splitting the droplets, mixing the droplets, cooling / heating the droplets (e.g., thermal cycling the droplets), and using magnetic particles within the droplets (e.g., to bind / remove materials from the droplets).

[0064] The devices described herein may include two parallel hydrophobic and oleophobic sheets spaced apart by a gap of a predetermined distance. The gap may be filled with air or with a fluid immiscible with the microfluidic droplets. The microfluidic droplets may be manipulated (e.g., moved, controlled, separated, mixed, etc.) by selectively reducing the gap, particularly near the microfluidic droplets. In some examples, the gap may be reduced by applying a force (e.g., a compressive force) to one or more of the parallel sheets.

[0065] The examples described in the present disclosure may be implemented as a microfluidic device. The microfluidic device may include a cartridge, the cartridge including: a first sheet having a hydrophobic and oleophobic first surface and a second surface; a second sheet having a hydrophobic and oleophobic first surface and a second surface, the first surface of the first sheet being disposed toward and separated from the first surface of the second sheet by a predetermined distance to form a gap between the first sheet and the second sheet; and at least one input port on the first sheet configured to introduce a first microfluidic droplet into the gap. The microfluidic device also includes a controller configured to selectively reduce the predetermined distance in one or more regions in the gap proximate to the first microfluidic droplet, where the reduced predetermined distance displaces the first microfluidic droplet in the cartridge.

[0066] In some examples, a controller of the microfluidic device may be configured to apply a compressive force to the second surface of the first sheet to selectively reduce the predetermined distance.

[0067] In some examples, the first sheet may be configured to deflect in response to a compressive force and the second sheet may be configured to resist deflection in response to a compressive force.

[0068] In some examples, the controller may be configured to apply a pinning compressive force to split the first microfluidic droplet, and to apply an actuation compressive force near the pinning compressive force to elongate and form a second microfluidic droplet from the first microfluidic droplet, where the pinning compressive force is greater than the actuation compressive force.

[0069] In some examples, the controller may be configured to apply a compressive force to the first sheet between two or more separate microfluidic droplets and to release the compressive force to combine the two or more separate microfluidic droplets into a single microfluidic droplet.

[0070] In some examples, the controller may be configured to alternately apply a first compressive force and a second compressive force different from the first compressive force to the first sheet to mix two or more separate microfluidic droplets together.

[0071] In some examples, the controller may be configured to mix two or more microfluidic droplets together by repeatedly applying and releasing a compressive force on the first sheet in proximity to the two or more microfluidic droplets.

[0072] In some examples, the controller may be configured to control the magnet to attract ferrous particles suspended within the first microfluidic droplet.

[0073] In some other examples, the controller may be further configured to resuspend the one or more ferrous particles in the first microfluidic droplet by applying and releasing a compressive force to the first microfluidic droplet and disabling the magnet.

[0074] In some examples, the first sheet may further include two or more pinning posts disposed on the first surface and extending into the gap configured to confine movement of the first microfluidic droplet, in which case the device may further include a heater disposed beneath the second surface opposite the two or more pinning posts.

[0075] In some examples, the cartridge may further include a well disposed through the opening on the second sheet and configured to confine the movement of the first microfluidic droplet, in which case the cartridge may further include a heating element disposed below the well and configured to heat the first microfluidic droplet.

[0076] In some examples, the second sheet may further comprise an electrode configured to create temporary pores in cell membranes of cells within the first microfluidic droplet.

[0077] In some other examples, the first microfluidic droplet is 10 -6 ~10 -15 It may have a volume in litres.

[0078] The examples described in the present disclosure may be implemented as a method for manipulating one or more microfluidic droplets. The method may include introducing a first microfluidic droplet into a gap, where the gap is formed between a first sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; the first surface of the first sheet being disposed toward and separated from the first surface of the second sheet by a predetermined distance to form the gap. The method may further include selectively reducing the predetermined distance by a control unit in one or more regions in the gap proximate to the first microfluidic droplet to move the first microfluidic droplet.

[0079] In some examples, the method may include applying a compressive force to the second surface of the first sheet to selectively reduce the predetermined distance.

[0080] In some other examples, the first sheet may be configured to deflect in response to a compressive force, and the second sheet is configured to resist deflection in response to the compressive force.

[0081] In some examples, the method may include forming a second microfluidic droplet from the first microfluidic droplet by applying a pinning compressive force to the first sheet to split the first microfluidic droplet; and applying an actuation compressive force near the pinning compressive force to elongate and form the second microfluidic droplet, where the pinning compressive force is greater than the actuation compressive force.

[0082] In some examples, the method may include applying a compressive force to the first sheet between two or more separate microfluidic droplets and releasing the compressive force to combine the two or more separate microfluidic droplets into a single microfluidic droplet.

[0083] In some examples, the method may include alternately applying a first compressive force and a second compressive force different from the first compressive force to the first sheet to mix two or more separate microfluidic droplets together.

[0084] In some other examples, the method may include repeatedly applying and releasing a compressive force to a second sheet in proximity to two or more microfluidic droplets to mix the two or more microfluidic droplets together.

[0085] In some examples, the method may include attracting iron particles suspended within the first microfluidic droplet with a controllable magnet. In another example, the method may include resuspending one or more iron particles in the first microfluidic droplet by applying a compressive force to the first microfluidic droplet and disabling the controllable magnet.

[0086] In some examples, the method may include constraining movement of the first microfluidic droplet with two or more pinning posts disposed on a first surface of the first sheet and extending into the gap, and the method may further include heating the first microfluidic droplet constrained by the two or more pinning posts with a heating element.

[0087] In some examples, the method may include restricting movement of the first microfluidic droplet through the well. The method may further include heating the first microfluidic droplet in the well with a heating element.

[0088] In some examples, the method may include creating temporary pores in the cell membranes of cells in the first microfluidic droplet with electrodes on the second sheet.

[0089] In some examples, the first microfluidic droplet is 10 -6 ~10 -15 It may have a volume in litres.

[0090] Other examples described in the present disclosure may be implemented as a non-transitory computer-readable storage medium comprising instructions that, when executed by one or more processors of the device, cause the device to perform operations. The operations may include sensing a first microfluidic droplet disposed within a gap, where the gap is formed between a first sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; the first surface of the first sheet disposed toward and separated a predetermined distance from the first surface of the second sheet to form the gap. The operations may further include selectively reducing the predetermined distance in one or more regions within the gap proximate to the first microfluidic droplet to move the first microfluidic droplet.

[0091] Other examples described in the present disclosure may be implemented as a device including a first sheet having a first surface and a second surface that are hydrophobic and oleophobic; a second sheet having a first surface and a second surface that are hydrophobic, the first surface of the first sheet being disposed toward and separated a predetermined distance from the first surface of the second sheet to form a gap between the first sheet and the second sheet; and a pressure actuator coupled to the first sheet and configured to apply pressure to the first sheet to selectively reduce the gap between the first sheet and the second sheet.

[0092] Other examples described in the present disclosure may be implemented as a device including a cartridge, where the cartridge includes a first sheet having a hydrophobic and oleophobic first surface and a second surface; a second sheet having a hydrophobic and oleophobic first surface and a second surface, the first surface of the first sheet being disposed toward and separated a predetermined distance from the first surface of the second sheet to form a gap between the first sheet and the second sheet; and a stylus configured to contact the first sheet to selectively reduce the predetermined distance in at least one region within the gap.

[0093] In some examples, the predetermined distance may be reduced by a compressive force provided by a stylus. Further, in some examples, the reduced predetermined distance may move the microfluidic droplet within the gap. The tip of the stylus may have a profile that is circular, elliptical, rectangular, square, or a combination thereof.

[0094] In some examples, the tip of the stylus may include a temperature control device configured to control a temperature of the microfluidic droplet in the gap. In some other examples, the tip of the stylus may include a light source, and the second sheet includes a light sensor configured to detect light transmitted or reflected through the microfluidic droplet. Additionally, the tip of the stylus may include an electrode configured to receive a voltage sufficient to attract the microfluidic droplet through the first sheet. In some examples, the first sheet may be a dielectric.

[0095] In some examples, the stylus of the device may include a magnet configured to attract ferrous particles suspended within a microfluidic droplet disposed in the gap, and the magnet may be further configured to provide a variable magnetic field strength to sort the ferrous particles from the microfluidic droplet.

[0096] Other examples described in the present disclosure may be implemented as a method of manipulating one or more microfluidic droplets. The method may include a method of introducing a microfluidic droplet into a gap, where the gap is formed between a first sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; the first surface of the first sheet is disposed toward and separated from the first surface of the second sheet by a predetermined distance to form the gap. The method may further include providing a compressive force by a stylus, thereby reducing the predetermined distance in at least one region in the gap proximate to the microfluidic droplet.

[0097] In some examples, the reduced predetermined distance may displace a microfluidic droplet within the gap. The tip of the stylus may be configured to have a profile that is circular, elliptical, rectangular, square, or a combination thereof.

[0098] Some examples of the methods described herein may include controlling the temperature of the microfluidic droplet with a temperature control device disposed at the tip of a stylus. Some examples of the methods described herein may include detecting light transmitted or reflected through the microfluidic droplet with a light sensor. In some examples, the method may include providing a voltage to an electrode disposed on the stylus that attracts the microfluidic droplet through the first sheet toward the stylus. The first sheet may be a dielectric.

[0099] In some examples, the method may include attracting ferrous particles suspended within the microfluidic droplet with a magnet in the stylus. The method may further include sorting the ferrous particles from the microfluidic droplet. The method may include dispersing the particles within the microfluidic droplet by repeatedly applying and removing a compressive force with the stylus.

[0100] In some examples, the method may further include aspirating the microfluidic droplet with a stylus through a first septum in the first sheet and receiving the microfluidic droplet with a lumen in the stylus coupled to the first septum. In some examples, the method may further include injecting the microfluidic droplet with the stylus into a second septum different from the first septum. In some further examples, the method may include controlling a temperature of the microfluidic droplet with a temperature control element in the stylus. In some examples, the method may include detecting light transmitted or reflected by the microfluidic droplet in the stylus. In some further examples, the method may include providing at least one of an acoustic wave and an ultrasonic wave to the microfluidic droplet in the stylus.

[0101] Other examples described in the present disclosure may be implemented as a non-transitory computer-readable storage medium including instructions that, when executed by one or more processors of a device, can cause the device to perform operations, including: sensing a first microfluidic droplet disposed within a gap, the gap being formed between a first sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface being hydrophobic and oleophobic; the first surface of the first sheet being disposed toward and separated a predetermined distance from the first surface of the second sheet to form a gap; and providing a compressive force with a stylus, thereby reducing the predetermined distance in at least one region within the gap proximate to the microfluidic droplet.

[0102] Also described herein are methods of sequencing by synthesis using mechanical compression to manipulate droplets in an air gap formed between a first elastically deformable sheet and a second sheet. Each of the first sheet and the second sheet may include a hydrophobic and oleophobic surface facing the air gap. Either or both surfaces may be functionalized to include a hybridization region, such as a primer hybridization region. Generally, these methods may include the use of a mechanical force actuator that can be actuated to locally deform the first elastically deformable sheet to form a localized region of smaller gap width adjacent to the droplet, and can be moved along the surface of the first sheet to translate the region of locally reduced gap width so that the droplet is pulled about it. The droplet follows the region of smaller gap width in the air gap by temporary capillary action. Also described herein are apparatus (e.g., devices and systems, including cartridges) for performing any of these methods for sequencing by synthesis.

[0103] Any of the methods and devices described herein may be adapted to perform sequencing by synthesis. For example, a method of sequencing by synthesis using mechanical compression is described herein, including the steps of: sequentially moving each droplet of a series of fluid droplets in a cartridge, the cartridge comprising an air gap formed between a first sheet having a first surface that is hydrophobic and oleophobic and a second sheet having a second surface that is hydrophobic and oleophobic, the first sheet and the second sheet being fixed opposite each other at a predetermined distance with the air gap therebetween, and further comprising a plurality of polynucleotide clusters (clusters of polynucleotides) on the first sheet or the second sheet, and the step of moving each droplet comprises one or moving a plurality of mechanical force applicators relative to the first sheet to elastically deform the first sheet to create regions of reduced spacing between the first and second sheets, with each droplet following the regions of reduced spacing on the plurality of polynucleotide clusters, wherein the series of fluid droplets includes droplets of a nucleotide polymerase reaction mixture, droplets of a first wash buffer, droplets of a dye / terminator cleavage mixture, and droplets of a second wash buffer; and imaging the plurality of polynucleotide clusters to detect addition or hybridization of nucleotides to clusters of the plurality of polynucleotide clusters.

[0104] Any of these methods may include repeating the steps of sequentially moving each droplet and imaging the plurality of clusters to generate sequence data for the plurality of polynucleotide clusters. These methods may also include introducing the series of fluid droplets into an air gap. For example, sequentially moving each droplet may include moving each droplet as it is introduced into the air gap. In any of these methods, imaging may include imaging the plurality of polynucleotide clusters after moving a droplet of a first wash buffer over the plurality of polynucleotide clusters. The plurality of polynucleotide clusters may be arranged in a patterned arrangement, or in some examples, in a non-patterned arrangement.

[0105] In any of these examples, moving the one or more mechanical force applicators relative to the first sheet to elastically deform the first sheet includes moving one or more styluses relative to the first sheet.

[0106] Any of these methods may include a mixing step. For example, the method may include a chaotic mixing step by repeatedly applying and releasing a compressive force to a first sheet on the plurality of polynucleotide clusters to mix one droplet of the series of fluid droplets on the plurality of polynucleotide clusters. Alternatively or additionally, the method may include a gentle mixing step by moving a mechanical force applicator relative to the first sheet in the plane of the first sheet, such as in the direction of the x-axis and / or y-axis.

[0107] Sequentially moving each droplet of the series of fluid droplets may include removing each droplet of the series of fluid droplets from the air gap after it has moved onto the plurality of polynucleotide clusters.

[0108] The methods described herein may be used with droplets of very small volume. For example, droplets of the series of fluid droplets may be 10 -6 ~10 -15 It has a liter volume (eg, microliter or less).

[0109] Generally, in any of these methods, the air gap may be open to atmospheric pressure and not pressurized, and thus the droplet is not moved by pressure but by a change in gap width (also called gap height) that results in a temporary capillary force pulling on the droplet.

[0110] Any of these methods may include hybridizing a library of polynucleotides to either the first sheet or the second sheet within an air gap to form clusters of polynucleotides.

[0111] In some examples, a method of sequencing by synthesis using mechanical compression may include the steps of: sequentially moving each droplet of a series of fluid droplets in a cartridge, the cartridge comprising a first sheet having a first surface that is hydrophobic and oleophobic and a second sheet having a second surface that is hydrophobic and oleophobic, the first sheet and the second sheet being fixed opposite each other at a predetermined distance with the air gap therebetween, and further comprising a plurality of polynucleotide clusters on the second sheet, the moving each droplet comprising moving one or more mechanical force applicators relative to the first sheet to elastically deform the first sheet and form a gap between the first and second sheets; creating an area of ​​reduced spacing such that each droplet follows the area of ​​reduced spacing on the plurality of polynucleotide clusters, the series of fluid droplets including a droplet of a nucleotide polymerase reaction mixture, a droplet of a first wash buffer, a droplet of a dye / terminator cleavage mixture, and a droplet of a second wash buffer; imaging the plurality of polynucleotide clusters to detect addition or hybridization of nucleotides to a cluster of the plurality of polynucleotide clusters; and repeating the steps of sequentially moving each droplet and imaging the plurality of clusters to generate sequence data for the plurality of polynucleotide clusters.

[0112] A method of sequencing by synthesis using mechanical compression may include the steps of: sequentially introducing a series of fluid droplets into an air gap formed between a first sheet having a hydrophobic and oleophobic first surface and a second sheet having a hydrophobic and oleophobic second surface, the first sheet and the second sheet being fixed opposite each other at a predetermined distance with an air gap therebetween, and further comprising a plurality of polynucleotide clusters on the second sheet; elastically deforming one or more of the first sheet to create regions of reduced spacing between the first and second sheets such that each droplet conforms to a region of reduced spacing on the plurality of polynucleotide clusters; the step of sequentially moving each droplet of the series of fluid droplets over the plurality of polynucleotide clusters by moving a plurality of mechanical force applicators relative to a first sheet, the series of fluid droplets including a droplet of a nucleotide polymerase reaction mixture, a droplet of a first wash buffer, a droplet of a dye / terminator cleavage mixture, and a droplet of a second wash buffer; imaging the plurality of polynucleotide clusters to detect addition or hybridization of nucleotides to clusters of the plurality of polynucleotide clusters; and repeating the steps of sequentially introducing the series of fluid droplets, moving each droplet, and imaging the plurality of clusters.

[0113] All of the methods and apparatus described herein, in any combination, are contemplated herein and may be used to realize the benefits as described herein. [Brief description of the drawings]

[0114] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0115] A better understanding of the features and advantages of the methods and apparatus described herein will be obtained by reference to the following detailed description illustrating exemplary embodiments and the accompanying drawings, in which:

[0116] [Figure 1] 1A-1C show a portion of a microfluidic device (eg, a portion of a mechanical microfluidic device or a cartridge for use in a mechanical microfluidic actuator).

[0117] [Diagram 2] 1 is a flow chart illustrating example operations for manipulating microfluidic droplets.

[0118] [Diagram 3] 3A-3C show a portion of another microfluidic device (eg, a mechanical microfluidic actuator).

[0119] [Figure 4] 1 is a flowchart illustrating example operations for splitting a microfluidic droplet into two or more microfluidic droplets.

[0120] [Diagram 5] 5A-5C show a portion of another microfluidic device.

[0121] [Figure 6] 1 is a flow chart illustrating an example operation for mixing microfluidic droplets.

[0122] [Figure 7] 7A-7C show a portion of another microfluidic device.

[0123] [Figure 8] 1 is a flow chart illustrating example operations for removing suspended iron particles from a microfluidic droplet.

[0124] [Figure 9] 9A-9B show a portion of another microfluidic device.

[0125] [Figure 10] 1 is a flowchart illustrating example operations for dispersing particles in microfluidic droplets.

[0126] [Figure 11] 11A-11C show a portion of another microfluidic device.

[0127] [Figure 12] 1 is a flowchart illustrating example operations for manipulating microfluidic droplets in conjunction with pinning posts.

[0128] [Figure 13A] 1 shows a portion of another microfluidic device. [Figure 13B] 1 shows a portion of another microfluidic device. [Figure 13C] 1 shows a portion of another microfluidic device. [Figure 13D] 1 shows a portion of another microfluidic device. [Figure 13E] 1 shows a portion of another microfluidic device.

[0129] [Figure 14] 1 is a flow chart illustrating example operations for manipulating microfluidic droplets in conjunction with wells.

[0130] [Figure 15] 15A-15C show a portion of another microfluidic device.

[0131] [Figure 16] 1 is a flow chart illustrating example operations for providing electroporation.

[0132] [Figure 17]1 illustrates an exemplary microfluidic device (eg, a mechanical microfluidic actuator).

[0133] [Figure 18A] 1 shows an example of a portion of a microfluidic device as described herein.

[0134] [Figure 18B] 18B shows possible relevant end profiles for a stylus such as that shown in FIG. 18A.

[0135] [Figure 19] 19A and 19B show a portion of another microfluidic device.

[0136] [Figure 20] 1 is a flow chart illustrating example operations for manipulating microfluidic droplets.

[0137] [Figure 21] 21A and 21B show a portion of another microfluidic device.

[0138] [Figure 22] 1 is a flow chart illustrating example operations for manipulating microfluidic droplets.

[0139] [Figure 23] 23A-23C show a portion of another microfluidic device.

[0140] [Figure 24] 1 is a flow chart illustrating example operations for removing suspended iron particles from a microfluidic droplet.

[0141] [Diagram 25] 25A and 25B show a portion of another microfluidic device.

[0142] [Figure 26]1 is a flowchart illustrating example operations for dispersing particles in microfluidic droplets.

[0143] [Figure 27] 27A-27C show a portion of another microfluidic device.

[0144] [Figure 28] 1 is a flow chart illustrating exemplary operations for aspirating liquid in a microfluidic device.

[0145] [Figure 29] 29A-29C show a portion of another microfluidic device.

[0146] [Diagram 30] 1 is a flow chart illustrating exemplary operations for manipulating liquids in a microfluidic device.

[0147] [Diagram 31] 31A-31C show a portion of another microfluidic device.

[0148] [Diagram 32] 1 is a flow chart illustrating exemplary operations for thermally treating a liquid in a microfluidic device.

[0149] [Diagram 33] 1 shows a portion of another microfluidic device.

[0150] [Diagram 34] 34A-34C show a portion of another microfluidic device.

[0151] [Diagram 35] 1 is a flow chart showing exemplary operations for performing a reaction measurement.

[0152] [Diagram 36] 1 shows a portion of another microfluidic device.

[0153] [Figure 37] 37A-37C show a portion of another microfluidic device.

[0154] [Figure 38] 1 is a flow chart illustrating exemplary operations for performing sonication.

[0155] [Figure 39] FIG. 1 shows a block diagram of a device that may be an example of a microfluidic device (e.g., a mechanical microfluidic actuator) as described herein.

[0156] [Diagram 40] Figures 40A-40D illustrate one exemplary cartridge for use with a mechanical microfluidic actuator. Figure 40A shows an example of a cartridge having three lanes. Figure 40B shows an example of a cartridge having eight lanes. Figure 40C shows an example of a portion of the cartridge of Figure 40A; Figure 40D shows an expanded view of the cross-section of Figure 40C.

[0157] [Diagram 41] FIG. 13 is an exploded view of another example of the cartridge.

[0158] [Diagram 42] 42A and 42B show partial cross-sectional views of a portion of the cartridge.

[0159] [Figure 43A] 13 shows a cross-sectional view through another embodiment of a cartridge.

[0160] [Figure 43B] Figures 43B and 43C illustrate the cartridge of Figure 43A mounted on a mechanical microfluidic actuator, and Figure 43C shows the cartridge of Figure 43B with a droplet in the air gap of the cartridge. [Figure 43C] See legend to Figure 43B.

[0161] [Fig. 43D] 1 shows an example of a cartridge mounted on an actuator assembly where three parallel lanes are simultaneously actuated by a roller stylus.

[0162] [Figure 43E] 1 shows an example of an 8-lane cartridge as described herein.

[0163] [Figure 44A] Figures 44A-44E illustrate examples of various footprints of a mechanical microfluidic device coupled with a cartridge as described herein: Figure 44A shows an example of a cartridge coupled to a mechanical microfluidic device having a footprint that includes a concave footprint. [Figure 44B] FIG. 44B shows a mechanical microfluidic device having a footprint with a raised section. [Figure 44C] FIG. 44C shows an example of a mechanical microfluidic device having thermally conductive cylindrical rails (forming the rail region) protruding from the footprint region. [Fig.44D] FIG. 44D shows an example of a mechanical microfluidic device having a thermally conductive flat platform rail (eg, rail region) protruding from a footprint region. [Figure 44E] FIG. 44E shows an example of a mechanical microfluidic device having a thermally conductive dome-shaped platform rail (eg, rail region) protruding from a footprint region.

[0164] [Figure 45A] Figures 45A-45C illustrate examples of footprints for mechanical microfluidic actuators. Figure 45A shows the footprint. [Figure 45B] FIG. 45B shows a mounting area made partially transparent. [Figure 45C] FIG. 45C shows the mounting area with the cartridge attached.

[0165] [Figure 45D] 45D and 45E illustrate enlarged cross-sectional views through the example footprints of FIGS. 45A-45C. [Figure 45E] See legend to Figure 45D.

[0166] [Fig.45F] 45F-45G illustrate another example of a cartridge mounted on a mechanical microfluidic actuator. [Figure 45G] See legend to Figure 45F.

[0167] [Diagram 46] 13A and 13B are schematic diagrams illustrating an example of a mounting portion of a cartridge coupled to a mounting area.

[0168] [Figure 47] Illustrates a method for transferring small volumes of fluid using solid (eg, non-vacuum / non-pipette) techniques.

[0169] [Figure 48] 1A-1C illustrate schematic diagrams of examples of mechanical microfluidic devices as described herein.

[0170] [Figure 49] 1 illustrates an example of an interior region of a mechanical microfluidic device, showing an array of magnetic elements.

[0171] [Figure 50] 1 shows an example of a mechanical microfluidic actuator.

[0172] [Figure 51] 1 illustrates an example of a multiplexed device that includes multiple mechanical microfluidic actuators.

[0173] [Figure 52A] 1 shows a schematic illustration of an example of an assembly forming a mechanical microfluidic actuator. [Figure 52B]1 shows a schematic illustration of an example of an assembly forming a mechanical microfluidic actuator.

[0174] [Diagram 53] 53A-53C illustrate a method of operating an apparatus as described herein.

[0175] [Figure 54] 1 illustrates a schematic diagram of a method of sequencing-by-synthesis (SBS) that may be implemented by the techniques described herein.

[0176] [Figure 55] Figures 55A-55B illustrate an example of a step of introducing a sequencing primer to a DNA template in a cartridge (e.g., a flow cell) having discrete clusters, which may be part of an SBN method performed using the methods and apparatus described herein.

[0177] [Figure 56A] 1A-1C are schematic diagrams illustrating the delivery of SBS reagents using the methods and devices described herein. [Figure 56B] 1A-1C are schematic diagrams illustrating the delivery of SBS reagents using the methods and devices described herein. [Figure 56C] 1A-1C are schematic diagrams illustrating the delivery of SBS reagents using the methods and devices described herein. [Figure 56D] 1A-1C are schematic diagrams illustrating the delivery of SBS reagents using the methods and devices described herein. [Figure 56E] 1A-1C are schematic diagrams illustrating the delivery of SBS reagents using the methods and devices described herein.

[0178] [Figure 57] 1 illustrates a cartridge as described herein.

[0179] [Figure 58]1 illustrates a schematic diagram of a method for extracting polynucleotides from a tissue sample (e.g., blood) that may be performed using the methods and devices described herein.

[0180] [Figure 59] 1 illustrates a schematic of how an RNAseq workflow can be performed using the methods and apparatus described herein.

[0181] [Figure 60] 1 illustrates a schematic diagram of a method for Twist exome target enrichment rapid hybridization that may be performed using the methods and apparatus described herein.

[0182] [Figure 61] 1 illustrates a schematic of the workflow for performing Amplisequ (2 primer pools) using the methods and apparatus described herein.

[0183] [Figure 62-1] 62A-62D illustrate the loading and unloading of droplets within a cartridge as described herein. [Figure 62-2] 62E-62J illustrate the loading and unloading of droplets within a cartridge as described herein.

[0184] [Figure 63] 1 illustrates evaporation prevention using the methods and apparatus described herein.

[0185] [Fig. 64A] 1 illustrates an example of a mechanical microfluidic device that includes a base configured to hold a cartridge. [Fig. 64B] 1 illustrates an example of a mechanical microfluidic device that includes a base configured to hold a cartridge. [Fig. 64C]1 illustrates an example of a mechanical microfluidic device that includes a base configured to hold a cartridge. [Fig.64D] 1 illustrates an example of a mechanical microfluidic device that includes a base configured to hold a cartridge.

[0186] [Figure 65] 65A-65C illustrate an example of a base of a mechanical microfluidic device having vacuum (suction) ports, well regions, and thermally conductive regions arranged in multiple lanes.

[0187] [Figure 66] 66A and 66B illustrate an example of droplet metering using a metering area that may be part of a cartridge as described herein.

[0188] [Figure 67] 67A and 67B illustrate an example of distributing microparticles (eg, beads, cells, etc.) within a cartridge of an apparatus as described herein.

[0189] [Figure 68] Figures 68A and 68B illustrate examples of combinatorial chemistry as described herein.

[0190] [Figure 69A] 1 is a table showing the efficiency of removing polynucleotide material using a mechanical microfluidic (eg, mechanical actuation on surfaces, MAOS) device as described herein compared to manual techniques.

[0191] [Figure 69B] Electrophoresis gel comparing polynucleotides purified using a mechanical microfluidic device to a manual control.

[0192] [Figure 70-1]70A-70E illustrate an example of the use of a mechanical microfluidic device for preparing a library from RNA, as described herein. [Figure 70-2] See description of Figure 70-1. [Figure 70-3] See description of Figure 70-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0193] Detailed Description Microfluidic droplets may be manipulated through mechanical manipulation by elastically deforming the upper (and / or lower) surfaces of an air gap formed between two otherwise relatively parallel surfaces. At least one of the surfaces may be formed of an elastically deformable material to which a mechanical force may be applied to selectively reduce the gap between the surfaces. This process may be referred to herein as Mechanical Actuation on Surfaces (MAOS). Generally, the devices and methods described herein use mechanical compression to alter the capillary forces between the two surfaces and draw the droplets toward the compressed region. Generally, these methods and devices may be performed in an open air gap such that temporary capillary forces, rather than pressure, drive the movement of the droplets. The applied compression force may be applied near or in close proximity to one or more droplets in the gap; the reduction of the gap causes the droplets to move, separate, combine, mix, or incubate, etc.

[0194] Generally, the upper and lower surfaces, including but not limited to the elastically deformable surfaces, may each be referred to herein as a sheet. As used herein, "elastically deformable" means that the deformation of the sheet is non-permanent and substantially recovers or returns to its original or initial shape upon release of the force (which may be mechanical, electromagnetic, or any other type of force). Furthermore, the elastically deformable material may recover its initial configuration after cyclical mechanical loading. For example, the elastically deformable material may be capable of accommodating large deformations (e.g., at least 5% strain, and sometimes much more) upon application of a suitable stress, and yet allow the material to substantially return to its initial configuration upon removal of the applied stress. Examples of sheets of elastomeric materials may include TPE (thermoplastic elastomer) thin films, silicone sheets, and the like.

[0195] A microfluidic apparatus (e.g., device or system) for controlled liquid manipulation may include a two-dimensional (planar) chamber. The chamber may include a first sheet and a second sheet separated by a gap therebetween. The gap may separate the first and second sheets by a workable distance (e.g., 0.1 mm to 5 mm, etc.). The first and second sheets may be hydrophobic or may include a hydrophobic coating at least on the inner (air gap) region. In some examples, the first and second sheets are both hydrophobic and oleophobic and / or include a hydrophobic and oleophobic coating. In some examples, multiple chambers may be formed in close proximity to one another.

[0196] The microfluidic droplets can be manipulated through mechanical manipulation by applying a force directly or indirectly to the first or second sheet to selectively reduce the gap. This process is sometimes referred to as mechanical actuation on surfaces (MAOS) and is also described as using mechanical compression to alter capillary forces. The applied force (which may include a compressive force) may be applied near or in close proximity to the droplets in the gap. In some aspects, the reduction of the gap may cause the droplets to move, separate, combine, mix, or incubate, etc.

[0197] In some examples, the force may be applied by a stylus, which may include electrodes and / or controllable magnets, and the microfluidic droplets may be manipulated by a combination of pressure exerted by the stylus in conjunction with a voltage provided by the electrodes and / or a magnetic field provided by the magnets.

[0198] FIG. 1A shows a portion of a microfluidic device 100. Any of the devices described herein may be implemented, in part or in whole, as a system or any other executable apparatus. The microfluidic device 100 may include a first sheet 110 and a second sheet 120 separated by a gap 130. In some examples, the gap 130 may be generally filled with air. In some examples, the microfluidic device 100 may be a cartridge that may be selectively coupled to a control unit or a base station. As shown, the first sheet 110 may be the "top" sheet and the second sheet 120 may be the "bottom" sheet. That is, the first sheet 110 may be higher or "above" the second sheet 120. The second sheet 120 may be closer to the ground than the first sheet 110. In other examples, the second sheet 120 may be above the first sheet 110.

[0199] The first sheet 110 and the second sheet 120 may form a planar structure occupying any feasible area. The first sheet 110 and the second sheet 120 are shown in the figures in an initial position. In the initial position, the first sheet 110 and the second sheet 120 are relatively parallel to one another, separated by a distance related to and / or determined by the gap 130.

[0200] Each sheet may include two surfaces. For example, the first sheet 110 may include a first surface 111 and a second surface 112, and the second sheet 120 may include a first surface 121 and a second surface 122. For ease of explanation, the first surfaces 111 and 121 may be arranged towards the gap 130, while the second surfaces 112 and 122 may be arranged on the opposite side of the first sheet 110 and the second sheet 120, respectively. In other words, the second surfaces 121 and 122 may be arranged away from the gap 130.

[0201] First surfaces 111 and 121 may be hydrophobic (water repellent). In some examples, first sheet 110 and second sheet 120 (and therefore first surfaces 111 and 121) may be formed from a hydrophobic and oleophobic material. In other examples, first surfaces 111 and 121 may be a hydrophobic and oleophobic coating or layer applied onto first sheet 110 and second sheet 120, respectively.

[0202] A droplet 140 may be introduced into the gap 130. In some cases, the droplet 140 may be introduced into the gap 130 through a port or opening (not shown) on the first sheet 110 and / or the second sheet 120. The droplet 140 may be mechanically manipulated by selectively reducing the gap near the droplet 140. In some examples, one or more of the sheets 110 and 120 may be flexible. A flexible sheet may deflect in response to one or more forces. For example, the first sheet 110 may be flexible and the second sheet 120 may be rigid or semi-rigid. A rigid or semi-rigid sheet may resist deflection in response to one or more forces. In other examples, the second sheet 120 may be flexible and the first sheet 110 may be rigid or semi-rigid. In still other examples, both the first sheet 110 and the second sheet 120 may be flexible. As used herein, the term flexible may describe any material that can bend, deform, bend, move, or the like.

[0203] The droplet 140 may have a predetermined volume. In some cases, the droplet 140 may have a volume of 10 -6 ~10 -15 The gap 130 may be a microfluidic droplet having a volume of the droplet 140 that may be liters, although in some examples the volume of the droplet 140 may have any other workable volume. The gap 130 may be determined, at least in part, by the volume of the droplet 140. In other words, the gap 130 may be chosen or selected such that the droplet 140 (e.g., the volume of the droplet 140) can touch both the first sheet 110 and the second sheet 120.

[0204] FIG. 1B shows another view of the microfluidic device 100. In this view, the first sheet 110 may be deflected by a compressive force near or proximate one side or end of the droplet 140. The compressive force creates a reducing gap 132 between the first sheet 110 and the second sheet 120 toward that end or side of the droplet 140. As the reducing gap 132 forms, the droplet 140 may deform asymmetrically and may pull toward the reducing gap 132. In some cases, the droplet movement may be caused by differential temporary capillary action and / or a differential pressure gradient within the droplet 140. The compressive force may be provided by any feasible means. For example, an array of electromechanical, mechanical, and / or pneumatic actuators may be disposed next to the first sheet 110 and / or the second sheet 120 to selectively provide the compressive force to form the reducing gap 132. In another example, the compressive force may be provided by a stylus that may contact the first sheet 110 and / or the second sheet 120 .

[0205] In some examples, microfluidic device 100 may include one or more optical sensors (not shown) that may detect the presence and / or position of droplet 140. In this manner, data from the optical sensors may be used to assist in applying a compressive force near or in proximity to droplet 140.

[0206] Figure 1C shows another view of the microfluidic device 100. In this view, the compressive force on the first sheet 110 has been removed or reduced, and the first sheet 110 and the second sheet 120 have returned to their initial positions (as shown in Figure 1A). The gap 130 may be similar to the gap 130 in Figure 1A. The droplet 140 is shown in a second position, having moved in response to the compressive force described with respect to Figure 1B.

[0207] Thus, any droplet may be manipulated to any area within the microfluidic device 100 by reducing the gap (width of the air gap) near one end of the droplet in the manner described in FIGS. 1A-1C. This method is advantageous in that it avoids the generation and control of high voltages associated with conventional microfluidic devices and also avoids the need for multiple electrodes. The compressive force described herein may be provided by any viable source. For example, a mechanical actuator such as a lever, ball, or roller may provide the compressive force to at least one of the first sheet 110 or the second sheet 120. In some examples, the compressive force may be computer-controlled or processor-controlled. Thus, the manipulation of the droplet 140 may be computer-controlled and / or processor-controlled.

[0208] The mechanical actuator may be configured to reduce or prevent damage (tearing, buckling, snagging, etc.) to the elastically deformable surface. For example, the mechanical actuator, or a portion of the mechanical actuator, may be configured to be soft (e.g., low durometer hardness, such as having a Shore 00 hardness of 70 or less (e.g., 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, etc.). Alternatively or additionally, the mechanical actuator may include a rolling contact portion ("roller"), e.g., a wheel, ball, etc. For example, the mechanical actuator may include a roller having a soft material (a material with a low durometer hardness).

[0209] 2 is a flow chart illustrating example operations 200 for manipulating microfluidic droplets. In some examples, the operations described herein can be performed with additional operations, fewer operations, operations in a different order, operations in parallel, and with some different operations. Although operations 200 are described below with respect to microfluidic device 100 of FIGS. 1A-1C, operations 200 may be performed by any other suitable system or device.

[0210] The operation 200 begins at block 202 where a microfluidic droplet is introduced into a gap between two hydrophobic and oleophobic sheets. For example, a droplet 140 may be loaded into a gap 130 between a first sheet 110 and a second sheet 120 of a microfluidic device 100. The gap 130 may be an initial separation distance between the first sheet 110 and the second sheet 120. The first sheet 110 and the second sheet 120 may be hydrophobic and oleophobic or may include surfaces covered with hydrophobic and oleophobic layers. The droplet 140 may be placed at an initial position. The gap 130 may be filled with any viable gas, such as air. In some examples, the gap 130 may be filled with an immiscible fluid (with respect to the droplet 140). The presence and / or location of the droplet 140 may be determined by an optical sensor (not shown). Exemplary optical sensors may include one or more digital cameras, or an array of visible and / or invisible light detectors, etc. Thus, the optical sensor may determine when a droplet 140 is introduced into the gap 130 .

[0211] Next, in block 204, a distance between the two hydrophobic and oleophobic sheets is selectively reduced by mechanical actuation near the microfluidic droplet. The reduced distance can result in a reduced gap 132 between the first sheet 110 and the second sheet 120. In some examples, a compressive force may be provided to the sheet 110 proximate (e.g., next to) one side of the droplet 140. The compressive force may reduce the gap 130 and move the droplet 140 toward the compressive force. In some examples, the compressive force may deform one end of the droplet 140.

[0212] Next, in block 206, the distance between the two hydrophobic and oleophobic sheets is restored. For example, the compressive force applied in block 204 may be removed, which allows the first sheet 110 and / or the second sheet 120 to return to the initial separation distance. In some examples, the distance between the first sheet 110 and the second sheet 120 returns to a distance similar to the initial separation distance of block 202. The microfluidic droplet may come to rest at a different position to which it has moved from the initial position of block 202.

[0213] The steps of blocks 202-206 may be repeated any number of times to manipulate one or more droplets to any location within gap 130 of microfluidic device 100. In some examples, different compressive forces (e.g., different force amplitudes) may be applied to the droplets to perform different manipulations.

[0214] Figure 3A shows a portion of another microfluidic device 300. Microfluidic device 300 may include a first sheet 310, a second sheet 320, and a gap 330, which may be examples of first sheet 110, second sheet 120, and gap 130 of Figure 1. A source droplet 340 (which may be similar to droplet 140) may be introduced into gap 330 as described above with respect to Figures 1A and 2.

[0215] A pinning compressive force may be applied to the source droplet 340. The pinning compressive force is shown in the figures as being applied to the first sheet 310, but in other examples may be applied to the second sheet 320. As shown, the pinning compressive force may be applied toward a general center or middle of the source droplet 340. The pinning compressive force may be provided by any technically feasible device or operation. The pinning compressive force may begin to split or separate the source droplet 340 into two droplets.

[0216] FIG. 3B shows another view of microfluidic device 300. As shown, an actuation compressive force may be applied to source droplet 340. The actuation compressive force may be less than the pinning compressive force applied in FIG. 3A. The actuation compressive force may be applied simultaneously with (concurrent with) application of the pinning compressive force or after. The actuation compressive force may separate and guide satellite droplet 341 away from source droplet 340. In some examples, the pinning compressive force may bring first sheet 310 into contact with second sheet 320, which helps separate satellite droplet 341 from source droplet 340.

[0217] Figure 3C shows another view of microfluidic device 300. As shown, the pinning and actuation forces have been removed or reduced, which allows first sheet 310 and / or second sheet 320 to return to an initial position, such as the initial position shown in Figure 3A. Source droplet 340 is shown separated from satellite droplet 341.

[0218] 4 is a flow chart illustrating an example operation 400 for splitting a microfluidic droplet into two or more microfluidic droplets. Operation 400 is described below with respect to microfluidic device 300 of FIGS. 3A-3C, although operation 400 may be performed by any other suitable system or device.

[0219] The operations 400 begin at block 402 where a pinning compressive force is applied to the source microfluidic droplet by mechanical actuation. The source microfluidic droplet (which may be similar to source microfluidic droplet 340) may have previously been introduced into the gap between first sheet 310 and second sheet 320, for example, as described above with respect to FIGS. 1A and 2. In some examples, the pinning compressive force may begin to split or separate the source microfluidic droplet into two or more droplets. The pinning compressive force may be a mechanical actuation force provided to first sheet 310 and / or second sheet 320 and may be provided by any feasible means. In some cases, the pinning compressive force may be applied toward the center or center of the source microfluidic droplet. In some examples, the pinning compressive force may bring first sheet 310 into contact with second sheet 320.

[0220] Next, in block 404, an actuation compressive force is applied to the source microfluidic droplet by mechanical actuation. The actuation force may be applied to separate, direct, and / or steer the satellite microfluidic droplet away from the source microfluidic droplet. The actuation compressive force may be another mechanical actuation force, in which case the force is less than the pinning compressive force. The actuation compressive force may be applied coincident with or subsequent to application of the pinning compressive force.

[0221] At block 406, the pinning and actuation compressive forces are removed or reduced. In the absence of compressive forces, the first sheet 310 and the second sheet 320 may return to their relative initial positions, as depicted in FIG. 3C. When the pinning and actuation compressive forces are removed or reduced, the satellite droplets may remain separated from the source droplets.

[0222] In some examples, compressive forces can be used to fuse two or more separate droplets. One such example is described in conjunction with Figures 5A-5C and 6. Droplets of different or same size can be fused. For example, volumes of 250 nL to 80 μL can be firmly actuated to fuse with equivalent, larger, or smaller volumes already present in the air gap.

[0223] FIG 5A illustrates a portion of another microfluidic device 500. As shown in FIG 5A, microfluidic device 500 may include a first sheet 510, a second sheet 520, and a gap 530, which may be other examples of first sheet 110, second sheet 120, and gap 130 of FIG 1A. A first droplet 540 and a second droplet 541 (which may be similar to droplet 140 of FIG 1A, or source droplet 340 and satellite droplet 341 of FIG 3C) may be introduced into gap 530 as described above with respect to FIGS 1A-1C, 2, 3A-3C, and 4.

[0224] A compressive force may be applied to first sheet 510 and / or second sheet 520. In some examples, a compressive force may be applied between first droplet 540 and second droplet 541, which causes the respective droplets to deform, move, and possibly merge. The compressive force may be a mechanical actuation force as described herein.

[0225] 5B shows another view of microfluidic device 500. The compressive force may be removed or reduced, thereby allowing first sheet 510 and / or second sheet 520 to return to their initial (uncompressed) positions. As shown, first droplet 540 and second droplet 541 may be combined into fused droplet 542. Even though combined into a single droplet, the components within the individual droplets, or first droplet 540 and second droplet 541, may not be well mixed within fused droplet 542.

[0226] 5C shows another view of the microfluidic device 500. A compressive force may be repeatedly applied and released to the first sheet 510 and / or the second sheet 520 to mix the contents of the fused droplets 542. The repeated application of the compressive force may cause the fused droplets 542 to repeatedly deform and recover, thereby causing the contents of the fused droplets 542 to mix. In some examples, the compression / relaxation cycle caused by the application and release of the compressive force may be repeated a prescribed number of times to mix the contents of the fused droplets 542.

[0227] FIG. 6 is a flow chart illustrating an example operation 600 for mixing microfluidic droplets. The operation 600 is described with respect to the microfluidic device 500 of FIGS. 5A-5C, however, the operation 600 may be performed by any other suitable system or device. The operation 600 begins at block 602, where a compressive force is applied to the first sheet 510 and / or the second sheet 520 between the first microfluidic droplet 540 and the second microfluidic droplet 541 by mechanical actuation. The compressive force may be a mechanical actuation force provided by any operable means. The compressive force may move the first microfluidic droplet 540 and the second microfluidic droplet 541 toward each other, and in some cases, may combine them into a single (fused) microfluidic droplet 542.

[0228] At block 604, the compressive force is reduced or removed from the first sheet 510 and / or the second sheet 520. In some examples, the reduction or removal of the compressive force may allow the first sheet 510 and / or the second sheet 520 to return to an initial position.

[0229] In some cases, additional agitation of the fused microfluidic droplets 542 may be desired to provide additional mixing. To provide additional agitation, application and removal (or reduction) of the compressive force may be repeated for a predetermined number of cycles. Thus, in block 606, the number of completed compression cycles is determined. A completed compression cycle may include application and removal or reduction of the compressive force. If the number of compression cycles is less than the predetermined number, the operation may return to block 602. On the other hand, if the number of compression cycles is greater than or equal to the predetermined number, the operation 600 may end.

[0230] In some examples, iron particles may be suspended within the microfluidic droplets to aid in processing or assays, and after one or more processing steps are completed, the iron particles may be removed from the droplets for further processing.

[0231] 7A shows a portion of another microfluidic device 700. Microfluidic device 700 may include a first sheet 710, a second sheet 720, and a gap 730; these may be examples of first sheet 110, second sheet 120, and gap 130 of FIG. 1. Droplet 740 may include one or more iron particles 741, which may be suspended within droplet 740. Microfluidic device 700 may also include a magnet 750 (not shown).

[0232] 7B shows another view of microfluidic device 700. Magnet 750 may be activated or enabled. For example, magnet 750 may be an electromagnet that may be enabled through the application of power. In another example, magnet 750 may be a permanent magnet that may be moved toward droplet 740. Additionally, a compressive force may be applied to first sheet 710 and / or second sheet 720 toward one side of droplet 740. The compressive force may be applied at or near the same time that magnet 750 is enabled or moved.

[0233] The magnet 750 may cause the iron particles 741 to aggregate into one or more iron beads 742, which may cause the iron particles 741 to move out of suspension in the droplet 740. In addition, the compressive force may move the droplet 740 away from the magnet 750. In some cases, the movement of the droplet may sort or remove the iron particles from the droplet 740.

[0234] Figure 7C shows another view of the microfluidic device 700. The compressive force is removed or reduced, causing the first sheet 710 and the second sheet 720 to return to their initial positions. The compressive force applied in Figure 7B and removed or reduced in Figure 7C may move the droplet 740 away from the magnet 750. The movement of the droplet may sort ferrous material from the droplet 740, as the magnet 750 may attract and / or restrict the movement of the ferrous beads 742.

[0235] 8 is a flow chart illustrating example operations 800 for removing suspended iron particles from a microfluidic droplet. Although operations 800 are described with respect to microfluidic device 700 of FIGS. 7A-7C, operations 800 may be performed by any other suitable system or device.

[0236] Operations 800 begin at block 802, where a microfluidic droplet with suspended iron particles is moved to a region proximate a magnet by mechanical manipulation. For example, microfluidic droplet 740 may be moved near magnet 750. In some examples, droplet 740 may be moved through application of a force to one or more sheets as described herein.

[0237] Next, the magnet is enabled at block 804. In some cases, this step is optional, as indicated by the dashed lines in FIG. 8. In some examples, the magnet 750 may be a permanent and stationary magnet. In other examples, the magnet 750 may be enabled by moving the magnet 750 toward the microfluidic droplet 740, or the magnet 750 may be an electromagnet and receive power. The magnet 750 may cause the iron particles to come out of suspension and assemble toward the magnet 750. In some examples, the iron particles 741 may assemble into one or more iron beads 742.

[0238] Next, in block 806, microfluidic droplet 740 may be moved away from magnet 750 through mechanical actuation. For example, a compressive force may be applied to first sheet 710 or second sheet 720 to move microfluidic droplet 740 away from magnet 750. Moving microfluidic droplet 740 away from the magnet may sort ferrous materials from microfluidic droplet 740.

[0239] In some instances, the ferrous material may be returned to suspension within the droplet through mechanical actuation, one such example being described below in conjunction with Figures 9-10.

[0240] 9A shows a portion of another microfluidic device 900. The microfluidic device 900 may include a first sheet 910, a second sheet 920, and a gap 930. The first sheet 910, the second sheet 920, and the gap 930 may be examples of the first sheet 110, the second sheet 120, and the gap 130 of FIG. 1A.

[0241] Droplets 940 may include undispersed ferrous or non-ferrous particles. A compressive force may be applied to first sheet 910 and / or second sheet 920 (not shown) that may compress or deform droplets 940. In some cases, the compressive force may be applied to the center or middle of droplets 940.

[0242] FIG. 9B shows another view of the microfluidic device 900. The compressive force may be removed or reduced from the first sheet 910 and / or the second sheet 920. Removal or release of the compressive force may allow the droplet 940 to return to an uncompressed or undeformed state. The transition from a compressed state to an uncompressed state (or vice versa) may suspend one or more ferrous or non-ferrous particles 941 within the droplet 940. In some cases, the compressive force may be applied and / or removed rapidly or abruptly. The sudden application and / or removal of the compressive force may help to disperse the ferrous and non-ferrous particles 941 throughout the droplet 940. In some cases, the compressive force may be repeatedly applied and removed to more evenly distribute the particles.

[0243] 10 is a flow chart illustrating example operations 1000 for dispersing particles in microfluidic droplets. Although operations 1000 are described below with respect to microfluidic device 900 of FIGS. 9A-9B, operations 1000 may be performed by any other suitable system or device.

[0244] The operations 1000 begin at block 1002 where a compressive force is applied by mechanical actuation to a microfluidic droplet containing suspended ferrous and / or non-ferrous particles 941. The compressive force may be provided by a mechanical actuation force that may be applied to the first sheet 910 and / or the second sheet 920, and also to the droplet 940. The compressive force may deform or stretch the droplet 940.

[0245] Next, in block 1004, the compressive force may be released or reduced to suspend the ferrous and / or non-ferrous particles 941 within the droplet 940. Removal or reduction of the compressive force may cause the microfluidic droplet 940 to return to a spherical or quasi-spherical shape, which at least partially suspends the ferrous or non-ferrous particles in the droplet 940.

[0246] In some cases, additional agitation of the microfluidic droplets may be desirable to enhance the distribution of particles in the microfluidic droplets 940. To provide the additional agitation, the compressive force may be repeatedly applied and removed (or reduced) for a predetermined number of cycles. Thus, in block 1006, the number of completed compression cycles is determined. A completed compression cycle may include the application and removal or reduction of the compressive force. If the number of compression cycles is less than the predetermined number, the operation 1000 may return to block 1002. On the other hand, if the number of compression cycles is greater than or equal to the predetermined number, the operation 1000 may end.

[0247] In some instances, it may be desirable to heat the droplets as part of an analysis or assay of the droplets. However, the droplets may move during the heating operation and may not remain centered or positioned on the heating element. In some cases, pinning posts may be used to control the position of the droplets.

[0248] 11A illustrates a portion of another microfluidic device 1100. As shown in FIG. 11A, the microfluidic device 1100 may include a first sheet 1110, a second sheet 1120, a gap 1130, and a heater 1140. The first sheet 1110, the second sheet 1120, and the gap 1130 may be examples of the first sheet 110, the second sheet 120, and the gap 130 of FIG. 1A. The first sheet 1110 may include one or more pinning posts 1150 attached to a first surface 1111 of the first sheet 1110. In some examples, the pinning posts 1150 may be attached to the second sheet 1120.

[0249] The second sheet 1120 may include a first surface 1121 and a second surface 1122. The first surface 1121 may be disposed toward (e.g., proximate to) the gap 1130. As shown, a heater 1140 may be disposed on the second surface 1122 of the second sheet 1120 opposite the pinning posts 1150. The pinning posts 1150 may provide features on the first surface 1111 to which the droplet 1160 may temporarily bind, thereby limiting the movement of the droplet 1160. The droplet 1160 may be initially disposed away from the heater 1140 and the pinning posts 1150.

[0250] 11B shows another view of the microfluidic device 1100. In FIG. 11B, a compressive force may be provided to the first sheet 1110 and / or the second sheet 1120 to reduce the gap 1130 and move the droplet 1160 towards the heater 1140 and pinning post 1150.

[0251] 11C shows another view of the microfluidic device 1100. In FIG. 11C, the droplet 1160 is positioned in contact with the pinning posts 1150 and the compressive force is removed. Thus, the first sheet 1110 and the second sheet 1120 may return to their initial positions and the droplet 1160 is positioned on the heater 1140. Because the pinning posts 1150 engage the droplet 1160, movement of the droplet 1160 may be reduced. Reduced movement may be particularly advantageous when the droplet 1160 is undergoing a procedure such as heating by the heater 1140.

[0252] 12 is a flow chart illustrating example operations 1200 for manipulating microfluidic droplets in conjunction with pinning posts. Although operations 1200 are described below with respect to microfluidic device 1100 of FIGS. 11A-11C, operations 1200 may be performed by any other suitable system or device.

[0253] The operations 1200 begin at block 1202 where a microfluidic droplet 1160 is moved by mechanical manipulation to a region of the microfluidic device 1100 that includes a pinning post 1150. The mechanical manipulation may include the use of a compressive force as described with respect to FIGS.

[0254] Next, in block 1204, the microfluidic droplet 1160 is heated in the region of the pinning posts 1150. In some examples, the heat is provided by heater 1140. Next, in block 1206, the microfluidic droplet 1160 may be moved away from the region of the microfluidic device that includes the pinning posts 1150 by mechanical manipulation.

[0255] In some examples, wells may be disposed within the microfluidic device to contain and process the microfluidic droplets, an example of which is described in conjunction with Figures 13A-13E and 14.

[0256] FIG. 13A shows a portion of a microfluidic device 1300. As shown in FIG. 13A, the microfluidic device 1300 may include a first sheet 1310, a second sheet 1320, and a gap 1330. The first sheet 1310, the second sheet 1320, and the gap 1330 may be examples of the first sheet 110, the second sheet 120, and the gap 130 of FIG. 1A. In addition, the second sheet 1320 may include an opening 1325 that connects the gap 1330 to a heater 1350. The heater 1350 may be formed in the shape of a well 1355. Thus, the well 1355 may be connected to the gap 1330 through the opening 1325. As shown, the droplet 1340 may be positioned away from the opening 1325.

[0257] For ease of use, the second sheet 1320 may be disposed below (e.g., closer to the ground) the first sheet 1310. Such a configuration may allow gravity to assist in receiving or moving the droplets 1340 into the wells 1355. In some other examples, the openings and wells may be disposed on the first sheet 1310. In such a configuration, surface tension and / or capillary action may cause the droplets 1340 to remain in the wells even though the wells 1355 are inverted.

[0258] Figure 13B shows another view of the microfluidic device 1300. In Figure 13B, a mechanical actuation moves a droplet 1340 towards an opening 1325 in the second sheet 1320. For example, a compressive force may be applied to the first sheet 1310 and / or the second sheet 1320 to reduce the gap 1330 and move the droplet 1340.

[0259] 13C shows another view of the microfluidic device 1300. In FIG. 13C, a droplet 1340 is in a well 1355 of a heater 1350. The compressive force may be removed or reduced, which allows the first sheet 1310 and the second sheet 1320 to return to their initial positions. The well 1355 may advantageously restrict movement of the droplet 1340, particularly during heating by the heater 1350.

[0260] Another view of the microfluidic device 1300 is shown in Figure 13D. In Figure 13D, a droplet 1340 has been pulled from a well 1355 of a heater 1350 by mechanical manipulation. For example, a compressive force may be applied to the first sheet 1310 and / or the second sheet 1320 to reduce the gap 1330 and contact the droplet 1340. In some examples, the compressive force may be applied to a region of the microfluidic device 1300 associated with an orientation to receive the droplet 1340.

[0261] 13E shows the compressive force being removed or reduced from the microfluidic device 1300. The first sheet 1310 and the second sheet 1320 may return to their initial positions, and the droplet 1340 may be positioned away from the well 1355 and the heater 1350.

[0262] 14 is a flow chart illustrating example operations 1400 for manipulating microfluidic droplets in conjunction with wells. Operations 1400 are described below with respect to microfluidic device 1300 of FIGS. 13A-13E, although operations 1400 may be performed by any other suitable system or device.

[0263] The operation begins at block 1402 where a microfluidic droplet is moved into a well by mechanical actuation. For example, a compressive force may be applied to first sheet 1310 or second sheet 1320 to place microfluidic droplet 1340 into well 1355.

[0264] Microfluidic droplet 1340 may then be heated within the well in block 1404. For example, heater 1350 may heat microfluidic droplet 1340 within well 1355. Microfluidic droplet 1340 is then moved away from the well by mechanical manipulation in block 1406. For example, a compressive force may be applied to first sheet 1310 and / or second sheet 1320 to reduce gap 1330 and force microfluidic droplet 1340 out of well 1355.

[0265] FIG. 15A shows a portion of a microfluidic device 1500. As shown in FIG. 15A, the microfluidic device 1500 may include a first sheet 1510, a second sheet 1520, a gap 1530, and an electrode 1550. The first sheet 1510, the second sheet 1520, and the gap 1530 may be examples of the first sheet 110, the second sheet 120, and the gap 130 of FIG. 1A. The electrode 1550 may be coupled to an electrical circuit or the like (not shown) that provides a high-energy electric field associated with causing electroporation (the creation of temporary pores or openings in a cell membrane). As shown, a droplet 1540 may be positioned away from the electrode 1550.

[0266] 15B shows another view of the microfluidic device 1500. In FIG. 15B, a compressive force may be applied to the first sheet 1510 and / or the second sheet 1520. The compressive force (e.g., mechanical actuation) may reduce the gap 1530, causing the droplet 1540 to move towards the electrode 1550.

[0267] Another view of the microfluidic device 1500 is shown in Figure 15C. In Figure 15C, the compressive force is removed or reduced, thereby allowing the first sheet 1510 and / or the second sheet 1520 to return to their initial positions. The mechanical actuation of Figure 15B positions the droplet 1540 on the electrode 1550. The electrode 1550 may be used to electroporate the droplet 1540.

[0268] 16 is a flow chart illustrating example operations 1600 for providing electroporation. Although operations 1600 are described with respect to microfluidic device 1500 of FIGS. 15A-15C, operations 1600 may be performed by any other suitable system or device.

[0269] Operations 1600 begin at block 1602, where a microfluidic droplet is moved onto an electroporation electrode by mechanical actuation. For example, a compressive force may be applied to first sheet 1510 and / or second sheet 1520 to move microfluidic droplet 1540 onto electrode 1550.

[0270] Next, in block 1604, electrodes 1550 provide an electric field to the microfluidic droplet 1540. The electric field may be a high power electric field provided by one or more circuits and devices. The electric field may temporarily provide openings or "pores" in the cell walls of the cellular material within the microfluidic droplet 1540.

[0271] The microfluidic droplet is then moved away from the electrode by mechanical manipulation in block 1606. For example, a compressive force may be used in conjunction with first sheet 1510 and second sheet 1520 to move the microfluidic droplet away from electrode 1550.

[0272] 17 illustrates an exemplary microfluidic system 1700. The microfluidic system 1700 may include a cartridge 1710, a pressure actuator 1720, a magnet 1760, a heater 1770, and a controller 1780. In some examples, the pressure actuator 1720, the magnet 1760, the heater 1770, and the controller 1780 may be included in a housing or base station that may be coupled to the cartridge 1710.

[0273] The cartridge 1710 may be an example of the microfluidic devices 100, 300, 500, 700, 900, 1100, 1300, and 1500 of FIGS. 1, 3, 5, 7, 9, 11, 13, and 15, respectively. The cartridge 1710 may include an input port 1711, a first sheet 1712, a second sheet 1714, an optical sensor 1718, one or more pinning posts 1730, one or more electrodes 1740, and a first heater 1750. The first sheet 1712 and the second sheet 1714 may be hydrophobic and oleophobic sheets or may include hydrophobic and oleophobic layers on one or more surfaces. The first heater 1750 may be coupled to the gap 1716 through an opening 1715 in the second sheet 1714. The first heater 1750 may form a well 1755. One or more droplets may be inserted into the cartridge 1710 through an input port 1711. Although only one input port 1711 is shown in the figure, in other examples, the cartridge 1710 may include any workable number of input ports.

[0274] The pressure actuator 1720 may be in contact with or otherwise coupled to the cartridge 1710. As shown, the pressure actuator 1720 may be coupled to the first sheet 1712. In other examples, the pressure actuator 1720 may be coupled to the second sheet 1714. The pressure actuator 1720 may also be coupled to a controller 1780. The controller 1780 may cause the pressure actuator 1720 to selectively apply one or more compressive forces to the first sheet 1712 and / or the second sheet 1714. The compressive forces may manipulate the position of any droplets in the gap 1716. An optical sensor 1718 may detect the presence and / or location of droplets (e.g., microfluidic droplets) in the gap 1716. The optical sensor 1718 may be coupled to the controller 1780. In this manner, data from the optical sensor may be used to guide or direct the pressure actuator 1720.

[0275] A magnet 1760 may be disposed adjacent to or on the cartridge 1710. The operation of the magnet 1760 may be controlled by a controller 1780. Similarly, a second heater 1770 may be disposed adjacent to or on the cartridge 1710 and may also be controlled by the controller 1780.

[0276] A controller 1780 may control the operation of the microfluidic system 1700. Thus, the controller 1780 may control the operation of the pressure actuator 1720, the magnet 1760, the first heater 1750 and the second heater 1770, and the one or more electrodes 1740 to perform one or more operations described herein.

[0277] FIG. 18A illustrates a portion of another microfluidic device 1800. The microfluidic device 1800 may include a first sheet 1810, a second sheet 1820, and a stylus 1850. The first sheet 1810 may be separated from the second sheet 1820 by a gap 1830. The first sheet 1810, the second sheet 1820, and the gap 1830 may be other examples of the first sheet 110, the second sheet 120, and the gap 130 of FIG. 1A. The stylus 1850 may selectively provide a compressive force to either the first sheet 1810 or the second sheet 1820 (as shown, the stylus 1850 is selectively providing a compressive force to the first sheet 1810). The compressive force from the stylus 1850 may selectively reduce the gap 1830 in some areas of the microfluidic device 1800 to move the droplet 1840 as described above in conjunction with FIGS. 1-17. For example, the position and compression force of the stylus 1850 may be controlled by the controller 1780 of FIG.

[0278] In some examples, the area of ​​the stylus tip may be selected to correspond to the expected size of the droplet 1840. The droplet 1840 may be any viable droplet, including any viable microfluidic droplet. Thus, the droplet 1840 may be 10-6 ~10 -15 liters. Additionally, in some examples, the tip of the stylus may be shaped or treated to avoid scratching and / or abrading the surface of the first sheet 1810 or the second sheet 1820. For example, the tip of the stylus 1850 may include a roller to apply a compressive force to the first sheet 1810. In some other examples, the tip of the stylus 1850 may be coated with a lubricant.

[0279] 18B illustrates a stylus 1850 and a possible associated end profile 1860. The end profile 1860 is intended to be exemplary, not limiting (e.g., the end profile 1860 is not an exhaustive enumeration of all possible end profiles). Thus, other end profiles for the stylus 1850 are possible. Some end profiles 1860 may be able to move or manipulate the droplet 1840 more effectively. For example, a circular, rectangular, or elliptical profile may be able to move the droplet 1840 more effectively.

[0280] 19A shows a portion of another microfluidic device 1900. The microfluidic device 1900 may include a first sheet 1910, a second sheet 1920, a gap 1930, and a stylus 1950; these may be examples of the first sheet 1810, the second sheet 1820, the gap 1830, and the stylus 1850 of FIG. 18A. The stylus 1950 may include an insulated and / or exposed electrode 1955. In some examples, the electrode 1955 may be disposed toward a tip of the stylus 1950, which may contact at least one of the first sheet 1910 or the second sheet 1920.

[0281] In some examples, a voltage (e.g., potential) may be provided to the electrode 1955 that attracts the droplet 1940. When the stylus 1950 is placed in contact with at least one of the sheets of the microfluidic device 1900 (here illustrated as first sheet 1910), the associated sheet may act as or be a hydrophobic and oleophobic dielectric that separates the electrode 1955 from the droplet 1940.

[0282] The applied or provided voltage may be sufficient to affect or control the surface tension of the droplet 1940. In this manner, the stylus 1950 may attract and / or move the droplet 1940 within the gap 1930 without the application of a compressive force, but instead by providing a voltage to the electrode 1955 and then moving the position of the stylus 1950 relative to the first sheet 1910 and the second sheet 1920.

[0283] Figure 19B shows another view of microfluidic device 1900. As shown, stylus 1950 may be moved planarly relative to first sheet 1910 and second sheet 1920. When stylus 1950 is moved planarly while electrode 1955 is energized with a sufficient voltage, droplet 1940 may move to follow stylus 1950. Thus, any of the droplet operations described with respect to Figures 1-17 may be performed by energizing electrode 1955 and moving stylus 1950 instead of using a compressive force.

[0284] FIG. 20 is a flow chart illustrating example operations 2000 for manipulating a microfluidic droplet. The operations 2000 are described with respect to the microfluidic device 1900 of FIGS. 19A and 19B, however, the operations 2000 may be performed by any other suitable system or device. The operations 2000 begin at block 2002, where a potential is provided to an electrode disposed on or coupled to a stylus. For example, a voltage may be provided to an electrode 1955 disposed on or near the tip of the stylus 1950. In addition, the stylus 1950 may be in contact with at least one sheet (e.g., the first sheet 1910 or the second sheet 1920) of the microfluidic device 1900. The voltage applied or provided may be sufficient to affect or control the surface tension of the droplet 1940.

[0285] Next, the stylus is moved in block 2004. For example, stylus 1950 may be moved relative to first sheet 1910 and second sheet 1920. A sufficient voltage has been applied or provided (in block 2002) to electrode 1955 so that droplet 1940 may move in response to movement of stylus 1950.

[0286] The voltage or potential is then removed from the electrode in block 2006. For example, stylus 1950 may be moved to position droplet 1940 within a predetermined processing zone in block 2004. Now that the movement is complete, the voltage or potential may be removed from electrode 1955.

[0287] Figure 21A shows a portion of another microfluidic device 2100. The microfluidic device 2100 may include a first sheet 2110, a second sheet 2120, a gap 2130, a stylus 2150, and an electrode 2155; these may be examples of the first sheet 1910, the second sheet 1920, the gap 1930, the stylus 1950, and the electrode 1955 of Figure 19A.

[0288] In contrast to microfluidic device 1900, microfluidic device 2100 may use a combination of compressive force and applied voltage to manipulate droplet 2140. As shown, stylus 2150 may be positioned on one side of droplet 2140. To move droplet 2140, stylus 2150 may provide a compressive force to reduce gap 2130 while a voltage is provided to electrode 2155. In this manner, droplet 2140 may be moved by a combination of compressive force and electromotive force (e.g., voltage). For example, stylus 2150 may be moved to change the position or location of droplet 2140 within gap 2130 while stylus 2150 provides a compressive force and a voltage is applied to electrode 2155. The compressive force may be provided to either first sheet 2110 or second sheet 2120. FIG. 21A shows an example where stylus 2150 provides a compressive force to first sheet 2110.

[0289] Figure 21B shows another view of the microfluidic device 2100. In this view, the stylus 2150 has been moved to a new position relative to the first sheet 2110 and the second sheet 2120. The droplet 2140 has moved in response to the compression previously provided by the stylus 2150 and the voltage provided to the stylus 2150. Thus, in Figure 21B, the compression force is removed from the first sheet 2110 and the voltage is removed from the electrode 2155.

[0290] FIG. 22 is a flow chart illustrating example operations 2200 for manipulating a microfluidic droplet. The operations 2200 are described with respect to the microfluidic device 2100 of FIGS. 21A and 21B, however, the operations 2200 may be performed by any other suitable system or device. The operations may begin at block 2202, where a compressive force is applied to at least one of the sheets of the microfluidic device by mechanical actuation. For example, a stylus 2150 may provide a compressive force to the first sheet 2110 to reduce a gap 2130 near the droplet 2140. The reduced gap may at least partially manipulate the droplet 2140 between the first sheet 2110 and the second sheet 2120.

[0291] Next, in block 2204, an electrical potential is provided to an electrode disposed on or coupled to the stylus. For example, a voltage may be provided to an electrode 2155 disposed on or near the tip of the stylus 2150. The stylus 2150 may be in contact with at least one sheet of the microfluidic device 2100. The voltage applied or provided may be sufficient to affect or control the surface tension of the droplet 2140.

[0292] The stylus is then moved in block 2206. For example, the stylus 2150 may be moved relative to the first sheet 2110 and the second sheet 2120. In this manner, the droplet 2140 may be manipulated or moved using a combination of a compressive force provided by the stylus 2150 and a voltage applied to the electrode 2155.

[0293] The potential is then removed from the electrodes in block 2208. For example, the voltage may be removed from electrode 2155. The compressive force is then removed from at least one sheet of the microfluidic device in block 2210. For example, stylus 2150 may be moved away from first sheet 2110 or second sheet 2120. Now that droplet 2140 has been moved into a predetermined (e.g., desired) position in block 2206, the compressive force and voltage may be removed from stylus 2150.

[0294] In some examples, iron particles may be suspended within the microfluidic droplets to aid in processing or assays, and after one or more processing steps are completed, the iron particles may be removed from the droplets for further processing.

[0295] FIG. 23A shows a portion of another microfluidic device 2300. The microfluidic device 2300 may include a first sheet 2310, a second sheet 2320, a gap 2330, and a stylus 2350; these may be examples of the first sheet 1910, the second sheet 1920, the gap 1930, and the stylus 1950 of FIG. 19A. In addition, the stylus 2350 may include a magnet 2356. The magnet 2356 may be controllable. For example, the magnet 2356 may be an electromagnet that can be enabled and disabled through a control voltage. In another example, the magnet 2356 may be movable. Thus, the magnet 2356 may be moved toward the tip of the stylus 2350 (as shown) or away from the tip of the stylus 2350 (not shown). In this manner, the magnetic field strength at or near the tip of the stylus 2350 may be controlled and / or variable. A plurality of iron particles 2341 may be distributed (suspended) throughout the droplet 2340 .

[0296] 23B shows another view of microfluidic device 2300, in which magnet 2356 has been activated or enabled. For example, magnet 2356 may be an electromagnet that can be enabled through the application of power. In another example, magnet 2356 may be a permanent magnet that can be moved towards droplet 2340. Magnet 2356 may attract or collect iron particles 2341 into one or more iron beads 2342. Thus, iron particles 2341 may be released from suspension in droplet 2340.

[0297] 23C shows another view of the microfluidic device 2300. The stylus 2350 is moved while the magnet 2356 is activated or enabled. The movement of the stylus 2350 may sort or remove the iron bead 2342 from the droplet 2340.

[0298] 24 is a flow chart illustrating example operations 2400 for removing suspended iron particles from a microfluidic droplet. Although operations 2400 are described with respect to microfluidic device 2300 of FIGS. 23A-23C, operations 2400 may be performed by any other suitable system or device.

[0299] The operations 2400 begin at block 2402 where a stylus with a magnet is moved to a region proximate a droplet with suspended ferrous particles. For example, a stylus 2350 with a magnet 2356 may be moved near a droplet 2340 containing suspended ferrous particles 2341.

[0300] Next, in block 2404, a magnet in or on the stylus is enabled. For example, magnet 2356 may be an electromagnet that can be enabled through the application of power. In another example, magnet 2356 may be moved toward the tip of stylus 2350. In this manner, magnet 2356 may cause ferrous particles 2341 to come out of suspension and aggregate toward magnet 2356. In some cases, the aggregated ferrous particles 2341 may form ferrous beads 2342.

[0301] The stylus may then be moved away from the droplet in block 2406. For example, movement of the stylus 2350 may move the iron beads 2342 out of the droplet 2340, thereby sorting the iron particles 2341 out of the droplet 2340.

[0302] In some instances, the ferrous material may be returned to suspension within the droplet through mechanical actuation, further examples of which are described below in conjunction with Figures 25-26.

[0303] FIG 25A shows a portion of another microfluidic device 2500. The microfluidic device 2500 may include a first sheet 2510, a second sheet 2520, a stylus 2550, and a magnet 2556. The first sheet 2510, the second sheet 2520, the stylus 2550, and the magnet 2556 may be examples of the first sheet 2310, the second sheet 2320, the gap 2330, the stylus 2350, and the magnet 2356 of FIG 23A. In some examples, the magnet 2556 may be deactivated by either moving the magnet 2556 away from the tip of the stylus 2550, or by removing power if the magnet 2556 is an electromagnet.

[0304] The droplets 2540 may include undispersed ferrous or non-ferrous particles. A compressive force may be applied to the first sheet 2510 and / or the second sheet 2520 (not shown) that may compress or deform the droplets 2540. In some cases, the compressive force may be applied by the stylus 2550 to the center or middle of the droplets 2540.

[0305] FIG. 25B shows another view of the microfluidic device 2500. The compressive force may be removed or reduced from the first sheet 2510 and / or the second sheet 2520. Removal or release of the compressive force may allow the droplet 2540 to return to an uncompressed or undeformed state. The transition from a compressed state to an uncompressed state (or vice versa) may suspend one or more ferrous or non-ferrous particles 2541 within the droplet 2540. In some cases, the compressive force may be applied and / or removed rapidly or abruptly. The sudden application and / or removal of the compressive force may help to disperse the ferrous and non-ferrous particles 2541 throughout the droplet 2540. In some cases, the compressive force may be repeatedly applied and removed to distribute the particles more evenly.

[0306] 26 is a flow chart illustrating example operations 2600 for dispersing particles in microfluidic droplets. Although operations 2600 are described below with respect to microfluidic device 2500 of FIGS. 25A and 25B, operations 2600 may be performed by any other suitable system or device.

[0307] The operations 2600 begin at block 2602 where the magnet is disabled. The magnet 2556 may be disabled, for example, by either moving the magnet 2556 away from the tip of the stylus 2550 or by removing power from the electromagnet that comprises the magnet 2556.

[0308] Next, in block 2604, a compressive force is applied to the microfluidic droplet containing the suspended ferrous and / or non-ferrous particles by mechanical actuation. For example, the compressive force may be provided by a mechanical actuation force that may be applied by a stylus 2550 to the first sheet 2510 and / or the second sheet 2520, and also to the droplet 2540. The compressive force may deform or stretch the droplet 2540.

[0309] The compressive force may then be released or reduced to suspend the ferrous and / or non-ferrous particles in the droplet at block 2606. For example, removal or reduction of the compressive force may cause the microfluidic droplet 2540 to return to a spherical or quasi-spherical shape, which at least partially suspends the ferrous or non-ferrous particles within the droplet 2540.

[0310] In some cases, additional agitation of the microfluidic droplet may be desirable to enhance the distribution of particles in the microfluidic droplet 2540. To provide the additional agitation, the compressive force may be repeatedly applied and removed (or reduced) for a predetermined number of cycles. Thus, at block 2608, the number of completed compression cycles is determined. A completed compression cycle may include the application and removal or reduction of the compressive force. If the number of compression cycles is less than the predetermined number, the operation 2600 may return to block 2604. On the other hand, if the number of compression cycles is greater than or equal to the predetermined number, the operation 2600 may end.

[0311] Next, in block 2604, a compressive force is applied to the microfluidic droplet containing the suspended ferrous and / or non-ferrous particles 2541 by mechanical actuation. The compressive force may be provided by a mechanical actuation force that may be applied to the first sheet 2510 and / or the second sheet 2520 through a stylus 2550. The compressive force may deform or stretch the droplet 2540.

[0312] Next, in block 2604, the compressive force may be released or reduced to suspend the ferrous and / or non-ferrous particles 2541 in the droplet 2540. Removal or reduction of the compressive force may cause the droplet 2540 to return to a spherical or quasi-spherical shape, which at least partially suspends the ferrous or non-ferrous particles within the droplet 2540.

[0313] In some examples, the stylus may include or be coupled to a reservoir or other liquid container that can be used to hold liquid aspirated from the gap of the microfluidic device. Exemplary devices are described below in conjunction with Figures 27-38.

[0314] 27A shows a portion of another microfluidic device 2700. The microfluidic device 2700 may include a first sheet 2710, a second sheet 2720, and a gap 2730. The first sheet 2710, the second sheet 2720, and the gap 2730 may be examples of the first sheet 1910, the second sheet 1920, and the gap 1930 of FIG. 19A. The first sheet 2710 may include a slit septum or other configurable opening (not shown) that may allow the droplet 2740 to be aspirated (removed) from the gap 2730.

[0315] FIG. 27B shows another view of the microfluidic device 2700. A septum 2711 may be included on one of the sheets of the microfluidic device 2700. As shown, the septum 2711 is located on (included in) the first sheet 2710. In some examples, the septum 2711 may remain closed under most operating conditions. For example, a compressive force may be applied to the first sheet 2710 near the septum 2711. However, the septum 2711 may remain substantially closed under the application of the compressive force. In other words, the septum 2711 may have a closing force to prevent liquid from "leaking" through the first sheet 2710. A droplet 2740 may be moved under the septum 2711.

[0316] 27C shows another view of the microfluidic device 2700. A stylus 2750 may be positioned substantially over the septum 2711. The stylus 2750 may include a lumen 2751 that may be coupled to the septum 2711 and configured to receive the droplet 2740. In some examples, negative pressure may be provided to and / or through the stylus 2750 to draw the droplet 2740 through the septum 2711 and into the stylus 2750. The stylus 2750 may then move the droplet 2740 to another location within the microfluidic device 2700.

[0317] 28 is a flow chart illustrating example operations 2800 for aspirating liquid in a microfluidic device. Although operations 2800 are described below with respect to microfluidic device 2700 of FIGS. 27A-27C, operations 2800 may be performed by any other suitable system or device.

[0318] Operations 2800 begin at block 2802 where a droplet is disposed proximate to a septum. For example, any executable operation described herein may be performed to move droplet 2740 substantially below septum 2711.

[0319] Next, the stylus is moved over the septum at block 2804. For example, stylus 2750 may be moved so that it is over septum 2711. Stylus 2750 may include a lumen 2751 that may be coupled to septum 2711.

[0320] The droplet is then aspirated into the stylus at block 2806. For example, negative pressure may be applied by or through the stylus 2750. The negative pressure may draw the droplet 2740 into the lumen 2751.

[0321] In some instances, liquid may be provided to or returned from the microfluidic device through a septum, some examples of which are described below in conjunction with Figures 29 and 30.

[0322] 29A illustrates a portion of another microfluidic device 2900. The microfluidic device 2900 may include a first sheet 2910, a second sheet 2920, a gap 2930, and a stylus 2950. The first sheet 2910, the second sheet 2920, the gap 2930, and the stylus 2950 may be examples of the first sheet 2710, the second sheet 2720, the gap 2730, and the stylus 2750 of FIG. 27A. Thus, the first sheet 2910 may include a septum 2911.

[0323] In some examples, liquid, which may be contained within the stylus 2950, ​​may be injected through the septum 2911 into the gap 2930. The stylus 2950 may be positioned over the septum 2911 prior to the injection of the liquid. In some examples, a lumen 2951 of the stylus 2950 may be coupled to the septum 2911. A positive pressure may be applied to the lumen 2951 to force the liquid out of the stylus 2950 and form a droplet 2940 in the gap 2930. Thus, the applied pressure may overcome the closing force of the septum 2911.

[0324] 29B shows another view of the microfluidic device 2900. The stylus 2950 may be moved away from the droplet 2940. Additionally, a compressive force may be provided by the stylus 2950 to the first sheet 2910 (and / or the second sheet 2920) to reduce the gap 2930. The reduced gap 2930 may cause the droplet 2940 to move relative to the first sheet 2910 and the second sheet 2920.

[0325] 29C shows another view of the microfluidic device 2900. A stylus 2950 may be moved to move the droplet 2940. Once movement of the droplet 2940 is complete, the stylus 2950 may then be positioned to remove the source of compression on the first sheet 2910 and / or the second sheet 2920. In this manner, the stylus 2950 may operate as a pipette to move liquid (e.g., the droplet 2940) within the microfluidic device 2900.

[0326] Figure 30 is a flow chart illustrating example operations 3000 for manipulating liquids in a microfluidic device. The operations 3000 are described below with respect to the microfluidic device 2900 of Figures 29A-29C, although the operations 3000 may be performed by any other suitable system or device.

[0327] The operations 3000 begin at block 3002, where a droplet is dispensed from a stylus of a microfluidic device, through a septum and into the gap. For example, stylus 2950 may be positioned over septum 2911. Positive pressure may be applied to stylus 2950 and / or lumen 2951 to inject liquid through septum 2911 and into gap 2930.

[0328] Next, in block 3004, a distance between the two sheets is selectively reduced by mechanical actuation near the microfluidic droplet. For example, the reduced distance may result in a reduced gap 2930 between the first sheet 2910 and the second sheet 2920. In some examples, a compressive force may be provided by a stylus 2950 proximate (e.g., next to) one side of the droplet 2940. The compressive force may reduce the gap 2930, which moves the droplet 2940 toward the compressive force. In some variations, the compressive force may deform one end of the droplet 2940.

[0329] The distance between the two sheets is then restored in block 3006. For example, the compressive force applied in block 3004 may be removed, allowing the first sheet 2910 and / or the second sheet 2920 to return to their initial separation distance. In some examples, the distance between the first sheet 2910 and the second sheet 2920 returns to a distance similar to the initial separation distance associated with block 3602.

[0330] In some examples, the stylus may include additional devices or systems that can be used to handle droplets within the stylus, some examples of which are described below in conjunction with Figures 31-38.

[0331] FIG. 31A shows a portion of another microfluidic device 3100. The microfluidic device 3100 may include a first sheet 3110, a second sheet 3120, a gap 3130, and a stylus 3150. The first sheet 3110, the second sheet 3120, and the gap 3130 may be examples of the first sheet 1810, the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3150 may include a temperature control element 3155. Through the temperature control element 3155, any liquid aspirated from the gap 3130 may be heated within the stylus 3150 as part of a chemical assay or other protocol or process. For example, a droplet 3140 may be disposed below a septum 3111, which may be included within the first sheet 3110. The stylus 3150 may be disposed on the septum 3111. In some examples, the temperature control element 3155 may provide heating or cooling to a surrounding area based on a control voltage or signal, etc.

[0332] FIG. 31B shows another view of the microfluidic device 3100. The droplet 3140 may be aspirated from the gap 3130 through the septum 3111 and into the stylus 3150. In this manner, the droplet 3140 may be subjected to a temperature treatment provided by the temperature control element 3155 while the droplet 3140 is in the stylus 3150. In some examples, a temperature sensor (not shown) may be included in the stylus 3150 and / or the temperature control element 3155. A controller (not shown) may monitor or control the temperature of the droplet 3140 as part of the temperature treatment. For example, the controller may monitor the temperature of the droplet 3140 and control the temperature provided by the temperature control element 3155.

[0333] 31C shows another view of the microfluidic device 3100. After temperature treatment, a droplet 3140 may be injected into the gap 3130 through the septum 3111 in the first sheet 3110.

[0334] Figure 32 is a flow chart illustrating example operations 3200 for thermally treating a liquid in a microfluidic device. The operations 3200 are described below with respect to the microfluidic device 3100 of Figures 31A-31C, although the operations 3200 may be performed by any other suitable system or device.

[0335] The operations begin at block 3202 where a stylus is positioned over a septum and a droplet. For example, stylus 3150 may be positioned over septum 3111, which is on droplet 3140.

[0336] The droplet is then aspirated through the septum and into the stylus at block 3204. For example, negative pressure may be provided to the stylus 3150. In response, the droplet 3140 may be drawn through the septum 3111 and into the stylus 3150.

[0337] The temperature of the droplet is then controlled in block 3206. For example, a temperature control element 3155 may be used to monitor and control the temperature of the droplet 3140 contained within the stylus 3150.

[0338] Next, in block 3208, a droplet is injected through the septum into the gap. For example, a temperature treatment may be completed. A positive pressure may be provided to the stylus 3150, which causes the droplet 3140 to be injected through the septum 3111 and into the gap 3130. In some examples, the stylus 3150 may be repositioned relative to the first sheet 3110 and / or the second sheet 3120 before transferring the droplet 3140 back into the gap 3130.

[0339] FIG. 33 shows a portion of another microfluidic device 3300. The microfluidic device 3300 may include a first sheet 3310, a second sheet 3320, a gap 3330, and a stylus 3350. The first sheet 3310, the second sheet 3320, and the gap 3330 may be examples of the first sheet 1810, the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3350 may include a temperature control element 3355. The temperature control element 3355 may be disposed toward a portion of the stylus 3350 that may contact the first sheet 3310. The stylus 3350 and the temperature control element 3355 are positioned on the first sheet 3310 above the droplet 3340. In this manner, the temperature control element 3355 may provide heat for any temperature controlled treatment to the droplet 3340 through the first sheet 3310.

[0340] In some examples, the temperature control element 3355 may include a temperature sensor (not shown). In this manner, a controller (also not shown) may control the temperature of the droplet 3340 to be within a desired temperature.

[0341] FIG. 34A shows a portion of another microfluidic device 3400. The microfluidic device 3400 may include a first sheet 3410, a second sheet 3420, a gap 3430, and a stylus 3450. The first sheet 3310, the second sheet 3320, and the gap 3330 may be examples of the first sheet 1810, the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3450 may include a light sensor 3455 and a light source 3456. In some examples, a reaction that has occurred or is occurring in the droplet 3440 may be monitored by sensing light transmitted or reflected through the droplet 3440. In this manner, the droplet 3440 may be drawn into the stylus 3450, where the transmitted and / or reflected light can be detected and / or measured. The amount of light detected may be correlated to the progress or completion of the reaction.

[0342] In some examples, to measure the reaction, a stylus 3450 may be positioned over a septum 3411 contained within the first sheet 3410. Additionally, a droplet 3440 may be positioned below the septum 3411.

[0343] Another view of the microfluidic device 3400 is shown in FIG. 34B. As shown, a droplet 3440 may be aspirated through a septum 3411 into a stylus 3450. A light source 3456 may emit light into the droplet 3440. The light source 3456 may be any viable solid-state light source and / or an incandescent light source. The light may be reflected and / or transmitted through the droplet 3440. This light may be detected by a light sensor 3455. The light sensor 3455 may be any viable light sensor or detector, such as a photodiode. The amount of light detected may be correlated with the progress or completion of a reaction. In this manner, the detected light may be indicative of the progress or completion of a reaction.

[0344] 34C shows another view of the microfluidic device 3400. As shown, the droplet 3440 may be returned to the gap 3430. In some examples, a stylus 3450 may inject the droplet 3440 through a septum 3411 in the first sheet 3410. In this manner, after determining the optical transmission or refraction of the droplet 3440, the droplet 3440 may be returned to the gap 3430 for further processing.

[0345] Figure 35 is a flow chart illustrating example operations 3500 for performing a reaction measurement. Although operations 3500 are described below with respect to the microfluidic device 3400 of Figures 34A-34C, operations 3500 may be performed by any other suitable system or device.

[0346] The operations begin at block 3502 where a stylus is positioned over a septum and a droplet. For example, stylus 3450 may be positioned over septum 3411, which is on droplet 3440.

[0347] The droplet is then aspirated through the septum and into the stylus at block 3504. For example, negative pressure may be provided to the stylus 3450. In response, the droplet 3440 may be drawn through the septum 3411 and into the stylus 3450.

[0348] The droplet is then monitored for a reaction in block 3506. For example, a light source 3456 may be used to emit light into the stylus 3450 and into the droplet 3440. A light sensor 3455 may detect light transmitted and / or reflected from the droplet 3440. In some cases, the transmitted and / or reflected light may be correlated with the amount of progress of a reaction occurring in the droplet 3440.

[0349] Next, in block 3508, a droplet is injected through the septum into the gap. For example, monitoring of a reaction may be completed. A positive pressure may be provided to the stylus 3450, which causes the droplet 3440 to be injected through the septum 3411 and into the gap 3430. In some examples, the stylus 3450 may be repositioned relative to the first sheet 3410 and / or the second sheet 3420 before transferring the droplet 3440 back into the gap 3430.

[0350] 36 illustrates a portion of another microfluidic device 3600. The microfluidic device 3600 may include a first sheet 3610, a second sheet 3620, a gap 3630, and a stylus 3650. The first sheet 3610, the second sheet 3620, and the gap 3630 may be examples of the first sheet 1810, the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3650 may include a light source 3655. The light source 3655 may be disposed toward a portion of the stylus 3650 that may contact the first sheet 3610.

[0351] The photodetector 3621 may face the light source 3655. As shown, the photodetector 3621 may be disposed on the second sheet 3620. When the droplet 3640 is disposed between the light source 3655 and the photodetector 3621, the photodetector 3621 may detect transmitted light and / or reflected light. Thus, the light source 3655 and the photodetector 3621 may monitor the progress of the reaction in a similar manner as described with respect to FIGS. 34 and 35. In some examples, the positions of the light source 3655 and the photodetector 3621 may be reversed. In other words, the light sensor 3621 may be included in the stylus 3650, and the light source 3655 may be disposed on the second sheet 3620.

[0352] FIG. 37A illustrates a portion of another microfluidic device 3700. The microfluidic device 3700 may include a first sheet 3710, a second sheet 3720, a gap 3730, and a stylus 3750. The first sheet 3710, the second sheet 3720, and the gap 3730 may be examples of the first sheet 1810, the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3750 may include a sonication probe 3755. In some examples, the sonication probe 3755 may deliver sonic and / or ultrasonic stimuli to the droplet 3740. The sonication probe 3755 may be any viable piezoelectric device.

[0353] In some examples, a stylus 3750 may be positioned over a septum 3711 contained within the first sheet 3710 to deliver sonic or ultrasonic stimulation. Additionally, a droplet 3740 may be positioned below the septum 3711.

[0354] 37B shows another view of microfluidic device 3700. As shown, droplet 3740 may be aspirated through septum 3711 and into stylus 3750. Once droplet 3740 is within stylus 3750, sonication probe 3755 may be activated or enabled, which allows sonic and / or ultrasonic waves to be provided to droplet 3740. Delivery of the sonic stimulus may be controlled by a controller (not shown).

[0355] 37C shows another view of the microfluidic device 3700. As shown, the droplet 3740 may be returned to the gap 3730. In some examples, a stylus 3750 may inject the droplet 3740 through a septum 3711 in the first sheet 3710. In this manner, after delivering an acoustic stimulus to the droplet 3740, the droplet 3740 may be returned to the gap 3730 for further processing.

[0356] Figure 38 is a flow chart illustrating example operations 3800 for performing sonication. Operations 3800 are described below with respect to the microfluidic device 3700 of Figures 37A-37C, although operations 3800 may be performed by any other suitable system or device.

[0357] The operations begin at block 3802 where a stylus is positioned over a septum and a droplet. For example, stylus 3750 may be positioned over septum 3711, which is on droplet 3740.

[0358] The droplet is then aspirated through the septum and into the stylus at block 3804. For example, negative pressure may be provided to the stylus 3750. In response, the droplet 3740 may be drawn through the septum 3711 and into the stylus 3750.

[0359] Sonication may then be delivered to the droplets in block 3806. For example, sonication probe 3755 may subject droplets 3740 to sonication and / or ultrasonication.

[0360] Next, at block 3808, a droplet is injected through the septum into the gap. For example, sonication may be completed. A positive pressure may be provided to the stylus 3750, which causes the droplet 3740 to be injected through the septum 3711 and into the gap 3730. In some examples, the stylus 3750 may be repositioned relative to the first sheet 3710 and / or the second sheet 3720 before transferring the droplet 3740 back into the gap 3730.

[0361] 39 shows a block diagram of a device 3900, which may be an example of any of the microfluidic devices or systems described herein. Device 3900 may include a pressure actuator 3920, one or more magnets 3922, one or more heaters 3924, one or more electrodes 3926, an optical sensor 3927, a light source and sensor 3928, an acoustic wave device 3929, a processor 3930, and a memory 3940.

[0362] Any of the present devices (cartridge, mechanical microfluidic device configured to hold the cartridge, integrated device, etc.) may include a heat spreader connected to a thermal output (heating, cooling, etc.) to distribute energy in a controlled manner within the air gap. The heat spreader may be a thermally conductive material (e.g., copper, etc.) that may be configured as a pad or line in thermal communication with the thermal output. In any of the devices and methods described herein, the thermal output may be a heater or equivalently a cooling (e.g., Peltier) device. In any of the examples and variations described herein in which a heater or heating element is used, the heater or heating element may be a general purpose thermal output (e.g., cooling and / or heating). In some examples, both heating and cooling thermal outputs may be used. In any of these examples, only a heating thermal output may be used.

[0363] In some examples, a pressure actuator 3920 coupled to the processor 3930 may be used to provide forces, including compression and actuation forces, to one or more sheets of the microfluidic cartridge. In some examples, the pressure actuator 3920 may use mechanical, pneumatic, and / or electrical actuators to provide the compression and / or actuation forces. The compression and / or actuation forces may be provided through a controllable stylus. In some other examples, the pressure actuator 3920 may provide positive and / or negative pressure to the lumen of the stylus. In this manner, droplets may be aspirated from the gap of the microfluidic device and drawn into the stylus.

[0364] One or more magnets 3922, also coupled to the processor 3930, may be used to selectively provide a magnetic field that may be used for and during manipulation of the microfluidic droplets. In some examples, the magnets may be disposed proximate to the sides of the hydrophobic and oleophobic sheet. In other examples, the magnets may be disposed within a stylus.

[0365] One or more heaters 3924, also coupled to the processor 3930, may be used to provide heat to one or more microfluidic droplets. The heat may be used during analysis or assay of the microfluidic droplets. In some examples, the heaters (e.g., heating elements) may be disposed proximate to the sides of the hydrophobic and oleophobic sheet. In other examples, the heaters may be disposed within the stylus.

[0366] One or more electrodes 3926, also coupled to the processor 3930, may be used to provide the electric field used for electroporation. In some examples, the processor 3930 may include one or more electrical circuits or devices to generate a large electric field for the one or more electrodes 3926.

[0367] An optical sensor 3927, also coupled to a processor 3930, may detect the presence and / or position of any droplets, such as any microfluidic droplets disposed between two or more hydrophobic and oleophobic sheets.

[0368] A light source and sensor 3928, also coupled to the processor 3930, may provide light and detect transmitted or reflected light associated with the microfluidic droplets. In some examples, the light source and sensor 3928 may be included in the stylus. In other examples, the light source and sensor 3928 may be disposed on the side of the hydrophobic and oleophobic sheet and on the stylus.

[0369] A sonic device 3929, also coupled to the processor 3930, may provide sonication or ultrasonication to the microfluidic droplets. In some examples, the sonic device 3929 may be included in a stylus. In some examples, the sone device 3929 may be a piezoelectric device.

[0370] The processor 3930, which is also coupled to the memory 3940, may be one or more suitable processors capable of executing scripts or instructions of one or more software programs stored within the device 3900 (such as in the memory 3940).

[0371] The memory 3940 may include septum position data 3941. In some examples, the septum position data 3941 may be a database of the positions of one or more septum openings, which may be located on the hydrophobic and oleophobic sheet. Thus, the processor 3930 may use the septum position data 3941 to position the stylus over any particular septum.

[0372] The memory 3940 may also include a non-transitory computer-readable storage medium (e.g., one or more non-volatile storage elements, such as EPROM, EEPROM, flash memory, hard drive, etc.) that may store the following software modules: a pressure actuator control module 3942 for controlling the pressure actuator 3920; a magnet control module 3944 for controlling the one or more magnets 3922; a heater control module 3946 for controlling the one or more heaters 3924; an electrode control module 3948 for controlling the one or more electrodes 3926; an optical sensor control module 3949 for controlling the optical sensor 3927; an acoustic control module 3950 for controlling the acoustic device 3929; and a light source and sensing control module 3951.

[0373] Each software module includes program instructions that, when executed by the processor 3930, cause the device 3900 to perform a corresponding function. Thus, the non-transitory computer-readable storage medium of the memory 3940 may include instructions for performing all or a portion of the operations described herein.

[0374] The processor 3930 may execute the pressure actuator control module 3942 to manipulate one or more microfluidic droplets disposed between at least two hydrophobic and oleophobic sheets by applying a force through the pressure actuator 3920. For example, execution of the pressure actuator control module 3942 may cause a compressive force, a pinning force, and / or an actuation force to be applied to at least one of the hydrophobic and oleophobic sheets. The forces may be selectively applied to move, separate, combine, and / or mix one or more microfluidic droplets. In some examples, execution of the pressure control module 3942 may move a stylus across at least one of the hydrophobic and oleophobic sheets and apply a compressive force to move the droplets.

[0375] The processor 3930 may execute a magnet control module 3944 to selectively control, enable, and / or disable one or more magnets 3922. In some examples, execution of the magnet control module 3944 may cause power to be applied to an electromagnet contained within the magnet 3922. In some examples, execution of the magnet control module 3944 may cause one or more magnets 3922 to move closer to or away from one or more microfluidic droplets.

[0376] The processor 3930 may execute the electrode control module 3948 to selectively provide an electric field to the one or more electrodes. For example, execution of the electrode control module 3948 may provide one or more large electric fields to the electrodes 3926 to cause electroporation on a cell membrane within the microfluidic droplet.

[0377] The processor 3930 may execute the optical sensor control module 3949 to control and process data from the optical sensor 3927. For example, execution of the optical sensor control module 3949 may cause the optical sensor 3927 to receive or capture image data, together with causing the processor 3930 to process the image data and determine the presence and / or location of any droplets.

[0378] In some examples, the processor 3930 may use image data from the optical sensor 3927 to control the operation of the pressure actuator 3920. For example, the processor 3930 may process the image data in conjunction with the pressure actuator control module 3942 and thereby guide the application of a compressive force onto one or more hydrophobic and oleophobic sheets.

[0379] The processor 3930 may execute an acoustic control module 3950 to control the acoustic device 3929. For example, execution of the acoustic control module 3950 may activate or enable the acoustic device 3929, thereby allowing or enabling the acoustic device 3929 to provide sonic and / or ultrasonic stimulation or treatment to the droplets.

[0380] The processor 3930 may execute the light source and sensing control module 3951 to control the light source and sensor 3928. For example, execution of the light source and sensing control module 3951 may cause light to be emitted by the light source and the transmitted or reflected light to be sensed by the light detector. In this manner, a reaction or process may be detected according to the detected light.

[0381] cartridge As mentioned above, the cartridges described herein may generally include a first (e.g., upper) sheet that is elastically deformable, a second (e.g., lower) sheet, and a frame that separates the two to form an air gap. The first sheet may be an elastomeric material such as polyester (e.g., TPE), natural rubber, synthetic rubber, nitrile rubber, silicone rubber, urethane rubber, chloroprene rubber, ethylene vinyl acetate (EVA), etc. The second sheet may be the same material or a different material. The cartridge sheets are generally planar structures and may be membranes, layers, etc. The first and second sheets may be held under tension on or against the frame. In some examples, multiple frames may be used. The frame may be formed of any suitable material, such as rigid or semi-rigid polyester.

[0382] FIGS. 40A-40D illustrate an example of a cartridge 4006, 4006′ as described herein. The cartridge includes a frame 4017, a first sheet 4007, and a second sheet 4009. In the example shown in FIGS. 40A, 40C, and 40D, the cartridge is divided into three lanes 4005, 4005′, 4005″. In this example, the frame is configured as a divider with three separate lanes. The example cartridge shown in FIG. 40B has eight lanes 4015. Any suitable number of lanes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, or more) may be used. The cross-section of the cartridge shown in FIGS. 40C-40D illustrates an example with three lanes formed by the frame / divider 4017. The first sheet 4007 is held in tension on top of the frame, for example by welding and / or adhesive 4013. The second sheet 4009 in this example is also held in tension on the frame and is welded and / or glued thereto. An air gap 4011 is formed between the first and second sheets. In Fig. 40C and 40D, a portion of the cartridge is shown attached to a mounting portion of a mechanical microfluidic device having a recessed area and multiple vacuum ports for sealing the second (lower) sheet into the mounting area to make a tight thermal connection between the second sheet and the mounting area. As shown in Fig. 40D, sealing the second sheet 4009 to the base also extends the air gap 4011 to a greater height compared to the unattached configuration of the cartridge.

[0383] In the examples shown in Figures 40A-40E, the initial height of the air gap may be about 0.5 mm to about 5 mm. In general, the height may be 0.1 mm to about 7 mm, and may be adjusted to be smaller or larger as in Figures 40C and 40D. For example, compressing the first and / or second sheets to form an air gap height of about 3 mm to 0.5 mm has been found to be very effective for droplet mobility. In some examples, the neutral height of the air gap may be about 3 mm high (spacing between the first and second sheets); this height has been found to be effective in moving the droplet without significant damage to the films tested, as compared to larger gap spacings. At a height of about 3 mm, larger droplets (e.g., 140 uL aqueous droplet + 80 uL drop gloss) can touch the top film with less compression. However, the gap height may be adjusted based on the volume of the droplet and the materials forming the first and second sheets.

[0384] FIG. 41 shows an exploded view of one example of a cartridge 4100. In this example, the cartridge includes a first (e.g., upper) frame 4117 onto which a first sheet 4107 is mounted under tension so that it is pulled flat. All four sides of the sheet may be held under tension. The sheet may be pinned, clamped, welded, glued, tacked, or otherwise secured to the frame. In this example, the first sheet 4107 also includes a pair of openings 4133, 4133′ configured as input / output windows ("windows") into which a fluidic substance (droplet, drop gloss, etc.) can be applied by manual or automated means (e.g., pipetting, etc.). A fluidic substance may be added to the air gap through the window adjacent to an edge of the window such that a mechanical microfluidic device can use a force applicator adjacent to the window to pull the droplet further into the air gap to manipulate the droplet (or droplets) within the cartridge. The window 4133, 4133' may be any suitable size. In some examples, the entire distal and / or proximal end of the cartridge may be open as a window (e.g., the first sheet may extend only between two opposing sides of the frame (e.g., the first frame)). The edges of the window may be reinforced and / or smoothed. In some examples, the edges may be thickened (e.g., doubled). The window may be any size or any ratio of the surface size of the first sheet. For example, the window may be 50%-100% of the width of the surface of the first sheet and about 0.1%-10% of the length of the surface of the first sheet. In some examples, the window is 1 mm-10 cm long and 1 mm-5 cm wide. Larger windows may be used. As mentioned above, in some examples, the cartridge may also or alternatively include one or more smaller openings for applying / removing fluid (e.g., by pipetting).

[0385] The cartridge shown in FIG. 41 also includes a second frame 4121 to which the second sheet 4109 is attached. The second sheet may be under tension or may be more loosely attached, as it may be configured to be fixed to the base of a mechanical microfluidic actuator. The second sheet 4109 may be attached to the second frame 4121 in any suitable manner. In this example, the first and second frames may be disposable, and the first and second sheets may be, for example, TPE films, FEP films, etc. The first sheet may be adhesively attached to the first frame, for example, by an adhesive, such as a double-sided adhesive film (e.g., 3M 300LSE, 2 mil thick, double-sided adhesive).

[0386] In Figure 41, the cartridge also includes a spacer frame 4119 sandwiched between the first and second frames and between the first and second sheets. The first and second sheets may or may not be attached to the spacer frame. The first and second frames may be secured to the spacer frame. In some examples, the spacer frame is formed of a hydrophobic and oleophobic material, such as PTFE.

[0387] Alternatively, in some examples, only a single frame may be used, which may not include spacers, and a first sheet may be attached to a first side of the frame, while a second sheet may be attached to a second side of the frame. The frame of the cartridge shown in this example may be rigid; in some examples, the frame may be flexible and / or hinged.

[0388] Another example of a portion of a cartridge is illustrated in Figures 42A-42B. In this example, the cartridge is shown as having a first frame 4217 to which a first sheet 4207 is attached. The first sheet may be attached to the top or bottom of the first frame. In Figures 41 and 42A, the sheet is shown attached to the bottom of the first frame. The frame may be formed in any suitable manner, including laser cutting, injection molding, etc., and may be of any suitable material (e.g., a polymeric material such as polyester, ABS, or POM (glass-filled)). Figure 42B shows an enlarged cross-sectional side view of the first frame 4217 and the first sheet 4207. In this example, the first sheet is a TPE film and is adhesively held to the first frame under tension using adhesive 4210 (e.g., 3M 300LSE, 2 mil thick, double-sided adhesive). In Figures 40 and 41A-41B, the first and second sheets may be approximately 20-60 µm thick (eg, 25-50 µm thick) and may be formed of an elastomeric material.

[0389] Any of these cassettes may include a hydrophobic frame (backbone), such as polypropylene, and may include one or more internal structures, including, but not limited to, a spacer frame. For example, the device may include posts (pinning posts) and / or absorbents (absorbent material). The absorbents may be used to remove waste (e.g., from rinsing / washing, drop gloss, etc.). In some cases, the edges of the frame may include absorbents. The frame may include markings, including computer readable markings (e.g., QR codes, bar codes, etc.) that may uniquely identify the cassette. The cassettes may be oriented, such as to allow positioning within the base of a mechanical microfluidic device in a preferred or exclusive orientation, or may be unoriented (as may be applied in any orientation). In some cases, the cartridges may include a specific "top" and "bottom" and may be marked or coded (including color coding) and / or keyed to fit within the base of a mechanical microfluidic device with the upper surface "up."

[0390] Figures 43A-43C illustrate the insertion of an example cartridge 4302 (see Figure 43A) into the seat of a mechanical microfluidic device (shown in Figures 43B-43C). In this example, the cartridge includes an elastically deformable upper sheet 4307 and an elastically deformable lower sheet 4309, 4309', both of which are attached to a frame 4304 (e.g., a molded polymer frame) to form an air gap 4312 having an initial air gap height 4319. The upper and lower sheets in this example are shown as being attached to the same frame 4304 and adhesively attached 4314.

[0391] In Fig. 43B, the cartridge is shown mounted on the base of a mechanical microfluidic device 4322. The mechanical microfluidic device in this example includes a recessed mounting area 4320 containing multiple vacuum ports connected to a vacuum manifold 4322; the vacuum manifold 4322 is connected to a negative pressure source and controlled by a controller of the mechanical microfluidic actuator. In Fig. 43B, a suction force is shown applied; it pulls the lower sheet 4309 of the cartridge into continuous contact with the mounting area and increases the height of the air gap 4312 to a larger height 4319' (compared to the neutral height 4319).

[0392] Figure 43C illustrates a cartridge in the mechanical microfluidic device shown in Figure 43B with droplet 4331 shown in air gap 4312. In this example, upper sheet 4307 and lower sheet 4309 may be, for example, an elastomeric polyester, and cartridge frame 4304 may be molded polyester.

[0393] 44A-44B show schematics of two alternative examples of a cartridge mounted and secured to a footprint of a mechanical microfluidic device. In FIG. 44A, the cartridge includes a frame 4406, with a first sheet 4407 and a second sheet 4409 attached to the frame 4406 and separated from each other to form an air gap 4421. In FIG. 44A, similar to FIGS. 43B-43C, the footprint 4419 of the mechanical microfluidic device includes a vacuum port that secures the lower (second) sheet 4409 against (in contact with) the surface of the footprint such that there is no gap between the footprint and the second sheet; the second sheet is held immobile against the footprint as shown. In FIG. 44A, a droplet 4412 may be heated / cooled or otherwise manipulated through the lower (second) sheet by applying thermal energy through a specific sub-region of the footprint of the mechanical microfluidic actuator. In this example, the entire footprint is shown as being concave; in other examples, only a portion of the footprint is concave, forming a well (described below) within which a droplet can be held.

[0394] FIG. 44B shows another example of a cartridge held within a mounting area of ​​a mechanical microfluidic actuator. In this example, the cartridge includes a frame 4406, and an upper sheet 4407 and a lower sheet 4409; the cartridge is secured to the mounting area by a fixture, such as a clamp 4420, which applies a fixing force to the frame to hold the cartridge stationary in place. In this example, the mechanical microfluidic device does not need to include a vacuum (e.g., a vacuum port) to ensure contact between the second sheet 4409 and the external mounting surface of the mechanical microfluidic actuator; in FIG. 44B, the external mounting surface may be raised along the length of the air gap 4421. Thus, any of the mechanical microfluidic actuators described herein may not include a vacuum. Any of the mechanical microfluidic devices described herein may include a fixture (e.g., a clamp, lock, etc.) that holds the cartridge against the mounting area of ​​the mechanical microfluidic device.

[0395] As described in more detail below, any of these mechanical microfluidic actuators may include a controller and one or more thermal regions; the thermal regions may locally heat / cool the mounting region and thus the droplet in the air gap of the cartridge over this portion of the mounting region.

[0396] One or both surfaces forming the air gap may include regions having an air gap height (between the upper and lower surfaces) that is smaller than the height of the other regions (e.g., the majority of the air gap) but greater than the height of the region of deformation formed by the mechanical actuator (e.g., a stylus, roller, etc.). The region of reduced height of the air gap may be an intermediate height of the air gap between the maximum height of the air gap and the height of the actuated deformation. These regions of lower height may act as guides or rails for directing and / or manipulating the droplet, and may therefore be referred to as guide or rail regions in the air gap. The guide or rail regions ("rails") may be formed as part of one of the surfaces (e.g., a thickened or enlarged region) or may be formed by an external part of the device (e.g., as part of the mounting region of a mechanical microfluidic device). The reduced height of the air gap in these regions may hold the droplet in this region of the air gap by temporary capillary action when the elastically deformable surface is not deformed.

[0397] For example, any of the footprints of the mechanical microfluidic actuator may include a shape to which the lower sheet of the cartridge can conform, which may have a number of advantages, such as fixing the droplet in a particular area (e.g., pinning the droplet) and / or enhancing thermal energy transfer. FIGS. 44C-44E are schematic illustrations of examples where the footprint of a mechanical microfluidic device includes a protrusion. In FIG. 44C, the cartridge is shown mounted on the footprint of the mechanical microfluidic device 4419″, which includes rails 4411 (e.g., rail areas). The cartridge may be held down by a fixture (not shown; clamp, magnet, etc.). Thus, the droplet 4412 between the first sheet 4407 and the second sheet 4409 is held in the center of the air gap 4421 area (e.g., by capillary forces) and may be moved within the channel (e.g., into and out of the cross-section shown) by a force applicator that applies a force to reduce the height of the air gap. FIG. 44D shows an example where the mechanical microfluidic device 4419''' includes a stepped rail 4411' that deforms the lower sheet 4409' slightly into the air gap 4421 but has a wider base than the rail in FIG. 44C. In FIG. 44E, the mechanical microfluidic device 4419'''' is similar to that shown in FIG. 44D but includes a dome-shaped rail 4411''. Any of these examples may, but need not, include a vacuum manifold with vacuum ports for securing the second sheet 4409, 4409', 4409'' to the exterior surface of the mounting area. Alternatively or additionally, these devices may include a fixture (clamp, lock, magnetic fixture, etc.) to hold the cartridge in place.

[0398] Generally, the methods and apparatus described herein may include the use of a rail region within the air gap. The rail region may generally have a gap width on either side of the rail region that is less than the gap width of the region around the rail region in the air gap, e.g., around the perimeter of the air gap. The rail region may form a raised bed. As described above, the rail region may be formed by bending a second (e.g., lower) sheet of the air gap; in some examples, the second sheet may be stiffer (e.g., formed of a relatively stiff material) than the first sheet, and the rail region may be formed of the stiffer second sheet. Surprisingly, the droplet may avoid the region at the perimeter (proximate to the rail region) that has a larger gap width, which may prevent loss of droplet volume, especially for droplets with smaller volumes (e.g., less than 15 μL, 10 μL or less, 5 μL or less, 1 μL or less, 1 μL or less, etc.).

[0399] Mechanical Microfluidic Devices As mentioned, any of these devices may include a mechanical microfluidic device. The mechanical microfluidic devices described herein drive actuators (e.g., stylus, rollers, etc., generally referred to as mechanical microfluidic actuators) to deform the elastically deformable surface of the air gap and to control the application of a temporary capillary action force to pull a droplet (or droplets) in the air gap. Generally, the mechanical microfluidic device may include a base for mounting one or more cartridges; a mechanical actuator ("mechanical microfluidic actuator"); one or more x,y actuators (e.g., one or more motors) for driving the mechanical actuator in x and y (e.g., across the surface of the device); one or more z actuators (e.g., motors) for driving the mechanical actuator up / down relative to the elastically deformable surface of the cartridge held by the mechanical microfluidic device; a frame for holding the mechanical actuator relative to (e.g., on) the cartridge; and one or more controllers for controlling the movement of the mechanical actuator by controlling the one or more actuators. The devices may also include one or more inputs (e.g., sensors, optical sensors, mechanical position sensors, encoders, etc.) for determining the relative position of the mechanical actuator and the air gap (or a cartridge containing the air gap). The devices may also include one or more outputs (e.g., displays, LEDs, etc.) for communicating with a user. Optionally, the devices may also include one or more (e.g., arrays) of thermal control regions (e.g., heaters, heat spreaders, coolers / Peltier elements, etc.); and / or one or more magnets, e.g., electromagnets, etc., for controllably applying (e.g., turning on / off, increasing or decreasing strength) a magnetic field to a region of the air gap.In some examples, the base may include one or more suction ports and / or a vacuum manifold for applying suction to the cartridge to secure the cartridge and in some cases to apply tension to conform the deformable lower surface of the air gap to a pattern or structure of the base, including, for example, forming one or more rail regions within the air gap. In some examples, the device may include one or more electrodes (e.g., electroporation electrodes, return electrodes, etc.) for applying energy to the droplets. In some examples, the device may include one or more sensors for detecting one or more properties of the droplets in the air gap and / or for sensing (and in some cases controlling) the operation of the mechanical microfluidic device. For example, the base region of the mechanical microfluidic device may include one or more thermistors for detecting the temperature of a region (e.g., a thermal control region); this information may be used as feedback to control the temperature of the sensed region and / or to control the operation of the device, including controlling the movement of a mechanical actuator (such that the applicator does not apply movement until a desired temperature profile is achieved).

[0400] Any of these mechanical microfluidic devices may include a light source for illuminating the cartridge, the droplets, and / or the material (cells, particles, etc.) within the cartridge and / or within the droplets. In some examples, light may be used to heat the device. One or more light sources may be used for imaging and / or for treatments applied to the particles. In addition to controlling and coordinating the operation of the mechanical actuators, a controller may control and / or coordinate the activity of all or some of these components, e.g., light sources, thermal controllers, suctions, magnets, etc.

[0401] Any of the devices described herein may be configured to determine the location of one or more droplets; this information may be used as feedback to control the operation of the device, including controlling the movement of the mechanical actuator. The droplets may be detected optically (e.g., using one or more optical sensors, cameras, etc. that can image the droplets through the cartridge, including imaging through an elastically deformable layer). The droplets may be detected electrically, including using capacitance and / or inductance to detect the droplets and / or particles (e.g., magnetic particles) within the droplets. In some examples, the base of the device may include one or more electrodes to determine an electrical property within the air gap, such as capacitance, to determine or approximate the location of the droplet. Alternatively or additionally, one or more sensors (e.g., optical detectors, electrodes, etc.) on the mechanical actuator may be used to detect or determine the location of the droplet. In some examples, the mechanical actuator may be scanned over the surface of the air gap without deforming the surface and applying a temporary capillary action force.

[0402] The devices described herein may also include one or more sensors for determining the force or contact and / or deflection applied by the mechanical actuator. In some examples, the sensor, also referred to as the actuator sessor, may be located on the actuator.

[0403] Any of the devices described herein may be configured to control multiple actuators and thus operate multiple different (and concurrent) lanes or regions within a single cartridge or multiple cartridges. In some examples, the device may be configured to independently control multiple different actuators. In some examples, the device may be configured to operate multiple different actuators that may operate in parallel.

[0404] As described herein, the device may divide the cartridge into different regions or zones, including regions for thermal regulation (e.g., temperature control zones), regions for applying magnetic fields (e.g., to pellet magnetic particles), regions for imaging or sensing the droplets, regions for metering the droplets, etc. A controller may coordinate these different regions or zones. In some examples, the device may move one or more droplets between different thermal regions to thermally cycle material within the droplets.

[0405] An example of a portion of a mechanical microfluidic device is illustrated in Figures 45A-45E. In Figure 45A, the mechanical microfluidic device 4500 includes a mounting area (pedestal) 4531 on which a cartridge may be held and / or secured. In the illustrated example, the mounting area defines a number of parallel lanes 4532 (eight are shown) running the length of the mounting area. In this example, the pedestal includes a number of vacuum ports connected to a vacuum manifold 4538 for applying suction to secure the lower (second) sheet of the cartridge in conformity with the mounting area. Additionally, each of the lanes of the mounting area includes a number of different zones: a zone 4533 for thermal control, a zone 4435 for application of a magnetic field, or a zone 4542 for magnetic and thermal control. The thermal control zone may be in thermal communication with a heater / cooler (e.g., a Peltier device, a resistive heater, etc.), and each of the magnetic control zones may include a localized electromagnet. This is illustrated in FIG. 45B where the footprint 4531 is made transparent to show the thermal control zone 4533, the electromagnet zone 4539, and the combined thermal control / magnetic zone 4542. In this example, the base of the mechanical microfluidic device may be a heat sink 4536 to allow for localized application of heating / cooling. FIG. 45C shows an example of a footprint of a mechanical microfluidic device with a cartridge 4506 attached to the footprint 4531. The cartridge 4506 is similar to that described in FIGS. 40A-43E and includes eight lanes with an upper transparent elastically deformable sheet and a lower elastically deformable sheet; each lane includes a window 4532 formed through the upper elastically deformable sheet to allow access into the air gap.

[0406] This example illustrates three types of zones arranged in an alternating pattern along the length of each lane of the footprint, which may correspond to lanes in cartridge 4506, as shown in FIG. 45C. Other patterns and / or types of zones (e.g., heating / cooling, magnetic, electrical energy, sensing / imaging, UV application, (ultrasonic) application, etc.) may be included and controlled by the device's controller (not shown). Examples of footprint topologies are shown in a little more detail in FIG. 45D and 45E. For example, in FIG. 45D and 45E, the footprint may include multiple wells 4541 formed therein that may be below thermal control regions. For example, in FIG. 54D, the footprint of the mechanical microfluidic device includes multiple thermal control regions configured as wells 4541 with shallow bowl-shaped recesses formed in thermally conductive material 4533. The bowl also includes suction ports 4555 in communication with suction manifold 4538 to hold second sheet 4509 downward. Suction force applied to a lower elastically deformable sheet that forms the lower surface of the cartridge's air gap creates this well or bowl at the lower surface of the air gap. The controller may pull the droplet (e.g., with a force applicator) into and / or out of the well by deforming the upper elastically deformable surface to temporarily increase the capillary action force. In some examples, a mechanical force applicator (not shown) may be lowered over the well once the droplet has been pulled into the well to pin the droplet within the well; this may reduce or limit evaporation, especially when heating the droplet (e.g., to thermocycle the droplet).

[0407] In the example cartridges and mechanical microfluidic devices shown in Figures 45A-45G, operations performed on droplets can be relatively sequential, and the applicator may move in a straight line along the long axis 4580 of each channel in the cartridge, so operations may be parallelized and simplified, which ensures robust automation. Generally, in any of these methods and devices, the device may adjust the height of the force applicator to control transient capillary action forces; in some examples, the height of the force applicator may be adjusted to account for local height changes in the air gap, including those caused by the pedestal 4531 (e.g., well, rail, etc.).

[0408] FIG. 45F illustrates another example of a cartridge illustrating the operation of a device with a cartridge that includes an air gap. In this example, a cartridge 4506′ is shown mounted on a pedestal 4531. This exemplary cartridge includes a window opening 4532 into each lane at one end of the cartridge; the window is an inlet into the air gap through which reagents / samples can be applied (e.g., by pipetting) and / or removed (e.g., to remove final material for product library recovery, etc.). The cartridge 4506′ may also include an absorbent pad 4539 in each lane at the opposite end from the inlet, which may wick and contain waste material from the air gap. The mounting area 4531 of the mechanical microfluidic device in this example also includes multiple thermal control (e.g., heating) zones 4533 and magnetic field application zones 4535. In Fig. 45F, the device is configured to initially direct the flow 4550 of sample / reagents from the window towards the opposite end of the cartridge after each input; droplets may be used to add samples and to add components (e.g. buffers, washes, enzyme mixtures, etc.) for performing sequential protocol steps. Droplets may be sequentially positioned on control regions (thermal control regions, magnetic field regions, etc.). The controller may then redirect the flow 4551 back towards the window (inlet) 4532 after completion of each step or the entire workflow, for example to remove substances such as intermediate products and / or final samples, for example for final extraction of substances.

[0409] Generally, the controller may control force applicators (stylus, rollers) etc. to move in the yz plane as needed during protocol execution to deliver fresh reagents; move reaction products to the next thermal zone (heater) or between thermal zones (cycling) or to magnet zones for different protocol steps; and / or move waste into the waste pad. As described herein, droplets may be combined (fused), split, metered, mixed etc. within the lanes. In variations including one or more particles (e.g. magnetic particles), the magnetic particles may be pelleted, washed, resuspended, mixed, split, washed, eluted etc.

[0410] The example shown in FIG. 45F is configured as a reverse workflow approach that allows for the use of a single inlet 4532, although in some examples multiple inlets may be used. The movement of droplets within the air gap is not limited to back and forth (e.g. only in the x direction) but may be in any direction within the plane (x, y) in some examples. In any of these examples, the lane may be divided into a forward side (e.g. left side) and a reverse side (e.g. right side) so that droplets can move forward or backward on one side or the other within the lane; this may in some examples minimize cross contamination from earlier steps in the protocol since droplets do not pass through the same region of the air gap. In some examples, the cartridge may include inlets / outlets in areas at both ends of the cartridge.

[0411] Figure 45G illustrates the end of a run (protocol); now the thermal control and magnetic field regions have all been used 4561 by both the forward and reverse directions 4551, and the absorbent pad 4539 contains waste from all steps of the workflow at the end of the run. The system may collect the product (e.g., library in some examples) from the air gap and place it into a corresponding 8-well strip tube or 96-well plate. In Figure 45G, the product is removed from the inlet 4532' (window), for example by pipetting.

[0412] Figure 46 illustrates a schematic of an example of a portion of a device (e.g., cartridge and mechanical microfluidic actuator) similar to that shown in Figures 45A-45E, including a vacuum port 4638 that secures the second sheet 4609 of the cartridge to the mounting area 4631 of the mechanical microfluidic device. In Figure 46, a droplet 4612 is shown in the air gap area and is coated with a drop gloss 4652 material. The drop gloss coating may be formed of a material that limits evaporation and is immiscible with the droplet.

[0413] Generally, any droplet of suitable size may be used, including microliter and sub-microliter droplets. However, in some cases, smaller ones (e.g., less than 2 μL) may be difficult to reliably transfer. It may also be beneficial to use fluid transfer of droplets of any size that does not require negative pressure (e.g., aspiration), e.g., does not involve pipetting. FIG. 47 illustrates an example of a method for reliably transferring very small droplets, including (but not limited to) transfer into a cartridge as described herein. In this example, a solid transfer member 4671 having a concave end region 4673 ("droplet void") may be included at the distal end of the device. The concave area / void may be configured to hold a particular droplet volume, such as less than a few microliters in volume (e.g., 0.1 μL, 0.2 μL, 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, 0.8 μL, 0.9 μL, 1 μL, 1.1 μL, 1.2 μL, 1.3 μL, 1.4 μL, 1.5 μL, 1.6 μL, 1.7 μL, 1.8 μL, 1.9 μL, 2 μL, etc.). Larger volumes may be used as wells (e.g., 1-50 μL, 0.1-50 μL, etc.). The droplet void area 4673 may be inserted into a solution of fluid 4675 to be transferred and removed, such that a droplet 4677 of expected size and volume becomes trapped within the end of the solid transfer member 4671, as shown. The droplet may be released, for example in an air gap, by immersion in a solution (e.g., another droplet) 4679 (e.g., a drop gloss) having a lower surface tension; as shown, this immersion results in the displacement and release of the droplet 4677.

[0414] Generally, these devices are able to handle smaller volume droplets by increasing the volume / amount of the immiscible fluid (drop gloss) so that the final volume is large enough for displacement in the air gap by use of a mechanical actuator as described herein.

[0415] Another schematic diagram of a mechanical microfluidic device 4800 is shown in Figure 48. In this example, the mechanical microfluidic device includes a force applicator 4878 (e.g., stylus, bearings, rollers, etc.) and a force applicator driver subassembly 4874 (e.g., force applicator subassembly). The force applicator subassembly may include one or more drivers (e.g., x and / or y motion drivers, z motion driver 4873, etc.) and / or a frame or gantry on which the force applicator is driven to reposition and / or apply forces to the cartridge 4877 when the cartridge is installed in the cartridge seat 4878 of the device. The force applicator subassembly may include one or more stepper motors, motion rails (e.g., gantry / frame), and / or a home switch.

[0416] The mechanical microfluidic device of FIG. 48 also includes a thermal subassembly 4879 for controlling the temperature of one or more regions of the air gap. In this example, the thermal subassembly may include a thermally conductive zone or region of the mounting area that may be in thermal communication with a heating and / or cooling element (e.g., a Peltier device); multiple heating / cooling elements may be included. Any of these mechanical microfluidic devices 4800 may also include a magnetic control subassembly 4885, 4885' for controllably applying a magnetic field within the air gap. FIG. 49 illustrates an example of an array of magnetic elements (electromagnets) 4985 within the base 4819 of a mechanical microfluidic actuator.

[0417] In some examples, the mechanical microfluidic device may include a cartridge fixture 4876, 4876' (e.g., holder, clamp, lock, etc.) for securing the cartridge to a cartridge seat or mounting area 4878 of the mechanical microfluidic actuator. In FIG. 48, the device includes a vacuum / aspiration subassembly (not shown) for applying a suction force to secure the cartridge within the mounting area. In some examples, the mechanical microfluidic device may include a fluid handling (e.g., pipetting) subassembly 4883 for adding and / or removing fluid from the air gap. Other subassemblies that form part of the mechanical microfluidic device may include an imaging subassembly (e.g., for imaging droplets in the air gap) and / or a sensing subassembly (e.g., for sensing droplets or other inputs from the air gap and mechanical microfluidic actuator). The mechanical microfluidic devices described herein may also include one or more control inputs (e.g., keyboards, touch screens, buttons, switches, etc.) and / or one or more outputs (e.g., displays, LEDs, wireless communication outputs / inputs, etc.) and hardware, software, and / or firmware for controlling the same. In some cases, the same features may be used for the control inputs and outputs. In general, the mechanical microfluidic actuators described herein may include one or more controllers 4899 for controlling and coordinating the operation of the various subassemblies.

[0418] Any of these devices may include leveling. For example, in FIG. 48, the device includes adjustable leveling feet 4881.

[0419] In general, the mechanical microfluidic actuators described herein may be single cartridge use (e.g., for use with a single cartridge at a time) or may be configured to use multiple cartridges. FIG. 50 illustrates an example of a mechanical microfluidic device 5001 at least partially enclosed within a housing 5095 and configured for use with a single cartridge 5077. The device includes a force applicator subsystem 5097 (e.g., 3-axis motor, encoder, home and limit sensors, solenoids, raises, shafts, couplings, bearing gears, belts, etc.); and an electrical subsystem 5094 for controlling the power requirements of the device, e.g., controller, power distribution, user interface board, touch screen, etc. (and in some examples, for applying power to one or more electrodes, e.g., for electroporation and / or electrochemical procedures on the cartridge). The device also includes a thermal subsystem 5092 (e.g., Peltier, high power TEC driver, heat spreader, heat sink, etc.), controller 5091, and input / output 5093 (e.g., display / touch screen). An example of a multiplex device 5101 that allows for handling of multiple cartridges in parallel is illustrated in Figure 51. As shown in Figure 51, either the single cartridge or the multiplex configuration may also include or be configured for use with a fluid handling system (e.g., liquid handler 5163).

[0420] 52A-52B are schematic diagrams illustrating an example of a mechanical microfluidic device as described herein, showing one possible arrangement of the subassemblies described herein. For example, in FIG. 52A, the device includes a liquid staging subassembly (e.g., temperature control, input, chip storage, chip waste, etc.) for adding / removing liquid from the cartridge, as well as a chassis subassembly (e.g., chassis, fan, leveling feet, switches / buttons, touch screen, etc.), and a power distribution subassembly (main power supply, power distribution circuitry, etc.). The controller (main control PCBA) may also include sensors (e.g., ambient temperature sensor, ambient humidity sensor, level sensor), and Wi-Fi or other inputs / outputs. The controller may receive inputs from the chassis subassembly and may output / control all of these subassemblies. Specifically, the controller may control a liquid handler subassembly including a motion controller (drivers, position sensors, etc.), and may also control submodules including a temperature subassembly (Peltier subassembly), a magnetic subassembly, a linear motion subassembly, and a cartridge receptacle (e.g., cartridge data), each of which may provide input to the controller.

[0421] 64A-64D illustrate another example of a mechanical microfluidic device, particularly a base portion of a mechanical microfluidic device including a pedestal region for holding a cartridge. FIG. 64A shows an example of a pedestal (or "bed") region of the device for holding a cartridge. In this example, the lower portion of the cartridge (e.g., a layer or film formed of an elastically deformable material) is in direct contact with a pedestal that includes a thermal control (heater) region and a magnetic field region (magnet). In this example, the pedestal is formed of a thermal insulating material. Using thermal insulating material in the mounting region may prevent or reduce temperature bleeding between the thermal control region and may also enable or facilitate maintaining a vacuum seal between the mounting region and the cartridge. In FIG. 64A, the pedestal region 6431 includes an upper surface with different heights to form wells and chambers in the lower surface of the air gap for a cartridge (not shown) mounted on the device. In this example, the device shows a well 6471 (having a doughnut shape); a thermal control (e.g., a heater) and a thermal sensor (thermistor) may be coupled to this region to provide thermal control. In FIG. 64A, the device also includes a magnet 6435, which may be an electromagnet and may be moved up / down to increase / decrease the magnetic field applied to this region. For example, FIG. 64B shows multiple magnets each attached to a z-actuator 6483. The material forming the base 6431 (set) may be formed of a thermal insulating material. In any of the devices described herein, magnetic shielding (e.g., mu material) may be used to change, reduce, or refine the applied magnetic field. For example, magnetic shielding may be used to localize the magnetic field within the air gap and / or to prevent the magnetic field from reaching the air gap until application is desired.

[0422] FIG. 64C shows a partial exploded view of the base of the mechanical microfluidic device of FIGS. 64A-64B. In this example, the base is formed of thermally insulating PCB (printed circuit board) material and a well 6471 (ring) is formed or attached thereto. The well may be connected to a heat spreader (e.g., a thermally conductive material) and / or directly to a thermal regulator 6433 (e.g., a TEC / heater). As shown in the cross-sectional view of FIG. 64B, the magnetic region 6435 may overlie one or more magnets 6435. The device may also include a vacuum manifold 6437 (illustrated as a vacuum chuck) for securing the cartridge onto the footprint such that the elastically deformable base (lower layer) of the cartridge conforms to the shape of the footprint. FIG. 65D illustrates a schematic example of a cross-section through the device showing the different layers (e.g., footprint PCB, TEC, heat sink, and cooling fan).

[0423] Figures 65A-65C illustrate an example of a footprint 6500 (shown in Figure 65A) including a heat sink area, and an upper footprint 6501 (shown in Figure 65B) similar to that shown in Figures 64A-64D. Figure 65C shows the upper footprint and heat sink 6536 assembled. EXAMPLES

[0424] 53A-53C illustrate an example of a method for moving aqueous droplets as described herein. In this example, one or more microfluidic droplets are manipulated to be moved virtually anywhere within an air gap 5321 formed between an elastic first sheet 5307 and a second sheet 5309. As described above, the second sheet may also be an elastic sheet. Both the first surface 5391 and the second surface 5392 (also referred to as the inner surface facing the air gap) of the first and second sheets may be hydrophobic and oleophobic. The sheets may be formed of a hydrophobic and oleophobic material or may be coated with a hydrophobic and oleophobic material.

[0425] A water-based fluid droplet 5312 may be introduced into an air gap formed between a first sheet 5307 having a hydrophobic and oleophobic first surface and a second sheet 5309 having a hydrophobic and oleophobic second surface. As described above, the first sheet and the second sheet may be fixed generally parallel facing each other at a predetermined distance with an air gap between them. The first sheet and / or the second sheet may be held under tension. At least the first sheet is formed of an elastomeric material such that it can deform when a force (e.g., a mechanical stylus as shown in Figs. 53B and 53C) is actuated thereon and returns to a generally parallel configuration when the force is released. For example, in Fig. 53A, a mechanical force applicator (stylus 5375) is positioned above the top of the first (e.g., upper) elastomeric sheet 5307. As described above, one or more motion drives (e.g., x, y stage / z motion, robotic stage or control) may be used to drive the movement of the mechanical force applicator relative to the upper sheet. The first and second sheets may be part of a cartridge, which may include one or more tensioners (e.g., tensioning frames, etc.) that hold the first and / or second sheets under tension.

[0426] As shown in Figure 53B, the mechanical force actuator 5375 may be actuated downwards relative to the first sheet to a region proximate to the droplet. Locally reducing the height of the air gap (in a continuous gradient as shown) may pull the droplet into that region of lower height due to the resulting increased capillary forces. Thus, a mechanical force actuator actuated across the top of the sheet (and relative to the top sheet) as shown in Figures 53B and 53C will move the droplet within the air gap.

[0427] In any of these examples, the height of the air gap may be reduced in a gradient, and the distance between the upper and lower (first and second) sheets is reduced but do not contact each other. For example, the height is reduced by about 5% to 90% (e.g., about 10% to 80%, about 20% to 60%, about 10% to 50%, etc.). In some cases, it may be advantageous to reduce the height by about 5% to 60%, but not more than 60% (e.g., not more than 55%, not more than 50%, not more than 45%, not more than 40%, not more than 35%, not more than 30%, not more than 25%, etc.). This may allow the gradient to drive the movement, but may limit the area to the area local to the droplet. This may allow the first (upper) elastic sheet to return to a parallel configuration (in the area where the stylet has moved away from the sheet, as shown in FIG. 53C). As shown in Figures 53B-53C, applying a force (by a mechanical force applicator / stylet) to elastically deform the first sheet reduces the air gap distance between the first sheet and the second sheet in a localized region within the air gap proximate to the fluid droplet, and causes the droplet to move within the air gap, following the region of reduced height formed by the stylet.

[0428] In any of the methods and apparatus described herein, the sheets forming the air gap are made of a hydrophobic and oleophobic material; materials that are not hydrophobic and oleophobic did not work in many of the examples shown. In addition, the material forming the inner surface of the air gap may be substantially non-porous.

[0429] As mentioned, any droplets may be coated with a layer of drop gloss, such as a gloss coat, which may be a substance with low surface tension (e.g., oil) and may be immiscible with the droplet; being immiscible with the droplet may also prevent or limit evaporation.

[0430] In general, the methods and apparatus described herein may include the use of at least 0.01% surfactant in or around the droplet to be moved. Surprisingly, the inventors have found that the use of surfactant in the droplet (e.g., 0.01% or more, 0.02% or more, 0.025% or more, 0.01%-1%, 0.01%-0.7%, 0.01%-0.5%, 0.01%-0.25%, 0.01%-0.1%, etc.) or in the gloss layer surrounding the droplet may result in more predictable movement of the droplet in the air gap when pulled through a reduced gap width as described herein. Without being bound by theory, this may be due to the effective surface tension of the droplet; therefore, the use of surfactant in either or both the drop gloss and / or the droplet may result in more predictable and robust movement of the droplet. Without the use of a surfactant, droplet movement may be less predictable and may in some cases not track the movement of the mechanical actuator across the surface. Any suitable surfactant may be used. For example, the drop gloss used includes a non-ionic surfactant (e.g., Brij-35) or other hydrophobic polymer. In some examples, the droplet may include a surfactant such as Pluronic, Tween-20, Tetronic, etc. Thus, in any of these methods and devices, either or both of the drop gloss and / or the droplet may include a surfactant (e.g., 0.01% or more surfactant). In some cases, the surfactant may be added prior to commencing any step of moving the droplet by locally reducing the gap width in the area proximate to the droplet.

[0431] In instances where a mechanical force applicator (e.g., a stylus) is used, the contact surface of the stylus may be sized proportionally to the size / volume of the air gap and / or droplet. Specifically, the aspect ratio of the stylus, e.g., the size of the stylus tip relative to the size of the droplet and / or the size of the stylus tip relative to the height of the air gap, may be selected to be between 1:0.5 and 1:20 (tip:droplet).

[0432] DNA sequencing and DNA synthesis The methods and devices described herein may be particularly used for enzymatic processing of polynucleotides, including (but not limited to) sequencing and / or synthesis.

[0433] For example, these methods and devices may be used to perform DNA sequencing by synthesis (SBS). SBS has provided many significant benefits to the scientific research community and enabled many new diagnostic applications; including increased output from sequencing instrumentation, faster turnaround time for results, and orders of magnitude lower cost than the leading prior sequencing method, Sanger sequencing. Sanger sequencing relies on electrophoretic separation of DNA fragments created by specially modified terminating nucleotides. SBS eliminates the need for separation, allowing for the implementation of massively parallel sequencing approaches. As a result of these cost and throughput improvements, several important clinical applications have been developed, including non-invasive prenatal testing (NIPT) for detecting aneuploidies such as Down's syndrome in the blood of pregnant women, genetic carrier testing to inform parents about potential genetic risks, stratification of tumor patients, and tumor profiling and early detection through sequencing of nucleic acids in blood (known as cell-free sequencing).

[0434] SBS is a flow-based sequencing technique in which a series of liquid formulations are introduced into a flow cell containing DNA templates that have been isolated, purified, and processed to create a sequencing "library" and then flowed into a sequencing flow cell. The flow cell is loaded with template DNA and rides on a random or structured array that creates a separate "cluster" for each DNA library fragment. Reagents are delivered in a sequential process that enzymatically adds a single fluorescently labeled nucleotide with each cycle. After each fluorescent nucleotide is added, the flow cell is imaged, with each unique fluorescent label representing a specific base (A, C, G, T); the flow cell is run through software that assigns the next base in the sequence. After imaging, the fluorescent dye and nucleotide blocking group are chemically cleaved and washed away in preparation for the next cycle. Typically, this process is repeated for 75-600 cycles, with each cycle adding another base that is recorded (captured) through the imaging process and software analysis. Many protocols include a process (in most cases during the run) to create a complementary strand of DNA, which is also sequenced to improve coverage and accuracy. The methods for creating this complementary strand use similar reagents to those used to create the newly synthesized DNA that is sequenced in SBS.

[0435] Current methods typically use a delivery method to provide a continuous flow of each reagent in sequence. These reagents are delivered by pumping or pressure mechanisms; each reagent floods the flow cell to completely fill it, and then is flushed away to replace the next reagent required to drive the cycle. Although these volumes are fairly small, carryover from step to step or cycle to cycle can impair the resulting sequencing, so extra volumes are required to ensure there is no carryover. Figure 54 shows a schematic diagram of the SBS sequencing cycle and overall process map. The microfluidic methods and devices described herein may be used to flow various components in a "flow cell" configured as described herein, including wash steps.

[0436] Conventional digital microfluidics (such as those using electrowetting) have been proposed for use in nucleic acid sample preparation as the front-end of sequencing processes and as an alternative to manual benchtop library creation or traditional robotic pipetting systems, but they suffer from a number of drawbacks. Electrowetting on Dielectric (EWOD) has been successfully applied to front-end processes such as DNA isolation from patient samples and sequencing library creation for loading into sequencing instruments. Although electrowetting has reasonable suitability for automating up-front processes, the complexity of the sequencing process itself presents several technical and practical economic challenges. Notably, the requirement for flow cell imaging after every nucleotide addition cycle. In an ideal embodiment, a fully integrated sequencing process would allow samples to be introduced into the system and provide DNA sequencing results as an output. EWOD is unlikely to be successful as a fluidics solution for such fully integrated processes.

[0437] The methods and devices described herein, also referred to as superficial mechanical actuation, in which a mechanical force (e.g., a compression force) is applied to pull one or more droplets, may offer significant advantages over other microfluidic techniques, including electrowetting, and may allow the use of a single fluidics technique throughout the entire sequencing process, including SBS. As discussed above, the use of mechanical compression to vary capillary forces to move droplets in two dimensions may have many advantages, particularly with respect to sample preparation, and may be used for virtually all required steps, such as nucleic acid isolation, library generation, cluster generation, primer loading and hybridization, and multi-cycle sequencing reactions, including the steps illustrated in FIG. 54.

[0438] 55A-55B illustrate an example of the use of mechanical compression to vary capillary forces to move droplets in a flow cell 5501 to introduce sequencing primers to clusters of DNA templates (e.g., as part of the sequencing-by-synthesis process described above). FIG. 55A shows the loading and washing steps associated with primer introduction to sequencing templates in the flow cell 5501. As with any method described herein, multiple mechanical force applicators (styluses 5575) may be used simultaneously on the same flow cell (e.g., cartridge). These mechanical force applicators may be independently or collectively controlled / actuated. In FIG. 55A, the cartridge / flow cell includes regions where primers are located, either unpatterned or patterned (e.g., as nanowells), where cluster formation occurs. The steps to generate clusters may also be performed using mechanical force applicators as described herein, or the steps may be performed by pipetting and washing.

[0439] A first droplet 5502 may first be drawn onto the clusters using a first stylet and incubated on the clusters (to allow hybridization) as shown in Figure 55B; and then drawn off therefrom, and in some cases out of the flow cell (e.g., to a waste depot). A second droplet 5504 (wash buffer) may then be drawn onto the clusters to wash the clusters.

[0440] 56A-56E illustrate the steps associated with each cycle of SBS performed within a cartridge (e.g., flow cell) 5501. As described above, either multiple mechanical force applicators (styluses 5575) or the same stylus may be used sequentially. In this example, the figure shows unidirectional movement of the droplet, e.g., from left to right. However, the methods and devices described herein may allow movement in two dimensions (e.g., movement across the plane of the air gap and in any direction within this plane). Thus, in some examples, reagent droplets may be moved to the side and reintroduced or reused (and further refilled with depleted components, e.g., nucleotides, enzymes, other chemicals, etc.).

[0441] In Figure 56A, primer hybridization and washing (see Figures 55A-55B) may be followed by SBS cycles of application of a nucleotide-polymerase reaction mix, washing, and extension followed by imaging for multiplex sequencing. Figure 56A shows the start of the first cycle, in which nucleotides and polymerase are added to a cluster in the air gap of the cartridge / flow cell 5501 by pulling a droplet containing the nucleotides and polymerase with a mechanical force actuator. After a suitable time, the droplet may be pulled off the cluster, and one or more wash droplets may be moved onto the cluster during imaging (to identify additional nucleotides) (Figure 56B). The wash droplet may again be moved off the cluster, and a droplet containing the dye / terminator cleavage components may be moved onto the cluster (Figure 56C); this droplet may then be moved off using a mechanical force actuator, and the same or a different mechanical force actuator may be used to move another droplet of nucleotides and polymerase to begin the second cycle (Figure 56E).

[0442] The microfluidic method and device described herein can also be used for other applications in addition to SBS sequencing.For example, these methods and devices can be used for enzymatic synthesis of DNA oligos (which is very similar to SBS processing), such as cyclic enzymatic addition of nucleotides with reversible terminators.Other non-limiting examples can include DNA oligo synthesis.

[0443] For example, the methods and devices described herein may be used for nucleic acid extraction (illustrated in Figures 57 and 58A-58B) and library preparation (Figures 59, 60, and 61). Figure 57 illustrates a schematic of the overall workflow from nucleic acid extraction to sequencing using a system for surface mechanical actuation (e.g., mechanical compression to alter capillary forces).

[0444] In FIG. 57, a tope and side view of an 8-lane cartridge similar to that described above may be integrated into a system containing an array of cooling regions (e.g., Peltier) and magnetic and / or resistive heating zones, which may allow the cartridge and system to perform DNA / RNA extraction, library preparation, and sequencing in designated areas. In FIG. 57, the cartridge contains an air gap with multiple different reaction wells (including thermal control / cooling). The cartridge is divided into lanes (8 lanes are shown) and includes areas for DNA / RNA sequencing, library preparation, and sequencing (e.g., SBS). Lane dividers may separate the lanes.

[0445] FIG. 58 illustrates the operation of the cartridge and system shown in FIG. 57 for extracting polynucleotides (e.g., DNA, RNA, etc.) from clinical samples. FIG. 58A shows a side view schematic of DNA / RNA extraction from clinical samples on a cartridge. In this example, a 250 μl droplet of clinical sample (e.g., blood, saliva, and tissue homogenate, etc.) containing a 45 μl drop gloss and a 310 μL droplet of lysis buffer is fused and mixed in a PCR reaction well for 5 minutes by the methods described herein and incubated at 70° C. for 10 minutes. The reaction (Rxn) droplet is then actuated to the magnet / resistive heater zone and fused with 400 μL of HDQ Binding Buffer and 20 μL of Mag-Bind® Particles HDQ and mixed for 10 minutes; the magnet is then actuated until a pellet is formed, the supernatant is discarded to waste, and the pellet is washed twice with 600 μL of VHB wash buffer. The pellet may be resuspended in 600 μL of SPM wash buffer and pulled by mechanical actuation over the surface to clear the magnet / resistive heater zone; the magnet may be actuated and the wash buffer may be removed to waste. Finally, the eluted nucleic acid is eluted in 30 μl of Eluent.

[0446] In FIG. 58, droplets of clinical samples (e.g., blood, saliva, and tissue homogenates) are fused with lysis buffer 581, mixed for 5 minutes in a reaction well by techniques described herein (e.g., mechanical actuation on the surface), and incubated at 70° C. for 10 minutes. The lysate droplets are then actuated to a magnet / resistive heater zone 582, fused with binding buffer and magnetic / binding bead particles; and may be mixed for 10 minutes. The magnet may then be actuated 583 until a pellet is formed, the supernatant may be discarded to waste, and the pellet may be washed twice with wash buffer. The pellet is resuspended in wash buffer and pulled by mechanical actuation on the surface to clear the magnet / resistive heater zone; the magnet may be actuated and the wash buffer may be removed to waste. Finally, an elution buffer is actuated to the pelleted beads to elute the nucleic acid off the beads.

[0447] The RNA sequencing workflow is illustrated in Figure 59. In Figure 59, a side view through the cartridge shows a schematic of the RNAseq workflow on the cartridge. In step 591, a 2 ul droplet of fragmented RNA and first strand synthesis master mix, along with a 45 μL drop gloss droplet, is actuated to a PCR reaction well by mechanical actuation on a surface as described herein (e.g., with a stylus) and incubated (25°C for 10 minutes, 42°C for 15 minutes, 70°C for 15 minutes). A 6 μl droplet of second strand synthesis master mix is ​​then added to the reaction (Rxn) droplet and incubated at 16°C for 60 minutes. The Rxn droplet is actuated to the magnet / resistive heater zone 592 and fused with the 11.2 ul bead droplet, mixed, and incubated at RT for 5 min; the magnet is actuated until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 25 ul 80% EtOH (not shown in schematic), and the cDNA sample is eluted off the beads in 6 μl elution buffer. Then, a 5 ul droplet of the cDNA sample is actuated as described herein to the PCR reaction well, fused with 1 μl EndPrep Master Mix (with 10 μl Drop Gloss), and incubated at 20°C for 30 min, followed by 65°C for 30 min. Next, 3.1 μl Adaptor Ligation Master Mix and 0.25 μl Adaptor droplet are actuated as described herein, mixed with the Rxn droplet, and incubated at 20°C for 15 min 593. The adaptor-ligated Rxn droplet is actuated to the magnet / resistive heater zone and fused with the 7.28 μl bead droplet, mixed and incubated at RT for 5 min; the magnet is actuated until a pellet is formed, after which the supernatant is discarded to waste, the pellet is washed twice with 25 μl 80% EtOH (not shown in schematic), and the DNA library is eluted in 6 μl nuclease-free water containing 5 μM TRUESEQ BARCODES594.

[0448] Next, a 5 ul droplet of purified DNA library sample may be actuated by mechanical actuation (e.g., with a stylus) into a PCR reaction well, fused with 10.9 ul of USER / PCR master mix (with 45 ul drop gloss), incubated at 37°C for 15 min, cycled 19X at 98°C for 30, then 98°C for 10 sec, 65°C for 75 sec595. The amplified DNA droplet may be actuated into a magnet / resistive heater zone, fused with a 12.72 ul bead droplet, mixed, and incubated at RT for 5 min; the magnet is actuated until a pellet is formed, the supernatant is discarded, the pellet is washed twice with 25 ul of 80% EtOH (not shown in schematic), and the DNA library is eluted in 25 ul of elution solution596.

[0449] In Figure 59, a droplet of fragmented RNA and first strand synthesis master mix, along with a drop gloss, is actuated by mechanical actuation (e.g., stylus) into a reaction well and incubated (25°C for 10 minutes, 42°C for 15 minutes, 70°C for 15 minutes) 591. A droplet of second strand synthesis master mix is ​​then added to the reaction (Rxn) droplet and incubated at 16°C for 60 minutes. The Rxn droplet is actuated into a magnet / resistive heater zone and fused with the SPRI or Ampure bead droplet, mixed, and incubated at room temperature (RT) for 5 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 80% EtOH (not shown in schematic), and the cDNA sample is eluted off the beads in an elution buffer 592. Next, the cDNA droplets are actuated (e.g., with a stylus) to the reaction wells, fused with the EndPrep Master Mix, and incubated at 20 °C for 30 min, followed by 65 °C for 30 min. Next, the Adaptor Ligation Master Mix and Adaptor droplets are actuated by mechanical actuation (e.g., with a stylus), mixed with the Rxn droplets, and incubated at 20 °C for 15 min. The Adaptor Ligation Rxn droplets are actuated to the magnet / resistive heater zone, fused with the SPRI or Ampure bead droplets, mixed, and incubated at RT for 5 min; the magnet is actuated until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 80% EtOH (not shown in the schematic), and the DNA library is eluted in nuclease-free water containing primers. The purified DNA library and primer mixture is mechanically actuated (by a stylus) into a PCR reaction well as described herein, combined with the USER / PCR master mix with drop gloss, and incubated at 37°C for 15 minutes, 98°C for 30, then 98°C for 10 seconds, 65°C for 75 seconds, for a maximum of 19 cycles.Actuate the amplified DNA droplets to the magnet / resistive heater zone and fuse with the SPRI / Ampure bead droplets, mix, and incubate at RT for 5 min; actuate the magnet until a pellet is formed, discard the supernatant to waste, wash the pellet twice with 80% EtOH (not shown in schematic), and elute the RNA-seq library in 25 μl of elution buffer.

[0450] Figure 61 is a side schematic diagram of the second part of the Twist exome target enrichment method using a cartridge as described herein. In this example, an 8.3 μl droplet of DNA and hybridization mix is ​​actuated as described herein with a 45 μL drop gloss droplet to a PCR reaction well and incubated at 95°C for 5 minutes and 60°C for 2 hours 601. The Rxn droplet is actuated to a magnet / resistive heater zone and fused with 33.3 μl of streptavidin beads and mixed at RT for 30 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded, the pellet is washed (not shown in the schematic diagram) first with a 50 μl buffer droplet preheated to 70°C, followed by a 50 μl buffer droplet preheated to 48°C, and the purified DNA library sample is eluted off the beads in 7.5 μl elution buffer 602. A 7.5 μl droplet of purified DNA library sample is then actuated to a PCR reaction well as described herein, fused with 0.83 μl primers and 8.3 μl master mix (with 10 μl drop gloss), and incubated at 97° C. for 45 seconds, followed by 8 cycles of 97° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for 30 seconds, and finally 72° C. for 1 minute 603. The amplified DNA droplet is actuated to a magnet / resistive heater zone, fused with a 30 ul magnetic bead droplet, mixed, and incubated at RT for 5 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded, the pellet is washed twice with 25 μl 80% EtOH (not shown in schematic), and the DNA library is eluted in 30 μl elution solution 604.

[0451] Thus, in FIG. 60, the method for Twist exome target enrichment is performed on a cartridge as described herein with mechanical actuation of the cartridge surface. In FIG. 60, a droplet of DNA and hybridization mix 601 with a drop gloss is actuated as described herein to a reaction well and incubated at 95° C. for 5 minutes and at 60° C. for up to 4 hours. The Rxn droplet is actuated to a magnet / resistive heater zone, fused with streptavidin beads, and mixed at RT for 30 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded for waste, the pellet is washed (not shown in the schematic), first with a buffer droplet preheated to 70° C., followed by a 50 μl buffer droplet preheated to 48° C., and the purified DNA library sample is eluted off the beads in an elution buffer 602. The purified DNA library sample droplets are then actuated by mechanical actuation of the surface as described herein into the PCR reaction wells, fused with primers and drop gloss, and incubated at 97°C for 45 seconds, followed by up to 18 cycles of 97°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and finally 72°C for 1 minute 603. The amplified DNA droplets are actuated into the magnet / resistive heater zone, fused with the SPRI / Ampure magnetic bead droplets, mixed, and incubated at RT for 5 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded, the pellet is washed twice with 80% EtOH (not shown in the schematic), and the DNA library is eluted in an elution buffer 604.

[0452] Figure 61 illustrates the workflow for Aplicon-seq. For example, Figure 61 shows a side view schematic of the Ampliseq (2 primer pool) workflow on a cartridge as described herein. First, three droplets of 13.5 μl DNA, 9 μl HiFi mix, and 22.5 μl water are fused by mechanical actuation of the surface (e.g., using a stylus), mixed at RT for 5 seconds, and split into two equal droplets using a liquid handler (not shown in the schematic) 611. Second, each of the droplets is fused with a 5 μl unique primer droplet with a 45 μL drop gloss droplet, and the Rxn droplets (1&2) are actuated by mechanical actuation of the surface (e.g., using a stylus) to the PCR reaction well zone and incubated (99°C for 2 minutes, then 17 cycles of 99°C for 15 seconds and 60°C for 4 minutes) 611'. Next, Rxn droplets 1 & 2 are fused by mechanical actuation of the surface (e.g., with a stylus), actuated to the magnet / resistive heater zone, fused with a 4 μl FuPa reagent droplet, mixed, and incubated for 10 minutes at 50° C., 10 minutes at 55° C., and 20 minutes at 60° C. Second, 8 μl of switch solution, 4 μl of barcode adapter mix, and DNA ligase droplets are added to the Rxn droplet by mechanical actuation of the surface (e.g., with a stylus) and incubated for 30 minutes at 22° C., 5 minutes at 68° C., and 5 minutes at 72° C. 612. A 90 μl droplet of beads is then added to the Rxn droplet, mixed and incubated for 5 min at RT; the magnet is turned on until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 150 ul droplets of 80% EtOH (not shown in schematic), and the library is eluted off the beads in a droplet of 50 μL Platinum™ PCR SuperMix HiFi and 2 μL Equalizer™ Primers.

[0453] At 613, a 50 μl droplet of the purified library is actuated by mechanical actuation of the surface (e.g., with a stylus) into a PCR reaction well, incubated at 98° C. for 2 minutes, and repeated for 9 cycles of 98° C. for 15 seconds and 64° C. for 1 minute. Second, a 10 μL Equalizer Capture droplet is added to the Rxn droplet and mixed at RT for 5 minutes. At 614, the Rxn droplet is actuated into a magnet / resistive heater zone, fused with 6 μL of washed Equalizer™ beads, mixed, and incubated at RT for 5 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 150 μl of 80% EtOH (not shown in the schematic), and the DNA library is eluted in a 100 μl elution droplet.

[0454] Thus, as shown in the Ampliseq (2 primer pool) workflow in Figure 61, in 611, three droplets of DNA, PCR master mix, and water are fused by mechanical actuation of the surface (e.g., using a stylus), mixed at RT for 5 seconds, and split into two equal droplets using a liquid handler (not shown in the schematic). Secondly, 611', each of the droplets is fused with a unique primer droplet with a drop gloss, and the Rxn droplets (1&2) are actuated by mechanical actuation of the surface (e.g., using a stylus) to the PCR reaction well zone and incubated (99°C for 2 minutes, then 17 cycles of 99°C for 15 seconds and 60°C for 4 minutes). In step 612, first, Rxn droplets 1 & 2 are fused by mechanical actuation of the surface (e.g., with a stylus), actuated to the magnet / resistive heater zone, fused with the FuPa reagent droplet, mixed, and incubated for 10 minutes at 50° C., 10 minutes at 55° C., and 20 minutes at 60° C. Second, droplets of switch solution, barcode adapter mix, and DNA ligase are added to the Rxn droplet by mechanical actuation of the surface (e.g., with a stylus), and incubated for 30 minutes at 22° C., 5 minutes at 68° C., and 5 minutes at 72° C. Third, the droplet of beads is added to the Rxn droplet, mixed and incubated at RT for 5 minutes; the magnet is turned on until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 80% EtOH droplets (not shown in schematic), and the library is eluted off the beads in a droplet of Platinum™ PCR SuperMix HiFi and Equalizer™ Primers. At 613, first, a 50 ul droplet of purified library is actuated by mechanical actuation of the surface (e.g., with a stylus) into a PCR reaction well and incubated at 98°C for 2 minutes, followed by 9 cycles of 98°C for 15 seconds and 64°C for 1 minute. Second, the Equalizer Capture droplet is added to the Rxn droplet and mixed at RT for 5 minutes.At 614, the Rxn droplet is actuated to a magnet / resistive heater zone and fused with the washed Equalizer™ beads, mixed, and incubated at RT for 5 minutes; the magnet is actuated until a pellet is formed, the supernatant is discarded to waste, the pellet is washed twice with 80% EtOH (not shown in schematic), and the DNA library is eluted into the elution droplet.

[0455] Liquid introduction and removal 62A-62J illustrate an example of a method of applying a liquid (droplet) into a cartridge, such as the cartridge described herein. For example, in FIG. 62A, a partial cross-sectional view through the cartridge shows an opening into which a pipette tip can be inserted. A standard pipette tip may be used. As an initial step, a droplet of the drop gloss material (as described above) may be pipetted into the air gap of the cartridge. For example, about 10-45 μL of the drop gloss may be pipetted into the air gap, and the pipette tip may be removed (FIG. 62B). Next, a droplet of an aqueous reactant may be inserted into the air gap in the same manner, as shown in FIG. 62C. The droplet may be pipetted onto or adjacent to the drop gloss (which is added first). In some instances, the drop gloss may be combined with the droplet before it is pipetted together with the droplet. Alternatively, the drop gloss may be added after the aqueous (reaction) droplet is added. In general, liquid materials may be introduced by pipette tips, which may use unique applications (dedicated single tip / sample) or universal applications (shared tips for multiple dispenses) to introduce reagents across one or more lanes of the cartridge. In Figure 62C, any volume of aqueous reaction mixture may be used, such as from about 250 nL to 80 μL.

[0456] In Figures 62C-62D, the pre-dispensed drop gloss encapsulates the aqueous reagents to protect them from surface contamination and evaporation during the workflow steps. Ethanol and wash buffers do not require a drop gloss. The volume of the drop gloss may be more, less, or the same as the volume of the aqueous droplet. In some examples, as shown in Figures 62A-62D, reaction droplets of very small volumes (e.g., as small as 250 nL) can be manipulated because the volume of the drop gloss may exceed the volume of the reaction droplet (e.g., 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 1-10x, 1-8x, 1-7x, 1-6x, 1-5x, 1-4x, etc., or more); the reaction droplet may be combined with excess drop gloss that encapsulates it and allows it to be manipulated (moved, combined, split, heated, mixed, etc.) as described herein even in channels with relatively large channel heights (e.g., 1.5 mm or more). Thus, in some examples, the systems described herein can dispense reagents / master mixes / samples in volumes as small as 250 nL and as large as 80 uL. In some examples, for drop gloss, the system can dispense volumes between 10-45 uL. The lanes illustrated in some of these examples can accommodate total volumes (drop gloss + reagent) of up to 150 uL or reagent volumes of up to 80 uL. Larger or smaller lane widths and / or heights may be used.

[0457] As shown above, during introduction of liquid into the inlets of each lane, the dispensing tip is lowered (straight down) against the lower film surface in a position that ensures that a portion of the droplet will be inside the channel (due to the inherent wetting properties of the liquid) upon dispensing. Capillary pressure may pull the droplet into the air gap and away from the opening (or to the edge of the opening) so that the droplet can be manipulated as shown in Figures 62E-62G. Thus, for a small volume such as 250 nL, a pre-dispensed droplet of carrier drop gloss (e.g., about 5-10 ul) may be used into which the 250 nL droplet is dispensed, and then a compressive force is applied to one side of the port to pull the droplet of carrier drop gloss containing that small volume. For example, as shown in Figs. 62E-62F, a mechanical manipulator (stylus) may be lowered to the elastically deformable upper sheet to reduce the height (on or, more preferably, adjacent to the droplet), and then the mechanical manipulator (stylus) may be drawn across the surface of the upper sheet to move the droplet surrounded by the drop gloss, as shown in Fig. 62G. As shown, the reagent is protected inside the droplet of the drop gloss. In Fig. 62F, the stylus presses against the film surface adjacent to the inlet hole (at a safe distance to avoid contamination of the stylus). The drop gloss / reagent droplet is then drawn into the narrower gap (by capillary action, including increasing capillary forces), and the droplet is then fully inserted into the lane, with its entire surface sandwiched between the upper and lower films. To perform different protocol steps, the stylus continues to drive the two-phase mixture (e.g., the drop gloss and the aqueous droplet) across the heating / cooling zones and / or magnet / isothermal heater zones.

[0458] Figures 62H-62J illustrate the removal of a droplet from a cartridge. The drop gloss material may first be removed from the droplet, for example, by contacting with an oleophilic material that may wick the drop gloss material, mechanically separating, etc. Alternatively, the droplet may contain the drop gloss along with an aqueous material. In Figure 62H, the stylus pulls a reaction droplet (e.g., containing a product library or other reaction products, as shown and described above) proximate to an opening (e.g., an inlet hole, etc.) through the upper sheet and into the air gap, at a safe distance to avoid contamination of the stylus. This is illustrated in Figure 62I. The droplet to be removed is near, but not at, the opening into the air gap. However, since the upper sheet is formed of an elastic material, it may be deformed by the pipette top for access, as shown in Figure 62J. In this example, the pipette tip is inserted into the inlet hole (Figure 62I) to reach a position above the bottom film. The pipette tip is then moved towards the droplet (Figure 62J), which temporarily deforms the top sheet (film) until the pipette tip reaches the location of the sample (so that the droplet is only partially sandwiched between the top and bottom sheets); the droplet is then aspirated into the pipette until it is completely removed or until a specific volume has been removed. The pipette tip is then moved back to the inlet hole opening and then raised and moved to the product destination (e.g. tube / plate) so that the operator can collect the material at the end of the run.

[0459] Evaporation Control Generally, these methods and devices may be configured to prevent or reduce evaporation. Generally, the drop gloss coating of the aqueous material, alone or in combination with applying a force (e.g., mechanical force) to the droplet, may provide both enhanced heating uniformity and prevention of evaporation. For example, in some variations of the methods and devices described herein, the aqueous droplet may undergo less than 10% evaporation (e.g., less than 9%, less than 8%, less than 7%, less than 6%, etc.) when heated to 95 degrees Celsius or higher for at least 30 minutes. In one example, a droplet (20 μL in a 45 μL drop gloss) of an aqueous reaction mixture heated to 95° C. for 30 minutes experienced a total of approximately 5.8% evaporation.

[0460] FIG. 63 illustrates an example where a droplet (e.g., Drop Gloss+aqueous droplet) is held in a reaction well formed in the lower layer by adapting the lower layer (which is elastically deformable like the upper layer) to form a well in the air gap upon application of suction force. Thus, the base of the drive system that holds the cartridge, including the mounting area, is shaped to form a well when the lower layer is attached to the mounting area by suction. This well is also a thermal control area that includes a heater to control the temperature of the droplet in the air gap. In FIG. 63, the droplet was heated to 95° C. for 40 minutes (20 μL in 45 μL Drop Gloss). A stylet (shown in this example as a roller stylus) may be held above the droplet, above the top of the sheet. This applied mechanical force may hold or pin the droplet in position relative to the heating area. This may also help insulate the droplet. In some examples, the portion of the stylet above the droplet may be thermally insulating. In some examples, the lower layer (sheet) may be more heat tolerant than the upper layer (sheet).

[0461] Thus, the methods and devices described herein may provide surprisingly good protection against evaporation in the air gap compared to other microfluidic systems.

[0462] cartridge Any of the cartridges described herein may be adapted to enhance one or more features, including droplet retention, isolation, and / or metering. For example, any of the methods and devices described herein, including the cartridges, may include one or more functional regions and / or may be used with structures (e.g., particles; including, but not limited to, magnetic particles or beads) that are functionalized, such as by attaching or linking a binding substance. The binding substance may be an antibody or a portion of an antibody (e.g., an antibody fragment) for binding or linking to a cell, particle, polynucleotide, etc. For example, the cartridge may include an antibody or an antibody fragment immobilized on a surface or region of the cartridge (e.g., a lower surface or an upper surface in an air gap) for binding to a component in an aqueous droplet. Other functional substances may include polymers (e.g., which may be covalently attached to a surface or portion of a surface of the cartridge). In some examples, the substrate may be a polymer coating. The functional region may be localized to a region of the cartridge, including localization to a shape (e.g., a spot, a strip, etc.).

[0463] Any of the methods and devices (e.g., cartridges) described herein may also or alternatively include a material in the air gap region to filter, absorb, or retain droplets or materials within the droplets. For example, the methods and devices described herein may include a filter material, such as a porous filter material, that may be added to the air gap. Examples of filters include filter paper, such as a semi-permeable paper barrier, that may be positioned in the air gap. These filters may be compressible so that the droplets can be pulled into and through the filter using the mechanical actuators described herein. In some examples, the filter material may be a stack of filter material that can be compressed when the upper and / or lower surfaces are elastically deformed by the mechanical actuator. In some examples, the filter material is connected to the upper and / or lower surfaces so that the filter material can deform and restore with the surfaces when the upper and / or lower surfaces are elastically deformed. In some examples, the filter material may include a stack that includes filter paper or filter material. In some examples, the filter may be disposed transversely to the air gap (and / or attached to the upper and / or lower surfaces). In some examples, the filter may have a predetermined grad or pore size. Any suitable pore size may be used. The filter may be a paper filter material, a glass fiber filter material, a quartz fiber filter material, a PTFE filter material, or the like. For example, any of these devices (cartridges, unitary devices) may include one or more filters for purification, extraction, separation, or the like. For example, any of these devices may include one or more filters for blood sample filtration, including filters or filtration regions for separating plasma from a blood sample, extracting cells such as red blood cells, or the like. Filtration may be passive, e.g., based on pore size, and / or active, e.g., based on functional regions (one or more binding agents on / in the filter).

[0464] In use, droplets may be driven by mechanical actuation to and / or through the filter material. In some cases, the droplets may be filtered leaving behind components, including liquid components, solid components, and / or both liquid and solid components. The controller may be configured to cause a mechanical actuator (e.g., a stylet) to slow down, compress more or less, etc., to filter one or more droplets through the filter material.

[0465] In some examples, the device (e.g., cartridge) may include a sponge-like material within the air gap that can hold and / or release droplets. The sponge material may be of the open-cell type.

[0466] As discussed above, any of these devices (e.g., cartridges) may include absorbent materials that absorb the aqueous and / or non-aqueous components of the droplets. For example, the cartridge may include absorbent materials (e.g., hydrophobic or adsorbent materials that preferentially absorb oil and may be water repellent) for wicking or removing the gloss coat. In some examples, the cartridge may include materials (e.g., hydrophilic materials) that preferentially absorb aqueous materials but not the drop gloss. Alternatively, in some examples, the cartridge may include materials (such as sponge-like materials) that absorb and / or retain both the gloss coat and aqueous materials. In some examples (as shown in Figures 45F-45G), the cartridge may include an absorbent pad that can wick waste into itself and retain materials to prevent dripping or crowding of the air gap.

[0467] In some examples, the cartridge may include dried or lyophilized substances on or within the air gap, including coating all or a portion of the upper and / or lower surfaces. For example, one or more reagents may be coated or included within the air gap. In some examples, lyophilized buffer substances, such as salts, chelators (such as calcium chelators), surfactants, etc., may be coated on one or more regions of the cartridge surface. In some examples, the cartridge may include a pre-formulated master mix (e.g., including one or more lyophilized substances) that can be rehydrated prior to addition to the droplets and / or by the droplets. The mix may include, for example, buffers, enzymes, etc.

[0468] Any of the cartridges described herein may be configured or adapted to meter droplets to release smaller, predefined volumes of droplets from larger volume droplets. In some cases, the device may include one or more metering regions. These regions may be calibrated, e.g., based on their size (surface area), to operate with a mechanical force actuator (e.g., a stylus, wand, roller, etc.) to separate a predefined volume of liquid from the droplet, typically forming a smaller droplet. For example, the cartridge may include one or more hydrophilic metering zones, as illustrated in Figs. 66A-66B. As illustrated in Fig. 66A, one surface of the device facing the air gap (e.g., the lower inner surface) may include one or more metering pads formed with a hydrophilic coating. The metering pads may be sized to leave behind a predefined volume of aqueous liquid when actuated on the metering pad by a mechanical actuator (e.g., a stylus, roller, etc.). 66A-66B illustrate how hydrophilic metering pads or patches 6601, 6603, 6605 can be used to meter an aqueous volume. For example, in FIG. 66A, a larger aqueous droplet 6607 may be pulled across the hydrophilic metering pads 6601, 6603, 6605 and leave a predetermined volume (droplet) behind as shown in FIG. 66B. In some examples, the metered droplets 6611, 6613, 6615 may be the same or different sizes (volumes). The metered droplets may be actuated off the metering pad by adding a gloss coat material (e.g., a hydrophobic coating material) and / or by moving a mechanical actuator nearby. In some examples, the mechanical actuator may be controlled to vary the amount of deformation such that the temporary capillary force pulling the droplet toward the deformation region is increased beyond the force holding the droplet to the metering pad.

[0469] In general, the devices, including the cartridges described herein, may be configured to culture cells or tissues on or within the cartridge for short periods of time (e.g., minutes, hours) or for longer periods (tens of hours, days; e.g., 8 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 24 hours or more, 2 days or more, 3 days or more, etc.). In some examples, the cells may be maintained suspended in an aqueous medium. In some examples, the cells may adhere to a specific substrate (e.g., beads). In some examples, the cells may be clusters or groups (e.g., organoids). Thus, any of these devices may be adapted to handle living cells or tissues; for example, the cartridge may include one or more regions (e.g., wells, chambers) that may be part of or adjacent to the air gap region, and may be accessible for transporting droplets in and out, for example, by including at least one surface (e.g., upper and / or lower surface) that is elastically deformable, and / or an elastically deformable wall formed within the air gap. The mechanical microfluidic device may be configured to form a well or chamber (e.g., by applying a suction force to fix a deformable surface (e.g., a bottom surface) in the shape of a well or chamber). The mechanical microfluidic device may further include temperature control, humidity / gas (e.g., CO2) control, etc., to maintain the air gap, and thus the droplet holding the cells and / or tissue, at the appropriate temperature, pH, etc. required for sustaining the cells and / or tissue.

[0470] Any of these devices may include one or more features (e.g., micro- or nano-features) that may capture particles (e.g., beads, cells, etc.), which may be useful for sorting, separating, and / or modifying particles. For example, Figures 67A-67B illustrate the use of nano- or micro-features to capture particles and / or cells (groups of cells, or in some examples, single cells). As shown in Figure 67A, a droplet 6705 containing particles (e.g., beads, cells, tissue, etc.) may be moved within the air gap by a mechanical actuator deforming a surface as described herein and forced through a constricted area of ​​the air gap; the constricted area may form, for example, a channel or lane including sidewalls 6703, which include one or more trapping features 6707 for filtering particles, such as beads or biological contents, e.g., cells. The walls and / or trapping features within the air gap may be formed of a material that is also elastically deformable. The walls may be formed of or coated with a non-absorbent material. The walls and / or trapping features may be hydrophobic (e.g., coated or formed with a hydrophobic material). The walls and / or trapping features may be functionalized to retain particles. As shown in FIG. 67B, one or more particles may be captured 6711 as shown after being driven through a lane / channel by a mechanical actuator as described herein. The particles may be removed from the droplet 6705′ or recaptured by passing the droplet through the lane / channel in the opposite direction. Thus, this method and structure may also be used to meter a specific number and / or size of particles.

[0471] Any of the devices (e.g., cartridges and / or integrated systems) that include an air gap with one or more elastically deformable surfaces as described herein may include regions of channels or lanes. These channels or lanes may be arranged to provide a linear flow of motion for the droplets (e.g., a flow cell); the air gap may be arranged as a linear channel with different regions arranged sequentially; the droplets may be pulled either backwards or forwards along the linear channel in the air gap (as described above). Multiple parallel channels may be included to allow parallel processing of droplet "samples." As described above, these channels may include a center or central rail region to prevent or restrict the droplets from contacting the sides of the channel; such contact may restrict or limit movement and / or cause some loss of liquid from the droplets.

[0472] In any of these devices, the channels may include regions of different widths, such as channels of different widths; this may help sort or group particles within the droplet, similar to the effect shown in Figures 67A-67B.

[0473] In general, the cartridges described herein may be adapted for use with one or more accessory components that may be present in or proximate to the air gap such that one or more droplets may be added / removed using a mechanical actuator by deforming one or more of the surfaces of the air gap (upper and / or lower surfaces) to alter the temporary capillary forces to allow for the pulling of the droplets in the air gap. For example, any of these devices may include an integrated gel electrophoresis area in communication with (or within) the air gap. A sample droplet containing a substance to be electrophoresed may be pulled to the gel, and a current may be applied to run the electrophoresis.

[0474] Any of these devices may also include one or more sensors integrated with or in communication with the air gap. For example, any of these cartridges may include or be adapted to be used with a sensor. As described above, the cartridge may be adapted for optical imaging of the air gap region, including optical imaging through an upper and / or lower surface, one or both of which may be optically transparent. The cartridge may include an optically transparent region (e.g., a viewing window). In some examples, the device includes an opening that forms an optical path or through which a sensor can be inserted.

[0475] For example, Figures 68A-68B illustrate a cartridge including an opening 3805 into the air gap 6801 for a sensor 6803. The opening may be through a sidewall of the cartridge or through a top or bottom surface, including an elastically deformable surface, of the cartridge. In Figure 68A, a droplet 6807 in the air gap may be actuated by applying a mechanical force to elastically deform the upper and / or lower surface of the air gap in front of the droplet, which pulls the droplet by temporary capillary forces into contact with the sensor as shown in Figure 68B. In some examples, the devices and methods described herein may be adapted for use in combinatorial chemistry, including combinatorial synthesis. In these examples, the device may include a sensor configured as a biosensor. Droplets of combinatorial synthesis may be pulled (by temporary / dynamic capillary forces as described herein) to a biosensor, such as a biosensor strip, for detection of smaller molecules and fast kinetic rate monitoring. Any suitable sensor may be used, including optical sensors (white light, UV light, fluorescent, etc.) with one or more lenses, filters, etc.; chemical sensors (including biosensors); electrochemical sensors (which may include one or more electrodes used as transducers to detect the presence or absence of an analyte); and the like.

[0476] As mentioned, any of the cartridges described herein may include one or more reagents as part of the cartridge, including a dry (lyophilized) reagent that can be hydrated by applying one or more liquid droplets; or a reservoir that is in communication with the air gap and is in close proximity to or includes an elastically deformable surface that can be actuated by a mechanical actuator (e.g., a stylus, roller, etc.) to apply a temporary capillary action force. The reagent may be included as a gel, sphere, powder, etc., and may be hydrated or released during an action on the cartridge for hydration. In some examples, the reservoir may include a liquid component (e.g., an aqueous material, a gloss coat material, etc.). The reservoir may be in or in close proximity to the air gap, and may be opened or released by a mechanical actuator. In some examples, the mechanical actuator may crush, break, or otherwise disrupt the reservoir to release the material. For example, the air gap may contain a packet or pocket of liquid components that may be released by a mechanical actuator (e.g., a stylus, roller, etc.) that applies pressure to burst the packet / pocket; the packet / pocket may be configured to burst in a predetermined manner (e.g., by including perforations / thinned areas on an outer surface of the packet / pocket).

[0477] Devices including the cartridges described herein may be loaded manually or automatically. As described above, loading may be performed by a pipette, or an array of pipettes, or other suitable means. In some examples, loading may be performed by a syringe or microsyringe. One or more plungers may be used to draw fluid into the air gap. As shown in Figures 62A-62J above, fluid may be added and / or removed from the air gap through openings in the upper and / or lower surfaces of the air gap, including openings through the elastically deformable material (sheet) forming the surface. Alternatively or additionally, fluid may be added and / or removed from a side port into the cartridge.

[0478] operation The devices and methods described herein may be configured to perform various operations on one or more droplets in the air gap region, which may be combined or chained together as part of a protocol. The operations may be automated and controlled by a controller of the device with direct or indirect user input. For example, the operations described herein may include controlling any of the components described above, including, among others, mechanical actuators (e.g., stylus, rollers, etc.), alone or in combination with controlling heating, magnetic fields, sonic / ultrasonic, electrical, light, etc., in the air gap, to manipulate the droplet or material within the droplet. Examples of these operations are described herein, and various steps and components of these operations may be combined, repeated, and / or reversed to implement a protocol. The devices described herein may generally include access to a local or remote library of operations, including commands (instructions) for controlling these devices. In some examples, the devices include wired or wireless communication circuitry that may be used to output data from the device (e.g., device status, droplet position, steps taken / to be taken, data output from one or more sensors, etc.) and / or to input control information (e.g., protocols) to the device. Any of these devices may include a user interface that allows a user to input, select, and / or modify protocols and / or operations.

[0479] For example, these devices may be configured to receive droplets into an inlet and to transport droplets away from the inlet. The devices may be configured to receive an aqueous material with a gloss coat material. This may be done as described above and illustrated in Figures 62A-62J. Alternatively or additionally, the devices may be configured to receive aqueous droplets without a gloss coat material. The droplets may be transported from the inlet to a starting location in the air gap.

[0480] The device may be configured to split or break up the droplets in the air gap as described above (e.g., FIGS. 3A-3C) and / or to dilute or otherwise adjust the concentration of the droplets by combining them with additional (e.g., diluting) droplets in the air gap as described and illustrated above (e.g., FIGS. 5A-5C). The controller may be programmed to perform each of these operations by controlling the position (x,y) and / or height (z) of the mechanical actuator. The device may also be configured to use the mechanical actuator to mix the droplets as described above, for example, by moving the mechanical actuator up / down on the droplets (pressurizing / releasing the droplets for chaotic mixing) and / or by moving the droplets back / forth (gentle mixing). In some examples, the device may pin the droplets in the air gap to perform an operation (e.g., for heating, applying magnetic forces, ultrasound, imaging, etc.). The droplet may be pinned by applying a gentle pressure (force) on the droplet by a mechanical actuator (e.g., pressing down to deform one or both elastically deformable surfaces / sheets of the air gap). In some examples, the droplet may be pinned by moving the droplet to a region of the air gap that includes one or more protrusions in the air gap (which apply a static capillary force); the protrusions may hold the droplet in place until it is moved by deflection of a nearby elastically deformable region (e.g., on the upper and / or lower surface) to move the droplet away by applying a dynamic capillary force on the droplet. In some examples, the device may be configured to move the droplet between regions of different thermal profiles, such as moving back and forth to perform rapid thermal cycling. The thermal control regions may be adjacent or nearby, and moving the droplet between them as described herein may be particularly useful.

[0481] As explained, the methods and devices described herein may be used with magnetic particles (e.g., magnetic beads) onto which substances may be linked (bound, immobilized, etc.). The magnetic beads may be suspended and mixed in aqueous droplets, carried to the bottom of a cartridge, washed / rinsed, resuspended, split / divided, fused, etc. Substances bound to the beads may be modified (e.g., by adding / attaching groups, etc.), cleaved, and / or eluted from the beads.

[0482] Any suitable size or number of magnetic particles may be used. Any of these methods and devices described herein may be configured for use with a single particle (including but not limited to a single particle). For example, in some variations, a single macroparticle (e.g., having a diameter larger than 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, etc.) may be used, to which a substance may be linked (and subsequently eluted). In some examples, a single cell or tissue portion may be processed.

[0483] For example, immobilization of magnetic particles for washing and separation is described herein. The systems and devices described herein can be used with magnetic particles with very little loss and very high efficiency. For example, the results of an assay using magnetic particles to process polynucleotide markers of various lengths (100 bp to 300 bp) and compared to manual polynucleotide processing techniques showing relatively high efficiency and repeatability are shown in Figures 69A-69B. For example, a table comparing the removal efficiency of 100 bp and 200 bp fragments using Ampure™ beads at a ratio of 0.7x, either prepared manually or using a system including a mechanical force applicator similar to that described above in Figure 43D, is shown in Figure 69A. In Figure 69A, a side-by-side comparison is made between the manual and mechanical microfluidic devices. In both cases, the DNA and Ampure beads were incubated, magnetically pelleted, and ethanol washed and eluted; these steps were performed on the bench (manual) or in the cartridge (MAOS). Figure 69B is a gel showing the results across triplicate bioanalyzer electropherograms. As shown, mixing and magnetic pelleting was performed efficiently using the mechanical microfluidic device, with efficient removal of 100bp and 200bp compared to manual preparation. Overall, the consistency of results was significantly better with the mechanical microfluidic device than with manual, as evidenced by the lower standard deviations.

[0484] In any of these methods and devices, multiple magnetic fields may be applied and sub-droplets may be dispensed with the magnetic beads. The beads (particles) may be isolated and bound materials may be removed, for example by washing and elution. In general, the beads may be functionalized in any useful manner. The particles (beads) may be repeatedly pelleted and resuspended, and washed while the beads are pelleted to remove all or nearly all of the fluid. The particles may be magnetically pelleted onto the lower surface of the cartridge, and the fluid may be removed by pulling the droplets off the pellet; the step of pulling the droplets is performed by moving the force applicator to deform the elastically deformable surface to dynamically / temporarily increase the capillary transfer force, and moving the force applicator away from the pellet. The washing and / or elution droplets may then be moved (using a mechanical force applicator) onto the pellet to resuspend the pellet.

[0485] Assay The devices and methods described herein may be used to perform one or more assays to qualitatively evaluate or quantitatively measure the presence, amount, or functional activity of a target entity. These assays may be qualitative (pass / fail), semi-qualitative, quantitative, or functional. Examples of these assays include enzyme assays, detection of one or more biomarkers (e.g., immunoassays, reporter assays, etc.), nucleic acid assays, B-gal assays, enzyme-linked immunosorbent assays (EILSA), etc. Thus, these devices may include or have access to protocols for performing various assays within the air gap; protocols include adding reporter molecules and washing, amplifying the signal (e.g., by adding enzymatic amplification components, polymerases, etc.), applying and detecting markers (e.g., fluorophores, etc.). These devices and methods may also be used for highly accurate generation of standard curves (e.g., dilutions), such as serial dilutions.

[0486] As mentioned above, in some examples, the methods and devices described herein may be used with cultured cells, including performing assays on cultured cells. The devices may control temperature, gas (air, CO2, etc.), etc.

[0487] Generally, the cartridges described herein may be adapted by including one or more functional regions (solid phase regions) for binding (directly or indirectly) to targets or the assay may be performed in conjunction with particles (including magnetic particles).

[0488] Also described herein are methods and devices for operating on polynucleotides (e.g., DNA, RNA, etc.). For example, these methods and devices may be used for amplification, including but not limited to polymerase chain reaction (PCR), rolling circle amplification, loop-mediated isothermal amplification (LAMP), ligase chain reaction, transcription-mediated amplification, etc. These methods and devices may be used for sequencing, including but not limited to next-generation sequencing (whole genome sequencing), targeted sequencing, whole exome sequencing, hybridization capture, amplicon sequencing, Sanger sequencing, fragment sequencing, etc. These methods and devices may be used for detection of polynucleotides and / or nucleic acids.

[0489] As described in the examples above, the methods and devices described herein may be used for library preparation for sequencing (e.g., NGS). Figures 70A-70O illustrate another example of a method of using a mechanical microfluidic device to prepare a library. In this example, a cartridge 7001 is shown mounted on a mechanical microfluidic device as described above. For example, in Figure 70A, the device is used to synthesize a first strand from a sample. The fragmented RNA and first strand sample "synthesis master mix" is pipetted 7001 (using the same pipette tip) to dispense into 5 μL droplets of gloss coat in each lane. Multiple parallel lanes may be used. Using a unique pipette tip for each sample, 2 μL of the fragmented RNA and first strand "synthesis mix" may be added into each corresponding lane. A mechanical actuator (e.g., stylus / roller) may then be used to pull the droplet of gross / RNA / first strand synthesis mix to the last heated zone 7004 by deforming the elastically deformable upper surface (sheet) to pull the droplet. The last heated zone is located at the far end of the cartridge length, as shown in FIG. 70A. The droplet may then be incubated at 25° C. for 10 minutes, at 42° C. for 15 minutes, and at 70° C. for 15 minutes (e.g., first strand synthesis incubation). Approximately 5 minutes before the end of the 70° C. step, the system may begin the second strand synthesis step FIG. 70B. For second strand synthesis, 10 μL of gross coat is pipetted into each lane (the same tip may be used). A new tip may be used to dispense 6 μL of second strand synthesis master mix into each lane 7006. The system may then drive a mechanical actuator (stylus / roller) to deform the elastically deformable upper surface (sheet) to pull the droplet of the gross / second strand synthesis mix by temporary and / or dynamic capillary action forces to the last heated zone 7003 for merging and mixing with the first strand synthesis. This fused droplet may then be incubated at 16° C. for 60 minutes (second strand synthesis incubation).Ten minutes prior to the end of the 16° C. step, the system may begin cleanup of the cDNA beads as illustrated in FIGS. 70C and 70E. During all of these incubation steps, the device (system) may control the temperature of the wells 7003 holding the droplets. The wells were formed as described above by applying a suction force that was maintained to deform the lower elastically deformable sheet of the cartridge to conform to the base of the device.

[0490] As shown in Figures 70C-70E, cleanup of the cDNA beads may include first pipetting (manually or automatically) 10 μL of gross coat into each lane, then dispensing 11.2 μL of suspended Ampure™ XP beads into each lane 7008. Discard the tips.

[0491] The system may then drive a mechanical actuator (stylus / roller) to deform an elastically deformable upper surface (sheet) to pull the droplet of gross / bead mix by temporary and / or dynamic capillary action forces to the magnetic zone 7006 at the lower end of the cartridge length. The mechanical actuator may then be lifted off the bead mix and lowered onto the cDNA products in the heater 7003 previously used to deform the upper surface to pull the cDNA droplet to the magnetic zone for merging with the beads and mixing thoroughly by tapping (e.g., by moving the actuator up and down). The device may then be incubated at room temperature (RT) for 5 minutes to allow the DNA to bind to the beads. At the end of the incubation, the system may activate the magnet to pellet the magnetic beads. The pellet may then be washed, for example, by pipetting 25 μL of 80% EtOH 7009 into each lane. The system may drive a mechanical actuator (stylus / roller) to deform an elastically deformable upper surface (sheet) to pull the droplets off the pellet and deliver the supernatant from the pelleted beads to a waste absorbent pad at the lower end of the cartridge length. The mechanical actuator may then be elevated and moved in close proximity to the 80% EtOH so that it can be used to elastically deform the upper sheet and move the deformation region to the magnetic zone to pull the 80% EtOH to rinse the pelleted beads for 30 seconds. These steps (3e-3f) may be repeated one or more times.

[0492] As illustrated in FIG. 70E, 10 μL of gross coat may then be pipetted into each lane, and 6 μL of elution buffer 7010 may be added into each lane. The system may then use a mechanical actuator to pull the gross coat / elution mix to the magnetic zone 7006 and to bring the pelleted beads back into suspension through tapping (e.g., chaotic mixing). The droplets may then be incubated at room temperature for 5 minutes. At the end of the incubation, the system may activate the magnet to form a pellet. The system may move the mechanical actuator to remove the supernatant from the pelleted beads to the next (closer to the inlet) heater zone 7012. Since that magnet zone will not be reused in the workflow, the beads may remain there pelleted, and waste from the next step may be flushed over it or resuspended and driven to an absorbent waste pad.

[0493] The preparation may be completed by adding Endoprep Master Mix 7014 as shown in Figures 70F-70G. Using the same pipette tip, 5 μL of Gross Coat may be dispensed into each lane, and 1 μL of Endoprep Master Mix 7014 may be dispensed into each lane. A mechanical actuator may pull the Gross Coat / Endoprep Mix to the next available / unused heater zone (closer to the inlet as the workflow progresses). A mechanical actuator may mix the elution products by tapping. The mix may be incubated at 20°C for 30 minutes and at 65°C for 30 minutes. Five minutes before the end of the 65°C step, the system may start as shown in Figure 70G.

[0494] 5 μL of gloss coat may be dispensed 7016 into each lane along with 3.1 μL of ligation master mix. A mechanical actuator may pull the gloss coat / ligation to the same heater where the end prep incubation took place. 5 μL of gloss coat may be dispensed into each lane. 250 nL of barcoded adapters 7018 may be pipetted into each corresponding lane, and the mechanical actuator may deflect the elastically deformable sheet to pull the gloss coat / adapter mix to the same heater where the end prep incubation took place. The system may mix the ligation master mix / end prep product and adapters by chaotic mixing using the mechanical actuator.

[0495] Ligation incubation may be performed at 20° C. for 15 minutes. Five minutes before the end of the 20° C. step, the system may start the post-ligation bead cleanup (0.8X) step shown in FIGS. 70I-70J. 10 μL of gross coat may be pipetted into each lane, and 7.28 μL of Ampure™ XP beads 7020 may be pipetted into each lane in suspension. A mechanical actuator may be driven by the controller to pull the gross coat / bead mix droplet to the next available / unused magnetic zone (closer to the inlet as the workflow progresses). The mechanical actuator may be lifted off the bead mix and moved down to contact the ligation products in the previously used heater and used to pull the droplet to the magnet zone to fuse with the beads and mix thoroughly through chaotic mixing (e.g., by moving the mechanical actuator). A 5-minute incubation may be performed at room temperature (RT) to allow the DNA to bind to the beads. At the end of the incubation, the system may activate the magnet (to form a pellet). The pellet may then be washed as shown in FIG. 70J. For example, 25 μL of 80% EtOH 7022 may be dispensed into each lane. A mechanical actuator may pull the supernatant from the pelleted beads to a waste absorbent pad at the bottom end of the cartridge length 7011. The mechanical actuator may then pull 80% EtOH to the magnetic zone to rinse the pelleted beads for 30 seconds. These steps (6e-6f) shown in FIG. 70J may be repeated one or more times.

[0496] As shown in FIG. 70K, 10 μL of gloss coat may be pipetted into each lane, and 6 μL of primer 7024 may be pipetted into each lane. The mechanical actuator may pull the gloss coat / primer mix into the magnetic zone, and the pelleted beads may be mixed back into suspension through chaotic mixing (lifting and tapping the mechanical actuator on the droplets). Incubate for 5 minutes at room temperature. At the end of the incubation, the controller may activate the magnet to form a pellet.

[0497] A mechanical actuator may then pull the supernatant from the pelleted beads to the next heater zone (closer to the inlet / outlet), where the beads may remain pelleted since that magnet zone will not be reused during the workflow, and waste from the next step may be poured over it or resuspended and driven to an absorbent waste pad.

[0498] FIG. 70 illustrates the steps of amplifying an adaptor-tagged library (USER / PCR reaction). 10 μL of gross coat may be pipetted into each lane, and 10.9 μL of premixed USER enzyme and Q5 PCR master mix 7030 may be pipetted into each lane. A mechanical actuator may then pull the gross coat / USER / PCR mix to the next available / unused heater zone (closer to the inlet / outlet as the workflow progresses). A mechanical actuator may mix this droplet with the elution product (containing primers) by chaotic mixing. The system may incubate at 37° C. for 15 minutes, then heat to 98° C. for 30 seconds, then PCR cycle at 98° C. for 10 seconds, 65° C. for 75 seconds for up to 19 cycles, then extend at 65° C. for 5 minutes. PCR cycling may be done by controlling a single heater, or by moving the droplet between multiple heating zones (with a mechanical actuator).

[0499] Figures 70M-70O illustrate PCR bead cleanup (0.8X). 10 μL of gross coat may be pipetted into each lane along with 12.72 μL of Ampure™ XP beads 7032 in suspension. A mechanical actuator may pull the gross coat / bead mix to the next available / unused magnetic zone (closer to the inlet / outlet as the workflow progresses). The system may lift the mechanical actuator off the bead mix, lower it onto the PCR products in the previously used heater, and drive it to the magnet zone to fuse with the beads and mix thoroughly through chaotic mixing. The system may incubate for 5 minutes at room temperature (RT) to allow the DNA to bind to the beads. At the end of the incubation, the system may activate the magnet to form a pellet. The pellet may be washed by pipetting 25 μL of 80% EtOH 7033 into each lane as shown in Figure 70N. A mechanical actuator may drive the supernatant from the pelleted beads to a waste absorbent pad at the bottom end of the cartridge length. A mechanical actuator may pull 80% EtOH to the magnetic zone to rinse the pelleted beads for 30 seconds. The system may repeat Figure 70N (steps 8e-8f) one or more times.

[0500] The product (library) may then be eluted and removed as shown in FIG. 70O. 10-25 μL of elution buffer 7037 may be pipetted into each lane. A mechanical actuator may pull the elution buffer into the magnetic zone, which may then mix the pelleted beads back into suspension through chaotic mixing. The system may incubate at room temperature for 5 minutes. At the end of the incubation, the system may activate the magnet to form a pellet. A mechanical actuator may pull the supernatant from the pelleted beads (product library) into the collection zone 7035 and out of the cartridge.

[0501] The method and device described herein may be used, inter alia, to rapidly and efficiently carry out one or more diagnostic assays.For example, the cartridge described herein may be adapted to carry out a particular diagnostic assay.Examples of such diagnostic assays may include lysosomal storage disorder assay, enzyme detection assay, etc.

[0502] It should be recognized that all combinations of the concepts discussed above, and the additional concepts described in more detail below (provided such concepts are not mutually inconsistent), are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0503] The process parameters and sequences of steps described and / or illustrated herein are provided by way of example only and may be varied as desired. For example, the steps illustrated and / or described herein may be shown or discussed in a particular order, but the steps do not necessarily have to be performed in the order illustrated or discussed. The various exemplary methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein or may include additional steps in addition to those disclosed.

[0504] Any of the methods described herein (including the user interface) may be implemented as software, hardware, or firmware and may be described as a non-transitory computer-readable storage medium having a set of instructions stored thereon; the set of instructions may be executed by a processor (e.g., a computer, tablet, smartphone, etc.) and, when executed by the processor, cause the processor to control or perform any step, including, but not limited to, displaying, communicating with a user, analyzing, modifying, determining, or changing a parameter (including timing, frequency, intensity, etc.). For example, any of the methods described herein may be performed, at least in part, by an apparatus that includes one or more processors having a memory having a non-transitory computer-readable storage medium having a set of instructions for the process of the method stored thereon.

[0505] Although various aspects have been described and / or illustrated herein in the context of a fully functional computing system, one or more of these exemplary aspects may be distributed as a program product in various forms, regardless of the specific type of computer-readable medium used to actually effect the distribution. The aspects disclosed herein may also be implemented using software modules that perform specific tasks. These software modules may include scripts, batches, or other executable files that may be stored on a computer-readable storage medium or within a computing system. In some aspects, these software modules may configure a computing system to perform one or more of the exemplary aspects disclosed herein.

[0506] As described herein, the computing devices and systems described and / or illustrated herein broadly represent any type or form of computing device or system capable of executing computer-readable instructions such as those contained within the modules described herein. In their most basic configuration, these computing devices may each include at least one memory device and at least one physical processor.

[0507] As used herein, the term "memory" or "memory device" generally refers to any type or form of storage device or medium, volatile or non-volatile, capable of storing data and / or computer-readable instructions. In one example, a memory device may store, load, and / or maintain one or more of the modules described herein. Examples of memory devices include, but are not limited to, random access memory (RAM), read-only memory (ROM), flash memory, hard disk drive (HDD), solid-state drive (SSD), optical disk drive, cache, variations or combinations of one or more of these, or any other suitable storage memory.

[0508] Additionally, the term "processor" or "physical processor" as used herein generally refers to any type or form of hardware-implemented processing unit capable of interpreting and / or executing computer-readable instructions. In one example, a physical processor may access and / or modify one or more modules stored in the memory device described above. Examples of physical processors include, without limitation, a microprocessor, a microcontroller, a central processing unit (CPU), a field programmable gate array (FPGA) implementing a soft-core processor, an application-specific integrated circuit (ASIC), a portion of one or more of these, a variation or combination of one or more of these, or any other suitable physical processor.

[0509] The method steps described and / or illustrated herein, although illustrated as separate elements, may represent portions of a single application. Additionally, in some aspects one or more of these steps may represent or correspond to one or more software applications or programs that, when executed by a computing device, may cause the computing device to perform one or more tasks, such as the method steps.

[0510] Additionally, one or more of the devices described herein may transform data, physical devices, and / or representations of a physical device from one form to another. Additionally or alternatively, one or more of the modules presented herein may transform a processor, volatile memory, non-volatile memory, and / or any other portion of a physical computing device from one form of computing device to another form of computing device by executing on the computing device, storing data on the computing device, and / or otherwise interacting with the computing device.

[0511] The term "computer-readable medium" as used herein generally refers to any form of device, carrier, or medium capable of storing or carrying computer-readable instructions. Examples of computer-readable media include, but are not limited to, transmissive media, such as carrier waves; and non-transitory media, such as magnetic storage media (e.g., hard disk drives, tape drives, and floppy disks); optical storage media (e.g., compact disks (CDs), digital video disks (DVDs), and BLU-RAY disks); electronic storage media (e.g., solid-state drives and flash media), and other distribution systems.

[0512] Those skilled in the art will recognize that any process or method disclosed herein can be modified in many ways. The process parameters and sequence of steps described and / or illustrated herein are given by way of example only and can be varied as desired. For example, the steps illustrated and / or described herein may be shown or discussed in a particular order, but these steps do not necessarily have to be performed in the order shown or discussed.

[0513] The various exemplary methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein or may include additional steps in addition to those disclosed. Furthermore, a step of any method as disclosed herein may be combined with one or more steps of any other method as disclosed herein.

[0514] A processor as described herein may be configured to perform one or more steps of any of the methods disclosed herein. Alternatively, or in combination, a processor may be configured to combine one or more steps of one or more methods as disclosed herein.

[0515] As used herein, when a feature or element is referred to as being "on" another feature or element, it may be directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It is also understood that when a feature or element is referred to as being "connected," "attached," or "coupled" to another feature or element, it may be directly connected, attached, or coupled to the other feature or element, or there may be intervening features or elements present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" to another feature or element, there are no intervening features or elements present. Features and elements described or illustrated with respect to one embodiment may apply to other embodiments as well. It will also be appreciated by those skilled in the art that a reference to a structure or feature that is "adjacent" to another feature may have portions that overlap or underlie the adjacent feature.

[0516] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. For example, the singular forms "a", "an" and "the" used herein are intended to include the plural forms as well, unless the context clearly indicates otherwise. It is further understood that the terms "comprise" and / or "comprising" used herein specify the presence of a stated feature, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. The term "and" used herein includes any and all combinations of one or more of the associated listed items, and may be abbreviated as " / ".

[0517] Spatial relationship terms such as "under," "below," "lower," "over," and "upper" may be used herein for ease of description in describing the relationship of one element or feature to another element or feature as depicted in the drawings. It is understood that the spatial relationship terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the drawings. For example, if a device in the drawings is inverted, an element described as being "under" or "beneath" another element or feature would be oriented "over" that other element or feature. Thus, the exemplary term "under" may encompass both an above and below orientation. The device may be otherwise oriented (rotated 90 degrees or to other orientations) and the spatial relationship descriptors herein interpreted accordingly. Similarly, terms such as "upwardly," "downwardly," "vertical," and "horizontal" are used herein for descriptive purposes only, unless specifically indicated otherwise.

[0518] Although the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element described below may be referred to as a second feature / element, and similarly, a second feature / element described below may be referred to as a first feature / element, without departing from the teachings of the present invention.

[0519] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" mean that various components may be used together in the methods and articles (e.g., compositions and apparatuses, including devices and methods). For example, the term "comprise" is understood to imply the inclusion of any stated element or step, but not the exclusion of any other elements or steps.

[0520] In general, any of the apparatus and methods described herein should be understood as being inclusive, although, alternatively, all or a subset of the components and / or steps may be exclusive and may be expressed as "consisting of" or, alternatively, "consisting essentially of" various components, steps, subcomponents, or substeps.

[0521] All numbers used in this specification and claims, including those used in the examples, unless otherwise specified, may be read as if they are preceded by the term "about" or "approximately", even if the term does not explicitly appear. The phrase "about" or "approximately" may be used when describing a size and / or location to indicate that the described value and / or location is within a reasonable expected range of value and / or location. For example, a numerical value may have a value that is + / - 0.1% of the stated value (or ...

Claims

1. 1. A method for microfluidic manipulation of droplets, comprising: introducing the droplet into an air gap formed between an elastically deformable first sheet and a second sheet, the first sheet facing the second sheet at a distance from each other to form an air gap having a gap width of a predetermined distance in a neutral state; applying a compressive force to the first sheet using a mechanical force applicator to form a region of locally reduced gap width in the air gap adjacent to the droplet, thereby drawing the droplet toward the region of locally reduced air gap; and moving the droplet in the air gap by translating the mechanical force applicator along an outer surface of the first sheet, translating the region of locally reduced gap width in the air gap so that the droplet follows the mechanical force applicator. A method comprising:

2. 2. The method of claim 1, wherein the step of moving the droplet comprises moving the droplet along a rail region of the air gap, the rail region having a gap width that is smaller than a gap width of a region of the air gap surrounding the rail region.

3. 10. The method of claim 1, wherein the moving step comprises moving the droplet into a well formed by the second sheet.

4. The method of claim 3, further comprising controlling the temperature of the well.

5. 4. The method of claim 3, further comprising moving the droplet out of the well by translating the mechanical force applicator along an outer surface of the first sheet, translating a region of locally reduced gap width in the air gap away from the well, thereby pulling the droplet out of the well.

6. The method of claim 1 , further comprising modifying the droplet in the air gap.

7. 8. The method of claim 7, wherein the modifying step comprises one or more of reacting one or more substances in the droplet, heating the droplet, adding a substance to the droplet, and applying energy to the droplet.

8. 10. The method of claim 1, wherein the first sheet has a first hydrophobic and oleophobic surface positioned opposite a second hydrophobic and oleophobic surface of the second sheet.

9. The method of claim 1 , wherein the air gap is open to atmospheric pressure and is not pressurized.

10. 2. The method of claim 1, wherein applying a compressive force to the first sheet using the mechanical force applicator draws the droplet toward the area where the air gap is locally reduced by temporary capillary action.

11. 10. The method of claim 1, wherein the introducing, applying, and transferring steps are part of one or more of the following methods: nucleic acid extraction, library preparation, sequencing, and protein synthesis.

12. 10. The method of claim 1, wherein the tip of the mechanical force applicator has a rounded profile, a circular profile, an elliptical profile, a rectangular profile, or a square profile.

13. The method of claim 1 , wherein the tip of the mechanical force applicator comprises a roller.

14. The method of claim 1 , further comprising detecting light transmitted through or reflected from the droplet.

15. The method of claim 1 , further comprising applying a voltage to the droplet from the mechanical force applicator or from a region below the second sheet.

16. The method of claim 1 , further comprising attracting magnetic particles suspended within the droplet with a magnet within the mechanical force applicator or within a region below the second sheet.

17. 10. The method of claim 1, further comprising mixing the droplets by repeatedly applying and removing a compressive force with the mechanical force applicator.

18. 10. The method of claim 1, further comprising mixing the droplets by moving the mechanical force applicator relative to the first sheet in the plane of the first sheet.

19. 10. The method of claim 1, further comprising splitting the droplet by applying a pinning compressive force to the first sheet and applying an actuation compressive force to the first sheet proximate the pinning compressive force to elongate and split the droplet, the pinning compressive force being greater than the actuation compressive force.

20. The method of claim 1 , further comprising removing all or a portion of the droplet from the air gap through an opening in the first sheet.

21. The method of claim 1 , wherein the step of introducing the droplet comprises passing the droplet from the mechanical force applicator through an opening in the first sheet.

22. 1. A method for microfluidic manipulation of droplets, comprising: introducing the liquid droplet into an air gap formed between an elastically deformable first sheet and a second sheet, the first sheet facing the second sheet at a distance so as to form an air gap having a gap width of a predetermined distance in a neutral state, the air gap being open to atmospheric pressure and not pressurized, and the liquid droplet being positioned within a rail region of the air gap, the rail region having a gap width smaller than the gap width of a region surrounding the rail region; applying a compressive force to the first sheet using a mechanical force applicator to form a region of locally reduced gap width in the air gap adjacent to the droplet, thereby drawing the droplet toward the region of locally reduced air gap by temporary capillary action; and moving the droplet along the rail region of the air gap by translating the mechanical force applicator along an outer surface of the first sheet, translating the region of locally reduced gap width within the air gap, thereby pulling the droplet within the air gap. A method comprising:

23. 1. A method for microfluidic manipulation of droplets, comprising: introducing the droplet into an air gap formed between a first sheet that is hydrophobic, oleophobic, and elastically deformable and a second sheet that is hydrophobic and oleophobic, the first sheet facing the second sheet at a distance so as to form an air gap having a gap width of a predetermined distance in a neutral state, the air gap being open to atmospheric pressure and not pressurized; applying a compressive force to the first sheet using a mechanical force applicator to form a region of locally reduced gap width in the air gap adjacent to the droplet, thereby drawing the droplet toward the region of locally reduced air gap by temporary capillary action; and moving the droplet into a well formed by the second sheet by translating the mechanical force applicator along an outer surface of the first sheet, translating the region of locally reduced gap width in the air gap, thereby pulling the droplet within the air gap into the well; modifying the droplets in the wells; and moving the droplet out of the well by translating the mechanical force applicator along the outer surface of the first sheet, translating the region of locally reduced gap width in the air gap away from the well, thereby pulling the droplet out of the well. A method comprising:

24. A cartridge comprising a first sheet and a second sheet, the first sheet and the second sheet being fixed parallel and facing each other at a predetermined distance with an air gap therebetween; and a controller configured to selectively reduce the predetermined distance at one or more regions within the air gap proximate to the fluid droplet positioned within the air gap to move the fluid droplet within the air gap of the cartridge. A microfluidic device comprising:

25. 25. The apparatus of claim 24, wherein the controller is configured to selectively reduce the predetermined distance in the one or more regions within the air gap without reducing the predetermined distance in one or more proximal regions.

26. 25. The device of claim 24, wherein the first sheet comprises a first surface facing the air gap, the first surface being hydrophobic and oleophobic.

27. 25. The device of claim 24, wherein the second sheet comprises a second surface facing the air gap, the second surface facing the air gap being hydrophobic and oleophobic.

28. 25. The device of claim 24, wherein the cartridge further comprises at least one input port on the first sheet and / or the second sheet configured to introduce a first fluid droplet into the air gap.

29. 25. The apparatus of claim 24, wherein the controller is configured to apply a compressive force to the sheet to selectively reduce the predetermined distance.

30. 25. The device of claim 24, wherein the first sheet is configured to elastically deform in response to a compressive force.

31. 25. The apparatus of claim 24, wherein the apparatus further comprises a force applicator positioned to apply a compressive force to an outer surface of the first sheet, the controller directing movement of the force applicator relative to the outer surface.

32. 32. The apparatus of claim 31, wherein the force applicator comprises a stylus.

33. 32. The apparatus of claim 31, wherein the force applicator comprises a source of pressurized fluid.

34. 25. The device of claim 24, wherein the first sheet is elastically deformable.

35. 25. The apparatus of claim 24, wherein the cartridge comprises a frame to which the first sheet is tensioned to maintain the sheet parallel to and opposite the second sheet.

36. 25. The device of claim 24, wherein the second sheet comprises an elastic material.

37. 25. The device of claim 24, wherein both the first sheet and the second sheet comprise a resilient material that is held taut in a frame of the cartridge.

38. 25. The device of claim 24, wherein the controller is configured to apply a pinning compressive force to split the first fluidic droplet and an actuation compressive force near the pinning compressive force to elongate and form a second fluidic droplet from the first fluidic droplet, the pinning compressive force being greater than the actuation compressive force.

39. The controller: applying a compressive force to the sheet between two or more separate fluid droplets; and releasing the compressive force to combine the two or more separate fluid droplets into a single fluid droplet.

25. The device of claim 24, wherein the device is configured

40. 25. The apparatus of claim 24, wherein the controller is configured to apply alternating first compressive forces and second compressive forces different from the first compressive force to the sheet to mix two or more separate fluid droplets together.

41. 25. The apparatus of claim 24, wherein the controller is configured to mix two or more fluid droplets together by repeatedly applying and releasing a compressive force to the sheet in proximity to the two or more fluid droplets.

42. 25. The apparatus of claim 24, wherein the controller is configured to control the magnet to attract ferrous particles suspended within the first fluid droplet.

43. 25. The apparatus of claim 24, wherein the controller is further configured to resuspend the one or more ferrous particles in the fluid droplet by applying and releasing a compressive force to the fluid droplet and disabling the magnet.

44. 25. The device of claim 24, wherein the sheet further comprises two or more pinning posts disposed on the surface and extending into the gap, the pinning posts configured to restrict movement of the first fluid droplet.

45. 45. The apparatus of claim 44, further comprising a heater disposed beneath the second surface opposite the two or more pinning posts.

46. 25. The device of claim 24, wherein the cartridge further comprises a well configured to restrict movement of the fluid droplet.

47. 25. The device of claim 24, further comprising a base having a cartridge seat configured to secure the cartridge, wherein the second sheet is held against the cartridge seat with at least one region of the cartridge in communication with a heating element disposed below the seat and configured to heat the fluid droplets in the air gap.

48. 1. A method for manipulating one or more microfluidic droplets, comprising: a first sheet having a hydrophobic and oleophobic first surface; a second sheet having a hydrophobic and oleophobic second surface, the first sheet and the second sheet being fixed in parallel facing each other at a predetermined distance with an air gap therebetween; introducing a first fluid droplet into an air gap formed between the first and second fluid droplets; and applying a force to elastically deform the first sheet to reduce the distance of the air gap between the first sheet and the second sheet in a region within the air gap proximate the fluid droplet to move the fluid droplet within the air gap. A method comprising:

49. Cartridge mounting surface; a force applicator configured to contact an elastically deformable outer surface of the cartridge when the cartridge is placed on the cartridge placement surface and apply a compressive force to the elastically deformable surface of the cartridge; a force applicator driver configured to move the force applicator across the deformable outer surface; and a controller coupled to control the force applicator driver to move the force applicator relative to the deformable exterior surface of the cartridge to dynamically reduce the height of the air gap within the cartridge to move a fluid droplet within the air gap of the cartridge. A microfluidic device comprising:

50. 1. A method for moving droplets by microfluidics, comprising: introducing a fluid droplet into an air gap formed between a first elastically deformable sheet and a second sheet, the first sheet facing the second sheet in parallel and spaced apart a predetermined distance to form the air gap; and applying a compressive force to the elastically deformable sheet using a force applicator, thereby reducing the predetermined distance in at least one region within the air gap that is proximate to the fluid droplet, thereby displacing the droplet within the air gap. A method comprising:

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