Microfluidic two-dimensional capillary manipulation apparatus and method

Abstract

Methods and apparatus for controlled fluid handling may include a two-dimensional (planar) fluid chamber. The chamber may include a first sheet and a second sheet separated by a gap between them. The first and second sheets may be hydrophobic and oleophobic, or may include a hydrophobic and oleophobic coating.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to U.S. Provisional Patent Application No. 63 / 512,576, “MICROFLUIDIC TWO-DIMENSIONAL CAPILLARY MANIPULATION DEVICES AND METHODS,” filed on 7 July 2023, which is incorporated herein by reference in its entirety.

[0002] Inclusion by reference All publications and patent applications referenced herein are incorporated herein by reference in whole, just as each individual publication or patent application is specifically and individually indicated as being incorporated by reference. [Background technology]

[0003] background Microfluidics involves as little as 10¹⁶ liters of fluid. 15 We deal with extremely small amounts of fluid, down to femtoliters (fL), which is one-tenth of a liter. Fluids at the micrometer scale behave very differently than they do in everyday life: these unique characteristics are key to new scientific experiments and innovations. Microfluidic devices may require ultra-miniature 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 manipulating microscale fluid droplets simply and precisely. DMF has rapidly gained popularity for chemical, biological, and medical applications because it allows for direct control of multiple reagents (no pumps, valves, or tubing required); it can easily handle both solids and liquids (no clogging channels); and its hydrophobic surface (typically Teflon-coated) that contacts the fluid droplets is chemically inert, making it suitable for handling difficult-to-handle reagents (e.g., organic solvents, corrosive chemicals). However, conventional DMF devices use relatively large electric fields selectively applied to electrode arrays to manipulate droplets. Generating and controlling these electric fields requires specialized and complex circuits capable of withstanding relatively high voltages. [Overview of the project]

[0005] Summary of this disclosure Methods and apparatus for mechanically actinguating (e.g., moving, mixing, etc.) one or more droplets within an air gap are described herein. These methods may include one or more wells and one or more regions for cell culture (e.g., one or more hydrophilic regions), and methods for using any of these cartridges in one or more medical or medical-related procedures. For example, the methods described herein may include steps of amplifying polynucleotides, performing an assay, culturing cells (and performing an assay on the cultured cells), and so on.

[0006] Each of the first and second sheets 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 involve the use of a mechanical force actuator, which can be driven to locally deform the first elastically deformable sheet to form a localized region with a smaller gap height (e.g., thickness) close to the droplet, and the droplet can be pulled along the surface of the first sheet to move parallel to the region with the locally reduced gap height. The droplet follows the region with the smaller gap height in the air gap by capillary action.

[0007] In general, these methods and apparatus can be used to prepare, manipulate, and / or analyze fluid droplets, such as microfluidic droplets. For example, microfluidic apparatuses that may be particularly useful for handling and analyzing clinical, laboratory, biological, or chemical samples are described herein. These apparatuses can 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 on a sub-region of the air gap to reduce the height of the air gap in the region adjacent to the droplet and move the droplet toward this reduced-height region. By controlling the relative height of the air gap near the droplet (for example, by controlling the application of force to deform the elastically deformable sheet), the droplet can be moved efficiently and rapidly around the air gap, enabling droplet processing, including droplet bonding, droplet splitting, droplet mixing, droplet cooling / heating (e.g., droplet thermal cycling), and the use of magnetic particles within the droplet (e.g., for binding to / extracting substances from the droplet).

[0008] Apparatuses (systems, devices, etc.) for controlling microfluidic movement on a surface, such as droplet movement, by mechanical means are described herein. These apparatuses may be referred herein to as mechanical microfluidic actuation devices ("mechanical microfluidic actuators") and may include force applicators for applying force to an elastically deformable sheet that at least partially seals an air gap in which one or more droplets reside. The elastically deformable sheet may be part of the mechanical microfluidic actuator apparatus or may be part of a separate or integrated cartridge operated by the mechanical microfluidic actuator apparatus. In some examples, the cartridge may include a first (e.g., upper) elastically deformable sheet and a second (e.g., lower) sheet, held apart from each other (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). The mechanical microfluidic actuator 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 actuator 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 the cartridge base or mounting area of ​​the mechanical microfluidic actuator. Optionally, the device may include a vacuum / suction subassembly for securing a cartridge to the mounting area of ​​the mechanical microfluidic actuator. In some examples, the mechanical microfluidic actuator device may include a fluid handling (e.g., pipetting) subassembly for adding and / or removing fluid from the air gap.Other subassemblies forming part of a mechanical microfluidic actuator device may include an imaging subassembly (for example, for imaging droplets in an air gap) and / or a sensing subassembly (for example, for sensing droplets or other inputs from the air gap and the mechanical microfluidic actuator). The mechanical microfluidic actuator devices described herein may also include one or more control inputs (e.g., keyboard, touchscreen, button, switch, etc.) and / or one or more outputs (e.g., display, LED, wireless communication output / input, etc.), as well as hardware, software, and / or firmware for controlling them. In some cases, the same features may be used for the control inputs and outputs. Generally, the mechanical microfluidic actuators described herein may include one or more controllers for controlling and coordinating the operation of the various subassemblies.

[0009] For example, a microfluidic device described herein includes: a cartridge comprising a first sheet and a second sheet fixed facing each other at a predetermined distance and generally parallel to one another, with an air gap between them; and a controller configured to selectively reduce the predetermined distance in one or more regions within the air gap that are close to the fluid droplets positioned within the air gap, in order to move the fluid droplets 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 actuator 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 into the mechanical microfluidic actuator.

[0010] The second sheet may be elastically deformable or non-deformable. In some examples, the second sheet is made of the same material as the first sheet (e.g., an elastic material). The second sheet may be configured to be fixed (by suction) to the system holding the cartridge so that the cartridge has consistent thermal contact with the second sheet, so that the temperature can be changed rapidly and efficiently by heating / cooling a localized area (thermal control area) of the second sheet in order to heat / cool droplets in the air gap in the area above the thermal control area. In some examples, droplets may be moved and / or pinned by deforming the lower sheet to change the height of the localized area of ​​the air gap; alternatively or additionally, droplets may be moved and / or pinned by deforming the upper sheet to change the height of the localized area of ​​the air gap. The sheets may return to a neutral, non-deformable state so that the air gap returns to approximately the same predetermined distance when the force that deforms the sheets (either or both of the first and second sheets) is removed.

[0011] In any of these examples, the controller may 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. The sheet may have a first surface facing the air gap. Generally, one 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.

[0012] In any of these examples, the air gap may include at least one input / output port. For example, a 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, the 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. This cut-out portion (opening) may be 1 mm to 10 cm in length and 1 mm to 10 cm in width (e.g., 5 mm to 7 cm in length and 5 mm to 4 cm in width). The opening through the first sheet may be offset from the edge of the sheet by, for example, 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 surrounding the inlet / outlet. Therefore, tension may be applied to the edges of the inlet / outlet.

[0013] The air gap may generally be any suitable height ("thickness") when no force is applied to the sheet. 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 described herein (including, but not limited to, cartridges) 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 sheet to selectively reduce the predetermined distance.

[0014] For example, a microfluidic device comprising: a cartridge comprising a first sheet and a second sheet fixed facing each other at a predetermined distance and generally parallel to each other, with an air gap between them; and a controller configured to selectively reduce the predetermined distance in one or more regions within the air gap that are close to the fluid droplets positioned within the air gap, in order to move fluid droplets within the air gap of the cartridge.

[0015] 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 the first fluid droplet into the air gap.

[0016] The controller may generally be configured to selectively reduce a predetermined distance by applying (and moving) a compressive force across the sheet, thereby dynamically changing the height of the air gap to draw in droplets to follow the reduced height of the air gap as the compressive force moves across the sheet. This movement may be continuous or periodic.

[0017] Generally, the first (and / or second) sheet is configured to deform elastically in response to a compressive force. In any of these methods and apparatus, the first and / or second sheet may, depending on the circumstances, be partially or completely clear (e.g., optically transparent) or opaque. In some cases, both the first and second sheets are clear. In some examples, only the second sheet is clear. In some examples, only the first sheet is clear.

[0018] The sheet used herein includes at least one surface extending in a plane (for example, in the x, y directions). In some examples, the sheet may be relatively thin, as is the case with the first elastically deformable sheet. In some examples, the sheet may include regions of varying thickness. 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 varying thickness. In some examples, the second sheet may be elastically deformable. In some examples, the second sheet may be called a layer, plate, or base, etc.

[0019] Any of these devices may include a force applicator (e.g., a stylus in some examples) positioned to apply force to the outer surface of a first sheet in order to reduce the height of the air gap in a local area, where the controller may be configured to direct the movement of the force applicator relative to the outer surface (e.g., so 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.) and / or a thermal actuator (e.g., one that applies thermal energy to deform the sheet) and / or an optical actuator (e.g., one that applies laser light to deform the sheet). The force applicator may be contact-type or non-contact-type. In some examples, the force applicator may be equipped with a source of pressurized fluid.

[0020] As mentioned, at least one of the first or second sheets may be elastically deformable. In some examples, the first sheet may be called the upper sheet or upper sheet, and the second sheet may be called the lower sheet or lower sheet. The second sheet may also be called the plate in some examples.

[0021] In a device including a cartridge, the cartridge may include a frame on which a first sheet (and / or a second sheet) is stretched taut, maintaining the first sheet generally parallel to the second sheet. The frame may include a tensioner. In some examples, tension may be applied during manufacturing, and the sheets may be attached to the frame under tension (e.g., mechanically and / or chemically, e.g., by adhesive). The frame may surround the outer periphery of the cartridge. In some examples, the cartridge may be divided into areas such as lanes, chambers, separated by one or more walls (e.g., partitions). The partitions may be fixed (e.g., glued, welded, etc.) to the upper and / or lower sheets. In some examples, the frame and spacers are combined into a single component, which is referred to herein as a spacer frame.

[0022] The second sheet may be of the same material (e.g., an elastic material) as the first sheet or 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 in some examples be referred to as a plate. 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 pinned and held to the frame of the cartridge. The first and second sheets may be of any suitable thickness and may be of the same thickness or different thicknesses. For example, the first and / or second sheets may be from 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.).

[0023] 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.).

[0024] One of these devices may include a controller configured to operate a force applicator to apply a force (e.g., a "compressive force") to the first or second sheet to move a droplet (e.g., by capillary action) to a region of reduced height, thereby reducing the height of the portion of the air gap proximate the droplet. The controller may generally control 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 be capable of driving the movement of the droplet as it moves along the upper sheet; the droplet may follow the region of the air gap of reduced height formed by the moving force applicator.

[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 instances, the force applicator includes one or more sensors. The controller may be configured to execute pre-programmed and / or dynamic steps; such steps include moving the force applicator in a pattern to effect one or more fluid handling operations such as splitting or dividing the droplet, combining the droplet, mixing the droplet, washing the droplet (or substances within the droplet such as magnetic materials, magnetic beads, etc.). For example, the controller may be configured to apply a pinning compressive force to divide a first fluid droplet and to apply an actuating compressive force in the vicinity of the pinning compressive force to elongate and form a second fluid droplet from the first fluid droplet, where the pinning compressive force is greater than the actuating compressive force. The controller may be configured to apply a pinning compressive force to divide a first fluid droplet and to apply an actuating compressive force in the vicinity of the pinning compressive force to elongate and form a second fluid droplet from the first fluid droplet, where the pinning compressive force is greater than the actuating compressive force.

[0026] In some examples, the controller is configured to apply a compressive force to a sheet between two or more separate fluid droplets and to release the compressive force to combine the two or more separate fluid droplets into a single fluid 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 in order to mix the two or more separate fluid droplets together. Thus, the controller may be configured to mix the two or more fluid droplets together by repeatedly applying and releasing compressive forces to the sheet in close proximity to the two or more fluid 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, the coordinator may control and / or coordinate the addition of fluid into the air gap (e.g., fluid dispensing which may be done manually, automatically, or semi-automatically). The controller may also control or coordinate the activation of one or more heaters, magnets. For example, in some examples, the controller may be configured to control a magnet to attract iron particles suspended in the first fluid droplet. The controller may further be configured to resuspend one or more iron particles in the fluid droplet by applying and releasing a compressive force to the fluid droplet, and by deactivating the magnet. The first sheet may further comprise two or more pinning posts positioned 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 positioned beneath a second surface opposite to the two or more pinning posts. Alternatively, the devices described herein may be used without posts.

[0028] Any of the devices described herein may include a well into which a droplet may move. 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 the base or mounting area of ​​the mechanical microfluidic actuator. For example, the cartridge may fit snugly into the mounting area of ​​the mechanical microfluidic actuator, or it may conform to the shape of the mounting area which includes one or more wells. In some examples, the well is part of a cartridge and is configured, for example, to restrict the movement of a fluid droplet.

[0029] Any of these devices (e.g., a mechanical microfluidic actuator device) may include a base having a cartridge pedestal configured to hold a cartridge, thereby holding a second sheet in contact with the cartridge pedestal, with at least one region of the cartridge in communication with a heating element located below the pedestal and configured to heat fluid droplets in an air gap. In some examples, the mechanical microfluidic actuator device may include a security, such as a clamp, for securing the cartridge to and / or within the pedestal. In some examples, the pedestal may include a plurality of suction ports for applying negative pressure to hold the cartridge (e.g., the second or lower sheet of the cartridge) to the pedestal. The use of suction force may be particularly beneficial for securing the cartridge tightly in place so as to maintain good thermal contact. The use of this suction force 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 with a well, a heater, magnet, etc., may be configured to bring the well. In some examples, the heater and / or magnet may be arranged to bring droplets into the air gap in areas that are not part of the well, including the rail area. Generally, the heater and / or magnet may be part of the cartridge or part of the actuation device (e.g., the drive unit).

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

[0032] For example, a method for microfluidically manipulating a droplet may include the following steps: introducing a droplet into an air gap formed between an elastically deformable first sheet and a second sheet, wherein the first sheet is spaced apart from the second sheet to form an air gap having a gap height of a predetermined distance in a neutral state; applying a compressive force to the first sheet using a mechanical force applicator to create a region in the air gap adjacent to the droplet where the gap height is locally reduced, thereby drawing the droplet toward the region where the air gap is locally reduced; and moving the droplet within the air gap by translating the mechanical force applicator along the outer surface of the first sheet to translate the region where the gap height is locally reduced within the air gap so 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 height smaller than the gap height of the region surrounding the rail region in the air gap.

[0034] For example, a method for microfluidically manipulating a droplet may include the following steps: introducing a droplet into an air gap formed between an elastically deformable first sheet and a second sheet, wherein the first sheet is spaced apart from the second sheet to form an air gap having a gap height of a predetermined distance in a neutral state, the air gap is open to atmospheric pressure and unpressurized, and the droplet is positioned within a rail region of the air gap, the rail region having a gap height smaller than the gap height of the surrounding region; applying a compressive force to the first sheet using a mechanical force applicator to form a region in the air gap adjacent to the droplet where the gap height is locally reduced, thereby drawing the droplet towards the region where the air gap is locally reduced by capillary action; and moving the droplet along the rail region of the air gap by translating the mechanical force applicator along the outer surface of the first sheet to translate the region where the gap height is locally reduced, thereby pulling the droplet within the air gap.

[0035] Any of these methods may include the step of moving a droplet into a well formed by a 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 to react the substance within the droplet. The methods described herein may include the step of controlling the temperature of the well. For example, a method of microfluidically manipulating a droplet may include the following steps: introducing a 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, wherein the first sheet is spaced apart from the second sheet to form an air gap having a gap height of a predetermined distance in a neutral state, and the air gap is open to atmospheric pressure and unpressurized; creating a region in the air gap adjacent to the droplet where the gap height is locally reduced, thereby drawing the droplet towards the region where the air gap is locally reduced by capillary action. To that end, the steps include: applying a compressive force to a first sheet using a mechanical force applicator; moving the droplet into a well formed by a second sheet by translating the mechanical force applicator along the outer surface of the first sheet in order to translate a region in the air gap where the gap height is locally reduced, thereby pulling the droplet into a well in the air gap; modifying the droplet in the well; and moving the droplet out of the well by translating the mechanical force applicator along the outer surface of the first sheet in order to translate a region in the air gap where the gap height is locally reduced away from the well, thereby pulling the droplet out of the well. Any of these methods may include the step of moving the droplet out of the well by translating the mechanical force applicator along the outer surface of the first sheet in order to translate a region in the air gap where the gap height is locally reduced away from the well, thereby pulling the droplet out of the well.

[0036] Therefore, generally speaking, any of these methods may include a step of modifying the droplet in the air gap. The modification step may include one or more of the following: reacting one or more substances in the droplet, heating the droplet, adding substances 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 the second hydrophobic and oleophobic surface of the second sheet.

[0038] In general, the methods and apparatus described herein may manipulate droplets within an air gap by reducing the gap height (e.g., the distance between the upper and lower sheets); reducing the gap height may pull the droplets into the reduced-height region by capillary action. Having an air gap that is open to atmospheric pressure and does not need to be pressurized may be particularly beneficial. This is in contrast to systems that drive droplets by pressure (e.g., squeezing the fluid between sheets) by attempting to push the droplets or, alternatively, by attempting to suck them in by negative pressure.

[0039] One of these methods may include a step in which a compressive force is applied to the first sheet using a mechanical force applicator, thereby attracting droplets towards areas where the air gap is locally reduced by 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 introduction step, the application step, and the transfer step may be part of one or more of the following methods: nucleic acid extraction, library preparation, sequencing, and protein synthesis.

[0041] In general, 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.

[0042] Any of these methods and apparatus may be configured to detect light transmitted or reflected through the droplet. Any of these methods or apparatus 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 the step of attracting magnetic particles suspended in the droplet with a magnet in the mechanical force applicator or in a region below the second sheet.

[0043] Generally, these methods may include a step of mixing droplets within an air gap. The mixing may be disorderly or gentle. For example, disorderly mixing may be performed on the droplets by repeatedly applying and removing compressive force by moving a mechanical force applicator (or any force applicator) along the z-axis across the first sheet. Alternatively or additionally, mixing of droplets may be performed by moving a mechanical force applicator in contact with the first sheet in the plane of the first sheet, for example, in the x-axis and / or y-axis directions.

[0044] The methods and apparatus described herein may be configured to separate droplets by applying a pinning compressive force to a first sheet and by applying an actuation compressive force to the first sheet near the pinning compressive force to stretch and divide the droplets, wherein the pinning compressive force is greater than the actuation compressive force. Any of these methods may include a step of removing all or part of the droplets from an air gap through an opening in the first sheet. Any of these methods may include a step of introducing droplets by passing them from a mechanical force applicator through the opening in the first sheet.

[0045] As mentioned herein, methods for joining droplets using mechanical actuation through an elastically deformable sheet are also described herein. For example, any of these methods may include the steps of: 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] Methods for separating droplets using mechanical actuation through an elastically deformable sheet are also described herein. For example, the method may include the step of alternately applying a first compressive force and a second compressive force different from the first compressive force to a first sheet in order to mix two or more separate fluid droplets in an air gap together.

[0047] Methods for mixing droplets using mechanical actuation through an elastically deformable sheet are also described herein. For example, any of these methods may include a step of repeatedly applying and releasing a compressive force to the outer surface of a first sheet in a region of the first sheet adjacent to two or more fluid droplets in an air gap in order to mix two or more fluid droplets together. Any of these methods and apparatus may perform mixing by moving the mechanical force applicator in the z-axis direction, or instead by moving the mechanical force applicator relative to the first sheet in the y-axis or x-axis direction (e.g., in the plane of the sheet), which may result in gentler mixing than the disorderly mixing resulting from moving the mechanical force applicator in the z-direction. Gentle mixing may be particularly preferred when mixing long polynucleotides in order to avoid shearing.

[0048] These methods may also be used with magnetic beads or particles, which may be condensed (e.g., using magnets as described herein), washed, and resuspended. The methods 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. Methods for attracting iron particles suspended in a fluid droplet with a magnet outside an air gap are also described herein. Any of these methods may also include the step of resuspending one or more iron particles in the fluid droplet by: applying a compressive force to the outer surface of a first sheet on or near the fluid droplet in the air gap; and disabling a controllable magnet.

[0049] In some examples, the methods described herein may include a step of restricting the movement of a first fluid droplet by two or more pinning posts positioned within an air gap. Alternatively or additionally, these methods may include a step of using a well to hold the droplet, particularly when adjusting the temperature (e.g., during thermal cycling). The step of holding the droplet in a well (and / or "pinning" the droplet with one or more pinning posts) can prevent unintended movement of the droplet during operation, particularly when heating the droplet. Any of the methods described herein may include a step of heating the droplet restricted by the well and / or pinning posts with a heating element. Any of these methods may include a step of heating the fluid droplet in the well with a heating element (e.g., a mechanical microfluidic actuator).

[0050] In use, any of these methods and apparatus may be used in conjunction with a coating substance, which may be referred to as a gloss coat (e.g., also called 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 droplet. If the droplet is an aqueous substance, the gloss coating substance may be a hydrophobic substance. The gloss coating may be applied before or after the droplet is applied to the air gap. The gloss coat may be removed, for example, by wicking it with a substance that is absorbent to the gloss substance. The gloss coat may be particularly useful in preventing the evaporation of droplets within the air gap.

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

[0052] In general, the methods and apparatus described herein may be particularly useful for processing fluid droplets of various volumes, from small to medium. For example, the methods described herein are about 10 -15 ~10 -6 This may be useful for fluid droplets with a volume of liters.

[0053] Mechanical microfluidic actuator devices are also described herein. These microfluidic devices may include: a cartridge mounting surface ("seat"); a force dispenser configured to contact the elastically deformable outer surface of a cartridge and apply a compressive force to the elastically deformable outer surface of the cartridge when the cartridge is mounted on the cartridge mounting surface; a force dispenser drive configured to move the force dispenser across the deformable outer surface; and a controller connected to the force dispenser drive for controlling the force dispenser drive to move the force dispenser across the deformable outer surface of the cartridge in order to dynamically reduce the height of the air gap within the cartridge in order to move fluid droplets within the air gap of the cartridge. The force dispenser drive may include one or more motors for moving the force dispenser in x, y, and / or z directions. Most of the examples shown herein are configured to move the force applicator while keeping the cartridge fixed (e.g., moving the force applicator relative to an elastically deformable sheet and air gap), but in some examples the device may be configured to move the cartridge (e.g., an elastically deformable sheet and air gap) while keeping the force applicator fixed; alternatively, both the force applicator and the cartridge (e.g., an elastically deformable sheet and air gap) may move relative to each other.

[0054] A mechanical microfluidic actuator may include one or more force dispensers. In some examples, multiple force dispensers may be controlled independently (in parallel or in series). In some examples, the mechanical microfluidic actuator may switch between one type of force dispenser and another type of force dispenser. In some examples, the mechanical microfluidic actuator may be configured to perform multiple simultaneous actuations using multiple different force dispensers.

[0055] The force applicator may have any suitable shape, and in particular, a shape that applies sufficient compression to an 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 including circular, elliptical, rectangular, or square. In some examples, the force applicator includes a wheel, ball point, or roller.

[0056] In some examples, the force applicator may be adapted to perform one or more additional functions in addition to applying force to an elastically deformable sheet of the air gap to reduce the height of the air gap in order to drive the movement of droplets in the air gap (e.g., by 2D capillary action). In some examples, the tip of the force applicator includes a thermal output unit configured to control the 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 comprises a photosensor configured to detect light transmitted or reflected through the fluid droplets.

[0057] In some examples, the force applicator includes electrodes configured to apply a voltage (for example, 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 comprises a sonication probe configured to emit at least one of sound waves and ultrasound.

[0058] Methods for moving a droplet using mechanical actuation through an elastically deformable sheet are also described herein. For example, these methods may include the steps of: introducing a fluid droplet into an air gap formed between a first elastically deformable sheet and a second sheet, wherein the first sheet is positioned substantially parallel to the second sheet at a predetermined distance apart to form the 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 region adjacent to the fluid droplet in the air gap.

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

[0060] Any of the methods described herein may also include a step of controlling the temperature of a region below a second sheet to control the temperature of a fluid droplet in an air gap. In some examples, the method may include a step of controlling the temperature of a force dispenser to control the temperature of a fluid droplet. In some examples, the method may include a step of 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 dispenser.

[0061] Any of these methods may include a step of applying a voltage from a force dispenser or from a region below the second sheet. The first sheet and / or the second sheet may be dielectrics. Any of these methods may include a step of attracting iron particles (e.g., magnetic beads) suspended in the fluid droplet by a magnet in the force dispenser or in a region below the second sheet. In some examples, the method may include a step of removing the iron particles from the fluid droplet. The method may include a step of mixing the liquid droplet by repeatedly applying and removing a compressive force using a force dispenser. Any of these methods may include a step of using a force dispenser to draw the fluid droplet through an opening in the sheet.

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

[0063] Therefore, 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 apparatuses that may be particularly useful for handling and analyzing clinical, laboratory, biological, or chemical samples are described herein. These apparatuses 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 on a sub-region of the air gap in order to reduce the height of the air gap in the region adjacent to the droplet and move the droplet toward this reduced-height region. By controlling the relative height of the air gap near the droplet (e.g., by controlling the application of force to deform the elastically deformable sheet), the droplet may be moved efficiently and rapidly around the air gap, which enables processing of the droplet, including combining droplets, splitting droplets, mixing droplets, cooling / heating droplets (e.g., thermal cycling droplets), and using magnetic particles in the droplet (e.g., to bind to / extract substances from the droplet).

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

[0065] Examples described herein may be implemented as microfluidic devices. The microfluidic device may include a cartridge comprising: a first sheet having a first hydrophobic and oleophobic surface and a second surface; a second sheet having a first hydrophobic and oleophobic surface and a second surface, wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom 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 within the gap adjacent to a first microfluidic droplet, wherein the reduced predetermined distance moves the first microfluidic droplet within the cartridge.

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

[0067] In some examples, the first sheet may be configured to deflect in response to a compressive force, while 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 a first microfluidic droplet, and to apply an actuation compressive force near the pinning compressive force to extend 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 a 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 a first sheet in order 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 to a first sheet while being in close proximity to the two or more microfluidic droplets.

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

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

[0074] In some examples, the first sheet may further comprise two or more pinning posts positioned on the first surface and extending into the gap, configured to restrict the movement of the first microfluidic droplets. In such cases, the device may further comprise a heater positioned beneath a second surface opposite to the two or more pinning posts.

[0075] In some examples, the cartridge may further comprise a well, which is located through an opening on a second sheet and configured to restrict the movement of a first microfluidic droplet. In such cases, the cartridge may further comprise a heating element, which is located below the well and configured to heat the first microfluidic droplet.

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

[0077] In several other examples, the first microfluidic droplet is 10 -6 ~10 -15 It may have a volume of liters.

[0078] Examples described herein 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, which is formed between a first sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom by a predetermined distance in order to form the gap. The method may further include the step of selectively reducing the predetermined distance by a control unit in one or more regions within the gap adjacent to the first microfluidic droplet in order to move the first microfluidic droplet.

[0079] In some examples, the method may include the step of applying a compressive force to the second surface of the first sheet in order 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, while the second sheet may be configured to resist deflection in response to a compressive force.

[0081] In some examples, the method may include the step of forming a second microfluidic droplet from a first microfluidic droplet by applying a pinning compressive force to a first sheet to divide the first microfluidic droplet and applying an actuation compressive force near the pinning compressive force to extend and form the second microfluidic droplet, wherein the pinning compressive force is greater than the actuation compressive force.

[0082] In some examples, the method may include the steps of applying a compressive force to a 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 the step of alternately applying a first compressive force and a second compressive force different from the first compressive force to a first sheet in order to mix two or more separate microfluidic droplets together.

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

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

[0086] In some examples, the method may include a step of restricting the movement of a first microfluidic droplet by two or more pinning posts positioned on a first surface of a first sheet and extending into a gap. Furthermore, the method may include a step of heating the first microfluidic droplet, which is restricted by the two or more pinning posts, with a heating element.

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

[0088] In some examples, the method may include the step of creating transient pores in the cell membrane of a cell in a first microfluidic droplet using electrodes on a second sheet.

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

[0090] Other examples described herein may be implemented as a non-temporary computer-readable storage medium comprising instructions that, when executed by one or more processors of the device, cause the device to perform an operation. The operation may include the step of sensing a first microfluidic droplet disposed within a gap, the gap formed between a first sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom by a predetermined distance in order to form the gap. The operation may further include the step of selectively reducing the predetermined distance in one or more regions within the gap adjacent to the first microfluidic droplet in order to move the first microfluidic droplet.

[0091] Other examples described in this disclosure may be implemented as a device comprising: a first sheet having a first hydrophobic and oleophobic surface and a second surface; a second sheet having a first hydrophobic surface and a second surface, wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom by a predetermined distance in order to form a gap between the first sheet and the second sheet; and a pressure actuator connected to the first sheet and configured to apply pressure to the first sheet in order to selectively reduce the gap between the first sheet and the second sheet.

[0092] Other examples described herein may be implemented as a device including a cartridge, the cartridge comprising: a first sheet having a first hydrophobic and oleophobic surface and a second surface; a second sheet having a first hydrophobic and oleophobic surface and a second surface, wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom by a predetermined distance in order to form a gap between the first sheet and the second sheet; and a stylus configured to contact the first sheet and selectively reduce the predetermined distance in at least one region within the gap.

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

[0094] In some examples, the tip of the stylus may include a temperature control device configured to control the 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 may include a photosensor configured to detect light transmitted or reflected through the microfluidic droplet. Furthermore, 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 iron particles suspended in a microfluidic droplet placed within the gap. The magnet may be further configured to provide a variable magnetic field strength to separate the iron particles from the microfluidic droplet.

[0096] Other examples described herein may be implemented as methods for manipulating one or more microfluidic droplets. The method may include introducing a microfluidic droplet into a gap, which is formed between a first sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom by a predetermined distance in order to form the gap. The method may further include providing a compressive force with a stylus to reduce the predetermined distance in at least one region within the gap adjacent to the microfluidic droplet.

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

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

[0099] In some examples, the method may include a step of attracting iron particles suspended in a microfluidic droplet using a magnet in a stylus. Furthermore, the method may include a step of separating the iron particles from the microfluidic droplet. The method may also include a step of dispersing the particles in the microfluidic droplet by repeatedly applying and removing compressive force with a stylus.

[0100] In some examples, the method may further include the steps of drawing a microfluidic droplet by a stylus through a first septum in a first sheet, and receiving the microfluidic droplet by a lumen in the stylus connected to the first septum. In some examples, the method may further include the step of injecting the microfluidic droplet by a stylus into a second septum different from the first septum. In some further examples, the method may include the step of controlling the temperature of the microfluidic droplet by a temperature control element in the stylus. In some examples, the method may include the step of detecting light transmitted or reflected by the microfluidic droplet in the stylus. In some further examples, the method may include the step of providing at least one of sound waves and ultrasound to the microfluidic droplet in the stylus.

[0101] Other examples described herein may be implemented as a non-temporary computer-readable storage medium which, when executed by one or more processors of the device, can cause the device to perform an operation, the operation comprising the steps of: sensing a first microfluidic droplet placed in a gap, the gap being formed between a first sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; and a second sheet having a first surface and a second surface, the first surface of which is hydrophobic and oleophobic; wherein the first surface of the first sheet is positioned toward the first surface of the second sheet and separated therefrom by a predetermined distance in order to form the gap; and providing a compressive force by a stylus to reduce the predetermined distance in at least one region of the gap adjacent to the microfluidic droplet.

[0102] For example, mechanical microfluidic actuator devices configured to be connected to a liquid handling robot are described herein. Any of these devices may include: a mounting area configured to be connected to a cartridge; a stylus configured to deform the upper surface of a cartridge when the cartridge is mounted within the mounting area; an applicator driver subassembly connected to the stylus and configured to control the lateral movement of the stylus along the upper surface of the cartridge while deforming the upper surface of the cartridge vertically; and a coupling configured to connect the microfluidic actuator device to a liquid handling robot.

[0103] As mentioned, any of these devices may include a controller. For example, the controller may be configured to control the operation of the applicator drive assembly and the liquid handling robot.

[0104] Any of these devices may include a liquid handler (e.g., a liquid handling robot).

[0105] The mounting area may comprise one or more suction ports configured therefor to secure the cartridge. The applicator driver subassembly may include one or more drivers. For example, the one or more drivers may comprise x and / or y motion drivers, and / or z motion drivers. In any of these devices, the applicator driver subassembly may be configured to move a stylus alternately to apply a first compressive force and a second compressive force different from the first compressive force to the cartridge in order to mix two or more separate fluid droplets together. The applicator driver subassembly may be configured to move a stylus to mix the two or more fluid droplets together by repeatedly applying and releasing compressive forces to the cartridge in close proximity to the two or more fluid droplets. The applicator driver subassembly may be configured to control a magnet to attract iron particles suspended in the fluid droplets within the cartridge. The applicator driver subassembly may be configured to resuspend one or more iron particles in the fluid droplets within the cartridge by applying and releasing compressive forces to the fluid droplets and by deactivating the magnet.

[0106] In any of these devices, the applicator driver subassembly may comprise a frame or gantry on which a stylus may be driven to change position and / or to apply force to the cartridge. The applicator driver subassembly may be configured to move the stylus to apply a pinning compressive force to divide a first fluid droplet in the cartridge, and to apply an actuation compressive force near the pinning compressive force to extend and form a second fluid droplet from the first fluid droplet, where the pinning compressive force is greater than the actuation compressive force. The applicator driver subassembly may be configured to move the stylus to apply a compressive force to the cartridge between two or more separate fluid droplets, and to release the compressive force to combine the two or more separate fluid droplets into a single fluid droplet.

[0107] In any of these devices, the installation area may include one or more protrusions configured to deform a film on a cartridge to form one or more channels. Any of these devices may include a thermal control area within the installation area.

[0108] A mechanical microfluidic actuator device also described herein includes: a mounting area configured to be connected to a cartridge; a stylus configured to apply mechanical pressure to deform the upper surface of the cartridge when the cartridge is mounted within the mounting area; an applicator driver subassembly connected to the stylus and configured to control the lateral movement of the stylus along the upper surface of the cartridge while deforming the upper surface of the cartridge vertically; one or more well-forming surfaces rising and extending from the upper mounting surface of the mounting area; and one or more suction ports configured to pull the lower surface of the cartridge toward the upper searing surface so that the lower surface of the cartridge confirms with the well-forming surfaces.

[0109] The well-forming surface may be configured to form a heating / cooling well that has a thermal contract with the thermal control area of ​​the installation area. As described above, the apparatus may include a controller configured to control the operation of the applicator drive assembly and the thermal control area. Either of these apparatuses may include a liquid handling robot.

[0110] As described above, the applicator driver subassembly may include one or more drivers. The one or more drivers may comprise x and / or y motion drivers, and / or z motion drivers. The applicator driver subassembly may comprise a frame or gantry on which a stylus may be driven to change position and / or apply force to the cartridge. The applicator driver subassembly may be configured to move the stylus to apply a pinning compressive force to divide a first fluid droplet in the cartridge, and to apply an actuation compressive force near the pinning compressive force to extend and form a second fluid droplet from the first fluid droplet, where the pinning compressive force is greater than the actuation compressive force. The applicator driver subassembly may be configured to move the stylus to apply a compressive force to the cartridge between two or more separate fluid droplets, and to release the compressive force to combine the two or more separate fluid droplets into a single fluid droplet. The applicator driver subassembly may be configured to alternately move the stylus to apply a first compressive force and a second compressive force different from the first to the cartridge in order to mix two or more separate fluid droplets together.

[0111] The applicator driver subassembly may be configured to move a stylus to mix two or more fluid droplets together by repeatedly applying and releasing a compressive force to the cartridge in close proximity to the droplets. The applicator driver subassembly may be configured to control a magnet to attract iron particles suspended in the fluid droplets within the cartridge. The applicator driver subassembly may be configured to resuspend one or more iron particles in the fluid droplets within the cartridge by applying and releasing a compressive force to the fluid droplets and by deactivating the magnet.

[0112] A method for forming a well within the air gap of a cartridge, comprising the steps of: placing the lower sheet of the cartridge on the installation area to connect the installation area to the cartridge; applying suction force to bring the lower sheet to the installation area so that a well-forming surface extending upward from the upper surface of the installation area forms a heating / cooling well within the air gap; and driving a stylus to apply mechanical pressure to deform the upper surface of the cartridge when the cartridge is installed within the installation area; is also described herein.

[0113] A cartridge for surface mechanical actuation (MAOS) is also described herein, comprising: an upper surface having an elastically deformable sheet having a hydrophobic and oleophobic surface; a lower surface; an air gap between the upper and lower surfaces configured to vary in distance from the upper and lower surfaces to allow droplets to move through the air gap; a frame between the upper and lower surfaces to which the sheet is mounted; and a tensioner receiver on the frame, covered by the sheet and configured to receive a tensioning projection of a tensioner to pull the sheet into the tensioner receiver and keep the sheet taut when engaged with the tensioner.

[0114] The lower surface may comprise a second elastically deformable sheet that is primarily hydrophobic and oleophobic. The second elastically deformable sheet may comprise a plurality of hydrophilic cell adhesion pad regions configured to allow cell adhesion for culture within the air gap. The air gap may be divided into a plurality of lanes. The sheet may be attached to the outer edge of the frame. The tensioner receiver may comprise a tensioner receiver channel. Any of these cartridges described herein may include a tensioner. The cartridge may comprise a plurality of openings in the upper sheet configured to provide access to the air gap.

[0115] For example, a cartridge for surface mechanical actuation (MAOS) may include: an upper surface comprising a first elastically deformable sheet having a hydrophobic and oleophobic surface; a lower surface comprising a second elastically deformable sheet that is primarily hydrophobic and oleophobic; an air gap between the upper and lower surfaces; a frame between the upper and lower surfaces to which the sheet is attached to the frame on the outer edge of the frame; and a tensioner receiving channel on the frame, which is covered by the sheet and configured to receive a tensioning projection of a tensioner in order to pull the sheet into the tensioner receiving channel and keep the sheet taut when engaged with the tensioner.

[0116] A mechanical microfluidic actuator for surface mechanical actuation (MAOS) is also described herein, which may include: a mounting area configured to be connected to a cartridge; a tensioner comprising a plurality of tensioning protrusions extending outward from the inner surface, configured to engage with one or more tensioner receivers on the upper surface of the cartridge to pull the upper elastic sheet of the cartridge into the tensioner receivers and thereby keep the sheet taut; a stylus configured to deform the upper elastic sheet of the cartridge when the cartridge is mounted within the mounting area; and an applicator driver subassembly connected to the stylus and configured to control the lateral movement of the stylus along the upper elastic sheet of the cartridge while deforming the upper elastic sheet of the cartridge vertically.

[0117] Any of these devices may include a controller configured to control the operation of the applicator drive assembly. The mounting area may have one or more suction ports configured therefor to secure a cartridge. The applicator driver subassembly may include one or more drivers. The one or more drivers may include x and / or y motion drivers, and / or z motion drivers. The applicator driver subassembly may include a frame or gantry on which a stylus may be driven to change position and / or to apply force to the cartridge. The applicator driver subassembly may be configured to move the stylus to apply a pinning compression force to divide a first fluid droplet in the cartridge, and to apply an actuation compression force near the pinning compression force to extend and form a second fluid droplet from the first fluid droplet, where the pinning compression force is greater than the actuation compression force. The applicator driver subassembly may be configured to move the stylus to apply a compressive force to the cartridge between two or more separate fluid droplets, and to release the compressive force to combine the two or more separate fluid droplets into a single fluid droplet. The applicator driver subassembly may be configured to move the stylus alternately to apply a first compressive force and a second compressive force different from the first compressive force to the cartridge in order to mix the two or more separate fluid droplets together. The applicator driver subassembly may be configured to move the stylus to mix the two or more fluid droplets together by repeatedly applying and releasing a compressive force to the cartridge in close proximity to the two or more fluid droplets. The applicator driver subassembly may be configured to control a magnet to attract iron particles suspended in the fluid droplets within the cartridge.

[0118] The applicator driver subassembly may be configured to resuspend one or more iron particles in the fluid droplets within the cartridge by applying and releasing compressive force to the fluid droplets and by disabling the magnets. The mounting area may have one or more protrusions configured to deform the film on the cartridge to form one or more channels. Any of these devices may include a thermal control area within the mounting area.

[0119] All methods and apparatus described herein in any combination may be used to achieve the benefits described herein. [Brief explanation of the drawing]

[0120] The patent or application file contains at least one drawing produced in color. Copies of the patent or patent application publication accompanied by the color drawing are available from the Japan Patent Office upon request and payment of the required fees.

[0121] A better understanding of the features and advantages of the methods and apparatus described herein will be obtained by referring to the detailed description below, which illustrates exemplary embodiments, and the accompanying drawings.

[0122] [Figure 1] Figures 1A to 1C show parts of a microfluidic device (for example, parts of a mechanical microfluidic actuator, or cartridges for use in a mechanical microfluidic actuator).

[0123] [Figure 2] This flowchart illustrates exemplary actions for manipulating microfluidic droplets.

[0124] [Figure 3] Figures 3A to 3C show parts of another microfluidic device (e.g., a mechanical microfluidic actuator).

[0125] [Figure 4] This flowchart illustrates an exemplary operation for separating a microfluidic droplet into two or more microfluidic droplets.

[0126] [Figure 5] Figures 5A to 5C show parts of another microfluidic device.

[0127] [Figure 6] This flowchart illustrates an exemplary operation for mixing microfluidic droplets.

[0128] [Figure 7] Figures 7A-7C show a portion of another microfluidic device.

[0129] [Figure 8] This flowchart illustrates an exemplary procedure for extracting suspended iron particles from a microfluidic droplet.

[0130] [Figure 9] Figures 9A and 9B show a portion of another microfluidic device.

[0131] [Figure 10] This flowchart illustrates an exemplary operation for dispersing particles in a microfluidic droplet.

[0132] [Figure 11] Figures 11A to 11C show a portion of another microfluidic device.

[0133] [Figure 12] This flowchart illustrates an exemplary operation for manipulating microfluidic droplets in conjunction with a pinning post.

[0134] [Figure 13-1] Figures 13A to 13E show parts of another microfluidic device. [Figure 13-2] See the explanation in Figure 13-1.

[0135] [Figure 14] This flowchart illustrates an exemplary operation for manipulating microfluidic droplets in conjunction with a well.

[0136] [Figure 15] Figures 15A to 15C show a portion of another microfluidic device.

[0137] [Figure 16] This flowchart illustrates the typical operation for providing electroporation.

[0138] [Figure 17] An exemplary microfluidic device (e.g., a mechanical microfluidic actuator) is shown.

[0139] [Figure 18A] An example of a part of the microfluidic apparatus described herein is shown. [Figure 18B] The possible relevant end profiles of the stylus are shown in Figure 18A.

[0140] [Figure 19] Figures 19A and 19B show parts of another microfluidic device.

[0141] [Figure 20] This flowchart illustrates exemplary actions for manipulating microfluidic droplets.

[0142] [Figure 21] Figures 21A and 21B show parts of another microfluidic device.

[0143] [Figure 22] This flowchart illustrates exemplary actions for manipulating microfluidic droplets.

[0144] [Figure 23] Figures 23A to 23C show a portion of another microfluidic device.

[0145] [Figure 24] This flowchart illustrates an exemplary procedure for extracting suspended iron particles from a microfluidic droplet.

[0146] [Figure 25] Figures 25A and 25B show parts of another microfluidic device.

[0147] [Figure 26] This flowchart illustrates an exemplary operation for dispersing particles in a microfluidic droplet.

[0148] [Figure 27] Figures 27A to 27C show a portion of another microfluidic device.

[0149] [Figure 28] This flowchart illustrates an exemplary operation for aspirating liquid from a microfluidic device.

[0150] [Figure 29] Figures 29A to 29C show a portion of another microfluidic device.

[0151] [Figure 30] This flowchart illustrates exemplary operations for manipulating liquids in a microfluidic device.

[0152] [Figure 31] Figures 31A to 31C show a portion of another microfluidic device.

[0153] [Figure 32] This flowchart illustrates an exemplary procedure for heat-treating a liquid in a microfluidic device.

[0154] [Figure 33] This shows a portion of another microfluidic device.

[0155] [Figure 34] Figures 34A to 34C show a portion of another microfluidic device.

[0156] [Figure 35] This is a flowchart illustrating the typical steps for performing reaction measurement.

[0157] [Figure 36] This shows a portion of another microfluidic device.

[0158] [Figure 37] Figures 37A to 37C show a portion of another microfluidic device.

[0159] [Figure 38] This is a flowchart illustrating an example of the operation required for sound wave processing.

[0160] [Figure 39] A block diagram of a device that may be an example of a microfluidic device (e.g., a mechanical microfluidic actuator) as described herein is shown.

[0161] [Figure 40] Figures 40A–40D illustrate one exemplary cartridge for use with a mechanical microfluidic actuator. Figure 40A shows an example of a cartridge with three lanes. Figure 40B shows an example of a cartridge with eight lanes. Figure 40C shows an example of a portion of the cartridge in Figure 40A; Figure 40D shows a magnified view of the cross-sectional view in Figure 40C.

[0162] [Figure 41] This is a disassembled view of another example of a cartridge.

[0163] [Figure 42] Figures 42A and 42B show partial cross-sectional views of a portion of the cartridge.

[0164] [Figure 43-1] Figure 43A shows a cross-sectional view through another example of a cartridge. Figures 43B and 43C illustrate the cartridge of Figure 43A installed in a mechanical microfluidic actuator. Figure 43C shows the cartridge of Figure 43B with a droplet in the air gap of the cartridge. Figure 43D shows an example of a cartridge installed in an actuator assembly in which three parallel lanes are actuated simultaneously by a roller stylus. Figure 43E shows an example of an 8-lane cartridge as described herein. [Figure 43-2] See the explanation in Figure 43-1.

[0165] [Figure 44-1] Figures 44A–44E illustrate examples of various mounting areas for a mechanical microfluidic actuator connected to a cartridge as described herein. Figure 44A shows an example of a cartridge connected to a mechanical microfluidic actuator having a mounting area including a concave mounting surface. Figure 44B shows a mechanical microfluidic actuator having a mounting area with a raised section. Figure 44C shows an example of a mechanical microfluidic actuator having a thermally conductive cylindrical rail (forming a rail area) protruding from the mounting area. Figure 44D shows an example of a mechanical microfluidic actuator having a thermally conductive flat platform rail (e.g., rail area) protruding from the mounting area. Figure 44E shows an example of a mechanical microfluidic actuator having a thermally conductive dome-shaped platform rail (e.g., rail area) protruding from the mounting area. [Figure 44-2] See the explanation in Figure 44-1.

[0166] [Figure 45-1]Figures 45A–45C illustrate examples of installation areas for mechanical microfluidic actuators. Figure 45A shows the installation area. Figure 45B shows the installation area made partially transparent. Figure 45C shows the installation area with connected cartridges. Figures 45D and 45E illustrate enlarged cross-sectional views through the examples of installation areas in Figures 45A–45C. See the description of Figure 45D. [Figure 45-2] See the explanation in Figure 45-1.

[0167] [Figure 46] A schematic diagram illustrates an example of the installation area for a cartridge connected to the installation area.

[0168] [Figure 47] A method for transferring small volumes of fluid using solid-state techniques (e.g., non-vacuum / non-pipette) is illustrated.

[0169] [Figure 48] An example of a mechanical microfluidic actuator device as described herein is schematically illustrated.

[0170] [Figure 49] An example of the internal region of a mechanical microfluidic actuator (showing a row of magnetic elements) is illustrated.

[0171] [Figure 50] An example of a mechanical microfluidic actuator is shown.

[0172] [Figure 51] An example of a multi-system device including multiple mechanical microfluidic actuators is illustrated.

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

[0174] [Figure 53] Figures 53A to 53C illustrate how to operate the apparatus described herein.

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

[0176] [Figure 55] Figures 55A-55B illustrate an example of the step of introducing sequencing primers into 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 56-1] Figures 56A to 56E schematically illustrate the delivery of SBS reagent using the methods and apparatus described herein. [Figure 56-2] See the explanation in Figure 56-1.

[0178] [Figure 57] A cartridge as described herein is illustrated.

[0179] [Figure 58] A schematic diagram illustrates a method for extracting polynucleotides from a tissue sample (e.g., blood), which may be carried out using the methods and apparatus described herein.

[0180] [Figure 59] A schematic diagram illustrates how to perform an RNA-seq workflow using the methods and apparatus described herein.

[0181] [Figure 60]A schematic diagram illustrates a method for rapid hybridization of Twist exome targets, which may be performed using the methods and apparatus described herein.

[0182] [Figure 61] A schematic diagram illustrates the workflow for performing Amplisequ (2-primer pool) using the methods and apparatus described herein.

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

[0184] [Figure 63] Evaporation prevention using the methods and apparatus described herein is illustrated.

[0185] [Figure 64] Figures 64A to 64C schematically illustrate an example of a mechanical microfluidic actuator device having a cartridge and an installation area configured to include a well as described herein.

[0186] [Figure 65] An example of a mechanical microfluidic actuator device (e.g., a module) connected to a liquid handler (e.g., a liquid handling robot / liquid handling subsystem) is shown.

[0187] [Figure 66] Figures 66A-66B illustrate an example of a method for amplifying polynucleotides using the apparatus described herein.

[0188] [Figure 67]A schematic diagram of the cartridge's cross-section is shown; illustrating the use of the apparatus (including the cartridge) in a proteomics method that employs mechanical actuation to move droplets.

[0189] [Figure 68-1] Figures 68A–68F schematically illustrate one method of performing the assay using mechanical actuation to move the droplet as described herein. [Figure 68-2] See the explanation in Figure 68-1.

[0190] [Figure 69-1] Figures 69A–69D schematically illustrate cartridges configured for culturing cells as part of an overall assay or method. [Figure 69-2] See the explanation in Figure 69-1.

[0191] [Figure 70] An example of a cartridge configured to enable cell culture (e.g., including a hydrophilic adhesive pad) is shown in a top view and a side view.

[0192] [Figure 71] Another example of a cartridge similar to the one shown in Figure 70 is shown.

[0193] [Figure 72] Figures 72A and 72B illustrate examples of clamp-type tensioners and cartridges configured for use with such tensioners, respectively.

[0194] [Figure 73]Figure 73A shows a cross-sectional view through an exploded assembly of the tensioner and cartridge. Figure 73B shows a magnified detail view of the area of ​​the tensioner (including the tensioning projection). Figure 73C shows a magnified detail view of the area of ​​the cartridge (including the tensioning receiver configured to mate with the tensioning projection shown in Figure 73B).

[0195] [Figure 74] Figure 74A shows an example of a tensioner engaged with a cartridge, applying tension to the upper sheet of the cartridge. Figure 74B shows a magnified view of the edge region of the cartridge engaged with the tensioner as shown in Figure 74A. [Modes for carrying out the invention]

[0196] Detailed explanation A microfluidic apparatus (e.g., a device or system) for controlled fluid manipulation may include a two-dimensional (planar) fluid chamber. The chamber may include a first sheet and a second sheet separated by a gap between them. The gap may separate the first and second sheets by a feasible distance. The first and second sheets may be hydrophobic or may include a hydrophobic coating. In some examples, the first and second sheets are hydrophobic and oleophobic and / or include a hydrophobic and oleophobic coating.

[0197] Microfluidic droplets can be manipulated through mechanical operations that selectively reduce the gap by applying force directly or indirectly to a first or second sheet. This process is sometimes called surface mechanical actuation (MAOS) and is also described as the use of mechanical compression to alter capillary forces. The applied force (which may include compressive forces) may be applied near or adjacent to the droplet within the gap. In some aspects, the reduction of the gap may cause droplet migration, separation, merging, mixing, or incubation, etc.

[0198] In some examples, the force may be applied by a stylus. The stylus may include electrodes and / or a controllable magnet. The microfluidic droplet may be manipulated by a combination of the pressure applied by the stylus and the voltage provided by the electrodes and / or the magnetic field provided by the magnet.

[0199] Figure 1A shows a portion of the microfluidic device 100. Any of the devices described herein may be implemented, in part or in whole, as a system or any other viable 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 can be selectively coupled to a control unit or base station. As shown, the first sheet 110 may be a “top” sheet and the second sheet 120 may be a “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.

[0200] The first sheet 110 and the second sheet 120 may form a planar structure occupying any viable area. The first sheet 110 and the second sheet 120 are shown in the figure in their initial positions. In the initial positions, the first sheet 110 and the second sheet 120 are relatively parallel to each other, separated by a distance related to and / or determined by the gap 130.

[0201] 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 the sake of clarity, the first surfaces 111 and 121 may be positioned toward the gap 130, while the second surfaces 112 and 122 may be positioned opposite the first sheet 110 and the second sheet 120, respectively. In other words, the second surfaces 121 and 122 may be positioned away from the gap 130.

[0202] The first surfaces 111 and 121 may be hydrophobic (water-repellent). In some examples, the first sheet 110 and the second sheet 120 (and therefore the first surfaces 111 and 121) may be formed from a hydrophobic and oleophobic substance (material). In some other examples, the first surfaces 111 and 121 may be a hydrophobic and oleophobic coating or layer applied to the first sheet 110 and the second sheet 120, respectively. For example, in any of these devices, the first and second sheets (upper and lower sheets) may be coated with a hydrophobic and oleophobic coating (or separate coatings). In any of these devices, the first and / or second sheets may be formed from a material that includes a hydrophobic and oleophobic surface. In one example, the upper and lower sheets may include the same material, such as Duraflex PS8000 TPU (thickness 125 μm), for example, an aromatic polyester polyurethane material.

[0203] Droplet 140 may be introduced into gap 130. In some cases, droplet 140 may be introduced into gap 130 through ports or openings (not shown) on first sheet 110 and / or second sheet 120. Droplet 140 may be mechanically manipulated by selectively reducing the gap near droplet 140. In some examples, one or more of sheets 110 and 120 may be flexible. The flexible sheet may bend in response to one or more forces. For example, first sheet 110 may be flexible and second sheet 120 may be rigid or semi-rigid. The rigid or semi-rigid sheet may resist bending in response to one or more forces. In other examples, second sheet 120 may be flexible and first sheet 110 may be rigid or semi-rigid. In still other examples, both first sheet 110 and second sheet 120 may be flexible. As used herein, the term flexible may describe any substance (material) that can bend, deform, curve, or move, etc.

[0204] Droplet 140 may have a predetermined volume. In some cases, droplet 140 may be a microfluidic droplet having a volume of 10 -6 ~10 -15 liters, but in some examples, the volume of droplet 140 may have any other executable volume. Gap 130 may be at least partially determined by the volume of droplet 140. In other words, gap 130 may be selected or chosen such that droplet 140 (e.g., the volume of droplet 140) can touch both first sheet 110 and second sheet 120.

[0205] Figure 1B shows another diagram of the microfluidic device 100. In this diagram, the first sheet 110 may bend due to a compressive force near or adjacent to one side or end of the droplet 140. The compressive force creates a reduction gap 132 between the first sheet 110 and the second sheet 120 toward its end or side of the droplet 140. As the reduction gap 132 is formed, the droplet 140 may deform asymmetrically and be pulled toward the reduction gap 132. In some cases, droplet movement may be caused by differential capillary action and / or differential pressure gradients within the droplet 140. The compressive force can be provided by any viable means. For example, an array of electromechanical, mechanical, and / or pneumatic actuators may be positioned next to the first sheet 110 and / or the second sheet 120 to selectively provide the compressive force necessary to form the reduction gap 132. In another example, a compressive force may be provided by a stylus that can contact the first sheet 110 and / or the second sheet 120.

[0206] In some examples, the microfluidic device 100 may include one or more optical sensors (not shown). The one or more optical sensors may detect the presence and / or location of droplets 140. In this configuration, data from the optical sensors may be used to assist in applying a compressive force near or adjacent to the droplets 140. Any of these devices may include one or more light sources for illuminating the air gap to determine the location, size, and / or presence of one or more droplets. In some examples, the light sources may be positioned to best illuminate the droplets, for example, by providing contrast. For example, the light sources may be configured to illuminate the droplets from the side. In some examples, the illumination may be configured with sensors for providing dark-field illumination (for example, the device may include an obstruction in the optical path to the lens of the optical sensor). The device may control when light is applied or not (including intermittent illumination) to prevent undesirable alterations of the droplets (including evaporation, heating, photofading, etc.). Light may be applied at a low level (for example, below the threshold for detection without assistance, but detectable by the sensor to detect and / or track the droplet).

[0207] As will be described in more detail herein, any of these droplets may contain a drop gloss material (e.g., a surfactant that coats the droplet and reduces its effective surface tension compared to pure water). Either or both of the drop gloss and / or aqueous droplets may contain a dye or color that increases the detectability by the one or more sensors. The dye may otherwise be inert. In some examples, the device may be configured to detect the interface between the aqueous droplet and the drop gloss. The device may be configured to detect, analyze (e.g., measure), and / or track one or more droplets automatically or semi-automatically. For example, the device may include one or more processors that receive sensor inputs and analyze those inputs to detect, analyze, and / or track droplets in an air gap.

[0208] Figure 1C shows another diagram of the microfluidic device 100. In this diagram, 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 the same as 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.

[0209] Therefore, in the manner described in Figures 1A-1C, any droplet may be manipulated to any area within the microfluidic device 100 by reducing the gap near one end of the droplet. 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 lever, ball, or roller may provide 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.

[0210] Figure 2 is a flowchart illustrating exemplary operations 200 for manipulating microfluidic droplets. In some examples, the operations described herein can be performed with additional operations, fewer operations, operations in different sequences, parallel operations, and several different operations. Operations 200 are described below with respect to the microfluidic device 100 shown in Figures 1A-1C, but operations 200 may be performed by any other suitable system or device.

[0211] Operation 200 begins in block 202, in which 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 the microfluidic device 100. The gap 130 may be the 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 a hydrophobic and oleophobic layer. The droplet 140 may be placed in 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 a fluid that is immiscible (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 arrays of visible and / or invisible light detectors, etc. Therefore, the optical sensor may determine when the droplet 140 was introduced into the gap 130.

[0212] Next, in block 204, near the microfluidic droplet, the distance between the two hydrophobic and oleophobic sheets is selectively reduced by mechanical actuation. The reduced distance may result in a reduced gap 132 between the first sheet 110 and the second sheet 120. In some examples, a compressive force may be applied to sheet 110 in close proximity to (e.g., next to) one side of droplet 140. The compressive force may reduce the gap 130 and move droplet 140 toward the compressive force. In some examples, the compressive force may deform one end of droplet 140.

[0213] 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 their 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 in block 202. The microfluidic droplets may then come to rest at different positions, having moved from their initial positions in block 202.

[0214] The steps in blocks 202-206 may be repeated any number of times to move one or more droplets to any location within the gap 130 of the microfluidic device 100. In some examples, different compressive forces (e.g., different force amplitudes) may be applied to the droplets to perform different operations.

[0215] Figure 3A shows a portion of another microfluidic device 300. The microfluidic device 300 may include a first sheet 310, a second sheet 320, and a gap 330; these could be examples of the first sheet 110, the second sheet 120, and the gap 130 in Figure 1. A source droplet 340 (which may be similar to droplet 140) can be introduced into the gap 330 as described above with respect to Figures 1A and 2.

[0216] A pinning compressive force may be applied to the source droplet 340. While the pinning compressive force is shown in the figure as being applied to the first sheet 310, in other examples it may be applied to the second sheet 320. As shown, the pinning compressive force may be applied towards the approximate 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 cause the source droplet 340 to begin to split or separate into two droplets.

[0217] Figure 3B shows another diagram of the microfluidic device 300. As shown, an actuation compressive force may be applied to the source droplet 340. The actuation compressive force may be less than the pinning compressive force applied in Figure 3A. The actuation compressive force may be applied simultaneously with (at the same time as) or after the pinning compressive force. The actuation compressive force may separate and guide the satellite droplet 341 away from the source droplet 340. In some examples, the pinning compressive force may cause the first sheet 310 to come into contact with the second sheet 320, which helps separate the satellite droplet 341 from the source droplet 340.

[0218] Figure 3C shows another diagram of the microfluidic device 300. As illustrated, the pinning and actuation forces have been eliminated or reduced, allowing the first sheet 310 and / or the second sheet 320 to return to their initial positions, such as the initial position shown in Figure 3A. The source droplet 340 is shown separated from the satellite droplet 341.

[0219] Figure 4 is a flowchart illustrating an exemplary operation 400 for separating a microfluidic droplet into two or more microfluidic droplets. Operation 400 will be described below with respect to the microfluidic device 300 shown in Figures 3A-3C, but operation 400 may be performed by any other suitable system or device.

[0220] Operation 400 begins in block 402, where a pinning compressive force is applied to the source microfluidic droplet by mechanical actuation. The source microfluidic droplet (which may be the same as the source microfluidic droplet 340) may have been introduced earlier into the gap between the first sheet 310 and the second sheet 320, as described above with respect to Figures 1A and 2, for example. In some examples, the pinning compressive force may begin to divide or separate the source microfluidic droplet into two or more droplets. The pinning compressive force may be a mechanical actuation force applied to the first sheet 310 and / or the second sheet 320, and may be provided by any viable means. In some cases, the pinning compressive force may be applied toward the center or middle of the source microfluidic droplet. In some examples, the pinning compressive force may bring the first sheet 310 into contact with the second sheet 320.

[0221] 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 satellite microfluidic droplets away from the source microfluidic droplet. The actuation compressive force may be another mechanical actuation force, in which case the force is smaller than the pinning compressive force. The actuation compressive force may be applied concurrently with or after the application of the pinning compressive force.

[0222] In 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 respective initial positions, as depicted in Figure 3C. While the pinning and actuation compressive forces are removed or reduced, the satellite droplets may remain separated from the source droplets.

[0223] In some cases, compressive force can be used to fuse two or more separate droplets. Such examples are illustrated in conjunction with Figures 5A-5C and 6. Droplets of different or the same size can fuse. For example, volumes of 250 nL to 80 μL can be firmly actuated and fuse with equivalent, larger, or smaller volumes already present in the air gap.

[0224] Figure 5A shows a portion of another microfluidic device 500. As shown in Figure 5A, the microfluidic device 500 may include a first sheet 510, a second sheet 520, and a gap 530; these could be other examples of the first sheet 110, the second sheet 120, and the gap 130 in Figure 1A. A first droplet 540 and a second droplet 541 (similar to droplet 140 in Figure 1A, or source droplet 340 and satellite droplet 341 in Figure 3C) may be introduced into the gap 530 as described above with respect to Figures 1A-1C, 2, 3A-3C, and 4.

[0225] A compressive force may be applied to the first sheet 510 and / or the second sheet 520. In some examples, a compressive force may be applied between the first droplet 540 and the second droplet 541, which deforms, moves, and possibly combines the respective droplets. The compressive force may be a mechanical actuation force as described herein.

[0226] Figure 5B shows another diagram of the microfluidic device 500. The compressive force may be removed or reduced, thereby allowing the first sheet 510 and / or the second sheet 520 to return to their initial (uncompressed) positions. As shown, the first droplet 540 and the second droplet 541 may be combined to form a fused droplet 542. Even if combined into a single droplet, the components of the individual droplets, or the components of the first droplet 540 and the second droplet 541, do not need to be well mixed in the fused droplet 542.

[0227] Figure 5C shows another diagram of the microfluidic device 500. Compressive forces 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. Repeated application of compressive forces can cause the fused droplets 542 to repeatedly deform and recover, thereby causing mixing of their contents. In some examples, the compression / relaxation cycles caused by the application and release of compressive forces may be repeated a specified number of times to mix the contents of the fused droplets 542.

[0228] Figure 6 is a flowchart illustrating an exemplary operation 600 for mixing microfluidic droplets. Operation 600 is described with respect to the microfluidic device 500 in Figures 5A-5C, but operation 600 may be performed by any other suitable system or device. Operation 600 begins in block 602 in which 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 viable means. The compressive force may move the first microfluidic droplet 540 and the second microfluidic droplet 541 toward each other, and in some cases, they may be combined to form a single (fused) microfluidic droplet 542.

[0229] In 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 their initial positions.

[0230] In some cases, additional agitation of the fused microfluidic droplets 542 may be desired to provide additional mixing. To provide additional agitation, the application and removal (or reduction) of compressive force may be repeated over a predetermined number of cycles. Thus, in block 606, the number of completed compression cycles is determined. A completed compression cycle may include the application and removal or reduction of 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, operation 600 may terminate.

[0231] In some examples, iron particles may be suspended in microfluidic droplets to assist in processing or assays. After one or more processing steps are completed, the iron particles may be removed from the droplets for further processing.

[0232] Figure 7A shows a portion of another microfluidic device 700. The microfluidic device 700 may include a first sheet 710, a second sheet 720, and a gap 730; these could be examples of the first sheet 110, the second sheet 120, and the gap 130 in Figure 1. The droplet 740 may include one or more iron particles 741 that may be suspended within the droplet 740. The microfluidic device 700 may also include a magnet 750 (not shown).

[0233] Figure 7B shows another diagram of the microfluidic device 700. A magnet 750 may be activated or enabled. For example, the magnet 750 may be an electromagnet that can be activated by the application of power. In another example, the magnet 750 may be a permanent magnet that can be moved toward the droplet 740. In addition, a compressive force may be applied to the first sheet 710 and / or the second sheet 720 toward one side of the droplet 740. The compressive force may be applied simultaneously with or approximately simultaneously with the activation or movement of the magnet 750.

[0234] The magnet 750 may aggregate the iron particles 741 into one or more iron beads 742. Thereby, the iron particles 741 may be removed from the suspended state in the droplet 740. In addition, a compressive force may move the droplet 740 away from the magnet 750. In some cases, the movement of the droplet may sort or extract iron particles from the droplet 740.

[0235] FIG. 7C shows another view of the microfluidic device 700. The compressive force is removed or reduced, and the first sheet 710 and the second sheet 720 return to their initial positions. The compressive force applied in FIG. 7B and removed or reduced in FIG. 7C may move the droplet 740 away from the magnet 750. Since the magnet 750 can attract and / or limit the movement of the iron beads 742, iron substances can be sorted from the droplet 740 by the movement of the droplet.

[0236] FIG. 8 is a flowchart showing an exemplary operation 800 for removing suspended iron particles from a microfluidic droplet. The operation 800 will be described with respect to the microfluidic device 700 of FIGS. 7A - 7C, but the operation 800 may be performed by any other suitable system or device.

[0237] The operation 800 begins at block 802 where a microfluidic droplet with suspended iron particles is moved by mechanical operation to a region near a magnet. For example, the microfluidic droplet 740 may be moved near the magnet 750. In some examples, the droplet 740 may be moved through the application of a force to one or more sheets as described herein.

[0238] Next, the magnet is activated in block 804. In some cases, this step is optional, as shown by the dashed line in Figure 8. In some examples, the magnet 750 may be a permanent magnet and a stationary magnet. In some other examples, the magnet 750 may be activated by moving it toward the microfluidic droplet 740, or the magnet 750 may be an electromagnet and receive power. The magnet 750 may dislodge the iron particles from their suspension and cause them to aggregate toward the magnet 750. In some examples, the iron particles 741 may aggregate into one or more iron beads 742.

[0239] Next, in block 806, the microfluidic droplets 740 may be moved away from the magnet 750 through mechanical actuation. For example, a compressive force may be applied to the first sheet 710 or the second sheet 720 to move the microfluidic droplets 740 away from the magnet 750. The step of moving the microfluidic droplets 740 away from the magnet may be used to separate iron material from the microfluidic droplets 740.

[0240] In some cases, the iron substance may be returned to a suspension state within the droplet through mechanical actuation. Such an example will be described later, along with Figures 9-10.

[0241] Figure 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 in Figure 1A.

[0242] The droplet 940 may contain undispersed iron or non-ferrous particles. A compressive force capable of compressing or deforming the droplet 940 may be applied to the first sheet 910 and / or the second sheet 920 (not shown). In some cases, the compressive force may be applied to the center or middle of the droplet 940.

[0243] Figure 9B shows another diagram 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 incompressible or undeformable state. The transition from a compressed state to an incompressible state (or vice versa) may suspend one or more iron particles 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 iron particles and non-ferrous particles 941 throughout the droplet 940. In some cases, the compressive force may be repeatedly applied and removed to disperse the particles more evenly.

[0244] Figure 10 is a flowchart illustrating an exemplary operation 1000 for dispersing particles in a microfluidic droplet. Operation 1000 will be described below with respect to the microfluidic device 900 shown in Figures 9A-9B, but operation 1000 may be performed by any other suitable system or device.

[0245] Operation 1000 begins in block 1002, where a compressive force is applied by mechanical actuation to a microfluidic droplet containing iron and / or non-ferrous particles 941 to be suspended. The compressive force may be provided by a mechanical actuation force, which may also 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.

[0246] Next, in block 1004, the compressive force may be released or reduced in order to suspend the iron particles and / or non-ferrous particles 941 within the droplet 940. The 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 iron particles or non-ferrous particles within the droplet 940.

[0247] In some cases, additional agitation of the microfluidic droplets 940 may be desirable to enhance the particle distribution within the microfluidic droplets 940. To provide this additional agitation, a compressive force may be repeatedly applied and removed (or reduced) over 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 a compressive force. If the number of compression cycles is less than the predetermined number, 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, operation 1000 may terminate.

[0248] In some cases, heating of droplets may be desirable as part of droplet analysis or assay. However, droplets may move during the heating process 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.

[0249] Figure 11A shows a portion of another microfluidic device 1100. As shown in Figure 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 in Figure 1A. The first sheet 1110 may include one or more pinning posts 1150 attached to the first surface 1111 of the first sheet 1110. In some examples, the pinning posts 1150 may be attached to the second sheet 1120.

[0250] The second sheet 1120 may include a first surface 1121 and a second surface 1122. The first surface 1121 may be positioned toward (for example, adjacent to) the gap 1130. As shown, the heater 1140 may be positioned on the second surface 1122 of the second sheet 1120, opposite the pinning post 1150. The pinning post 1150 may provide on the first surface 1111 a feature that allows the droplet 1160 to be temporarily bound, thereby restricting the movement of the droplet 1160. The droplet 1160 may initially be positioned away from the heater 1140 and the pinning post 1150.

[0251] Figure 11B shows another diagram of the microfluidic device 1100. In Figure 11B, compressive force may be applied to the first sheet 1110 and / or the second sheet 1120 to reduce the gap 1130 and move the droplet 1160 toward the heater 1140 and pinning post 1150.

[0252] Figure 11C shows another diagram of the microfluidic device 1100. In Figure 11C, the droplet 1160 is positioned in contact with the pinning post 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. Since the pinning post 1150 engages with the droplet 1160, the movement of the droplet 1160 may be reduced. Reduced movement may be particularly advantageous when procedures such as heating by the heater 1140 are performed on the droplet 1160.

[0253] Figure 12 is a flowchart illustrating an exemplary operation 1200 for manipulating a microfluidic droplet in conjunction with a pinning post. Operation 1200 is described below with respect to the microfluidic device 1100 shown in Figures 11A-11C, but operation 1200 may be performed by any other suitable system or device.

[0254] Operation 1200 begins in block 1202, where a microfluidic droplet 1160 is moved by mechanical operation into the area of ​​the microfluidic device 1100, including the pinning post 1150. The mechanical operation may include the use of compressive force, as described with respect to Figures 1A-1C and 2.

[0255] Next, in block 1204, the microfluidic droplet 1160 is heated in the region of the pinning post 1150. In some examples, the heating is provided by a heater 1140. Then, in block 1206, the microfluidic droplet 1160 may be moved away from the region of the microfluidic device including the pinning post 1150 by mechanical operation.

[0256] In some examples, wells may be arranged within a microfluidic device for containing and processing microfluidic droplets. An example is illustrated in conjunction with Figures 13A-13E and 14.

[0257] Figure 13A shows a portion of the microfluidic device 1300. As shown in Figure 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 in Figure 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.

[0258] 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 enable gravity to assist in receiving or moving the droplet 1340 into the well 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 keep the droplet 1340 within the well even though the well 1355 is upside down.

[0259] FIG. 13B shows another view of the microfluidic device 1300. In FIG. 13B, the droplet 1340 is moved toward the opening 1325 in the second sheet 1320 by mechanical operation. 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.

[0260] FIG. 13C shows another view of the microfluidic device 1300. In FIG. 13C, the droplet 1340 is within the well 1355 of the heater 1350. The compressive force may be removed or reduced, which enables the first sheet 1310 and the second sheet 1320 to return to their initial positions. The well 1355 may advantageously restrict the movement of the droplet 1340, especially during heating by the heater 1350.

[0261] FIG. 13D shows another view of the microfluidic device 1300. In FIG. 13D, the droplet 1340 is withdrawn from the well 1355 of the heater 1350 by mechanical operation. 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, a compressive force may be applied to the region of the microfluidic device 1300 related to the direction for receiving the droplet 1340.

[0262] Figure 13E shows the state in which the compressive force on the microfluidic device 1300 has been removed or reduced. The first sheet 1310 and the second sheet 1320 may be returned to their initial positions, and the droplet 1340 may be positioned away from the well 1355 and the heater 1350.

[0263] Figure 14 is a flowchart illustrating an exemplary operation 1400 for manipulating microfluidic droplets in conjunction with a well. Operation 1400 is described below with respect to the microfluidic device 1300 shown in Figures 13A-13E, but operation 1400 may be performed by any other suitable system or device.

[0264] The operation begins in block 1402, where the microfluidic droplet is moved into the well by mechanical actuation. For example, a compressive force may be applied to the first sheet 1310 or the second sheet 1320 to move the microfluidic droplet 1340 into the well 1355.

[0265] Next, in block 1404, the microfluidic droplets 1340 may be heated within the well. For example, a heater 1350 may heat the microfluidic droplets 1340 within well 1355. Next, in block 1406, the microfluidic droplets 1340 are moved away from the well by mechanical operation. 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 microfluidic droplets 1340 out of well 1355.

[0266] Figure 15A shows a portion of the microfluidic device 1500. As shown in Figure 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 in Figure 1A. The electrode 1550 may be connected to an electrical circuit (not shown) that provides a high-energy electric field associated with electroporation (creation of temporary pores or openings in the cell membrane). As illustrated, the droplet 1540 may be positioned away from the electrode 1550.

[0267] Figure 15B shows another diagram of the microfluidic device 1500. In Figure 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 and move the droplet 1540 toward the electrode 1550.

[0268] Figure 15C shows another diagram of the microfluidic device 1500. 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. Mechanical actuation in Figure 15B positions the droplet 1540 on the electrode 1550. Electroporation may be performed on the droplet 1540 using the electrode 1550.

[0269] Figure 16 is a flowchart illustrating an exemplary operation 1600 for providing electroporation. Operation 1600 is described with respect to the microfluidic device 1500 shown in Figures 15A-15C, but operation 1600 may be performed by any other suitable system or device.

[0270] Operation 1600 begins in block 1602, where a microfluidic droplet is moved onto the electroporation electrode by mechanical actuation. For example, a compressive force may be applied to the first sheet 1510 and / or the second sheet 1520 to move the microfluidic droplet 1540 onto the electrode 1550.

[0271] Next, in block 1604, the electrode 1550 provides 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 cellular material within the microfluidic droplet 1540.

[0272] Next, in block 1606, the microfluidic droplets are moved away from the electrodes by mechanical operation. For example, a compressive force may be used in conjunction with the first sheet 1510 and the second sheet 1520 to move the microfluidic droplets away from the electrode 1550.

[0273] Figure 17 shows 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 contained in a housing or base station that can be coupled to the cartridge 1710.

[0274] Cartridge 1710 may be an example of the microfluidic devices 100, 300, 500, 700, 900, 1100, 1300, and 1500 shown in Figures 1, 3, 5, 7, 9, 11, 13, and 15, respectively. 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 a hydrophobic and oleophobic layer on one or more surfaces. The first heater 1750 may be connected to a 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 the input port 1711. Although only one input port 1711 is shown in the figure, in other examples the cartridge 1710 may include any number of input ports.

[0275] The pressure actuator 1720 may be in contact with or otherwise connected to the cartridge 1710. As shown in the illustration, the pressure actuator 1720 may be connected to the first seat 1712. In another example, the pressure actuator 1720 may be connected to the second seat 1714. The pressure actuator 1720 may also be connected to a controller 1780. The controller 1780 may cause the pressure actuator 1720 to selectively apply one or more compressive forces to the first seat 1712 and / or the second seat 1714. The compressive forces may manipulate the position of any droplet in the gap 1716. An optical sensor 1718 may detect the presence and / or location of a droplet (e.g., a microfluidic droplet) in the gap 1716. The optical sensor 1718 may be connected to the controller 1780. In this configuration, data from the optical sensor may be used to guide or orient the pressure actuator 1720.

[0276] The magnet 1760 may be located in close proximity to or on top of the cartridge 1710. The operation of the magnet 1760 may be controlled by the controller 1780. Similarly, the second heater 1770 may be located in close proximity to or on top of the cartridge 1710 and may also be controlled by the controller 1780.

[0277] The 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 in order to perform one or more operations as described herein.

[0278] Figure 18A shows 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 in Figure 1A. The stylus 1850 may selectively provide compressive force to either the first sheet 1810 or the second sheet 1820 (as shown, the stylus 1850 selectively provides compressive force to the first sheet 1810). To move the droplet 1840 as described above in conjunction with Figures 1-17, the compressive force from the stylus 1850 may selectively reduce the gap 1830 in some areas of the microfluidic device 1800. For example, the position and compression force of the stylus 1850 may be controlled by the controller 1780 shown in Figure 17.

[0279] 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 is 10-6 ~10 -15 It may also be in liters. Furthermore, 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 for applying compressive force to the first sheet 1810. In some other examples, the tip of the stylus 1850 may be coated with a lubricant.

[0280] Figure 18B shows the stylus 1850 and possible associated end profiles 1860. The end profiles 1860 are intended to be illustrative, not limiting (for example, end profiles 1860 are not an exhaustive list of all possible end profiles). Therefore, other end profiles for the stylus 1850 are also possible. Some end profiles 1860 may allow the droplet 1840 to be moved or manipulated more effectively. For example, circular, rectangular, or elliptical profiles may allow the droplet 1840 to be moved more effectively.

[0281] Figure 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, second sheet 1820, gap 1830, and stylus 1850 shown in Figure 18A. The stylus 1950 may include an insulated and / or exposed electrode 1955. In some examples, the electrode 1955 may be positioned toward the tip of the stylus 1950 so as to be in contact with at least one of the first sheet 1910 or the second sheet 1920.

[0282] In some examples, a voltage (e.g., potential) that attracts the droplet 1940 may be provided to the electrode 1955. When the stylus 1950 is placed in contact with at least one of the sheets of the microfluidic device 1900 (illustrated here as the first sheet 1910), the sheet in question may act as, or be, a hydrophobic and oleophobic dielectric that separates the electrode 1955 from the droplet 1940.

[0283] The applied or supplied voltage may be sufficient to affect or control the surface tension of the droplet 1940. In this configuration, the stylus 1950 may attract and / or move the droplet 1940 in the gap 1930 by providing a voltage to the electrode 1955 without applying a compressive force, and then by moving the position of the stylus 1950 relative to the first sheet 1910 and the second sheet 1920.

[0284] Figure 19B shows another diagram of the microfluidic device 1900. As shown, the stylus 1950 may move planarly relative to the first sheet 1910 and the second sheet 1920. While the stylus 1950 is moved planarly, the droplet 1940 may move following the stylus 1950 while the electrode 1955 is energized with a sufficient voltage. Thus, any of the droplet operations described with respect to Figures 1-17 may be performed by energizing the electrode 1955 and moving the stylus 1950, instead of using compressive force.

[0285] Figure 20 is a flowchart illustrating an exemplary operation 2000 for manipulating a microfluidic droplet. Operation 2000 is described with respect to the microfluidic device 1900 in Figures 19A and 19B, but operation 2000 may be performed by any other suitable system or device. Operation 2000 begins in block 2002, in which a potential is provided to an electrode positioned on or connected to a stylus. For example, a voltage may be provided to an electrode 1955 positioned on or near the tip of a stylus 1950. In addition, the stylus 1950 may be in contact with at least one sheet of the microfluidic device 1900 (e.g., the first sheet 1910 or the second sheet 1920). The applied or provided voltage may be sufficient to affect or control the surface tension of the droplet 1940.

[0286] Next, the stylus is moved in block 2004. For example, the stylus 1950 may be moved relative to the first sheet 1910 and the second sheet 1920. Since a sufficient voltage is applied to or provided to the electrode 1955 (in block 2002), the droplet 1940 can move in accordance with the movement of the stylus 1950.

[0287] Next, in block 2006, the voltage or potential is removed from the electrode. For example, in block 2004, the stylus 1950 may be moved to position the droplet 1940 within a predetermined processing zone. Since the movement is complete, the voltage or potential may be removed from the electrode 1955.

[0288] 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 could be examples of the first sheet 1910, second sheet 1920, gap 1930, stylus 1950, and electrode 1955 shown in Figure 19A.

[0289] In contrast to the microfluidic device 1900, the microfluidic device 2100 may use a combination of compressive force and applied voltage to manipulate the droplet 2140. As shown, the stylus 2150 may be positioned on one side of the droplet 2140. The stylus 2150 may provide a compressive force to reduce the gap 2130 while a voltage is provided to the electrode 2155 to move the droplet 2140. In this configuration, the droplet 2140 may be moved by a combination of compressive force and electromotive force (e.g., voltage). For example, while the stylus 2150 provides a compressive force and a voltage is applied to the electrode 2155, the stylus 2150 may be moved to change the position or location of the droplet 2140 within the gap 2130. The compressive force may be provided to either the first sheet 2110 or the second sheet 2120. Figure 21A shows an example in which the stylus 2150 provides a compressive force to the first sheet 2110.

[0290] Figure 21B shows another diagram of the microfluidic device 2100. In this diagram, the stylus 2150 has been moved to a new position relative to the first sheet 2110 and the second sheet 2120. The droplet 2140 is moving in response to the compression previously provided by the stylus 2150 and the voltage provided to the stylus 2150. Thus, in Figure 21B, the compressive force is removed from the first sheet 2110 and the voltage is removed from the electrode 2155.

[0291] Figure 22 is a flowchart illustrating an exemplary operation 2200 for manipulating a microfluidic droplet. Operation 2200 is described with respect to the microfluidic device 2100 of Figures 21A and 21B, but operation 2200 may be performed by any other suitable system or device. The operation may begin in block 2202 in which 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 the 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.

[0292] Next, in block 2204, a potential is supplied to electrodes positioned or connected to the stylus. For example, a voltage may be supplied to an electrode 2155 positioned 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 applied or supplied voltage may be sufficient to affect or control the surface tension of the droplet 2140.

[0293] Next, the stylus is 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 configuration, the droplet 2140 may be manipulated or moved using a combination of the compressive force provided by the stylus 2150 and the voltage applied to the electrode 2155.

[0294] Next, in block 2208, the potential is removed from the electrodes. For example, the voltage may be removed from electrode 2155. Next, in block 2210, the compressive force is removed from at least one sheet of the microfluidic device. For example, the stylus 2150 may be moved away from the first sheet 2110 or the second sheet 2120. Since the droplet 2140 has been moved into a predetermined (e.g., desired) position in block 2206, the compressive force and voltage may be removed from the stylus 2150.

[0295] In some examples, iron particles may be suspended in microfluidic droplets to assist in processing or assays. After one or more processing steps are completed, the iron particles may be removed from the droplets for further processing.

[0296] Figure 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 could be examples of the first sheet 1910, the second sheet 1920, the gap 1930, and the stylus 1950 in Figure 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 activated and deactivated 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 toward the tip of the stylus 2350 (not shown). In this form, the magnetic field strength at or near the tip of the stylus 2350 may be controlled and / or variable. Multiple iron particles 2341 may be distributed (suspended) throughout the entire droplet 2340.

[0297] Figure 23B shows another diagram of the microfluidic device 2300. In this diagram, the magnet 2356 is activated or enabled. For example, the magnet 2356 may be an electromagnet that can be activated by the application of power. In another example, the magnet 2356 may be a permanent magnet that can be moved toward the droplet 2340. The magnet 2356 may attract or aggregate the iron particles 2341 to form one or more iron beads 2342. Thus, the iron particles 2341 may escape from their suspension in the droplet 2340.

[0298] Figure 23C shows another diagram 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 beads 2342 from the droplet 2340.

[0299] Figure 24 is a flowchart illustrating an exemplary operation 2400 for extracting suspended iron particles from a microfluidic droplet. Operation 2400 is described in relation to the microfluidic device 2300 shown in Figures 23A-23C, but operation 2400 may be performed by any other suitable system or device.

[0300] Operation 2400 begins in block 2402, where the stylus with the magnet is moved to a region near a droplet containing suspended iron particles. For example, the stylus 2350 with the magnet 2356 may be moved near a droplet 2340 containing suspended iron particles 2341.

[0301] Next, in block 2404, a magnet inside or on the stylus is activated. For example, magnet 2356 may be an electromagnet that can be activated by the application of power. In another example, magnet 2356 may be moved toward the tip of the stylus 2350. In this configuration, magnet 2356 may dislodge the iron particles 2341 from their suspension and cause them to aggregate toward magnet 2356. In some cases, the aggregated iron particles 2341 may form iron beads 2342.

[0302] Next, in block 2406, the stylus may be moved away from the droplet. For example, the movement of the stylus 2350 may cause the iron beads 2342 to move away from the droplet 2340, thereby separating the iron particles 2341 from the droplet 2340.

[0303] In some cases, the iron substance may be returned to a suspension state within the droplet through mechanical actuation. Such other examples will be discussed later, along with Figures 25-26.

[0304] Figure 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 in Figure 23A. In some examples, the magnet 2556 may be released by either moving the magnet 2556 away from the tip of the stylus 2550, or by removing the power if the magnet 2556 is an electromagnet.

[0305] The droplet 2540 may contain undispersed iron or non-ferrous particles. A compressive force capable of compressing or deforming the droplet 2540 may be applied to the first sheet 2510 and / or the second sheet 2520 (not shown). In some cases, the compressive force may be applied to the center or middle of the droplet 2540 by a stylus 2550.

[0306] Figure 25B shows another diagram 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 incompressible or undeformable state. The transition from a compressed state to an incompressible state (or vice versa) may suspend one or more iron particles 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 iron particles and non-ferrous particles 2541 throughout the droplet 2540. In some cases, the compressive force may be repeatedly applied and removed to disperse the particles more evenly.

[0307] Figure 26 is a flowchart illustrating an exemplary operation 2600 for dispersing particles in a microfluidic droplet. Operation 2600 is described below with respect to the microfluidic device 2500 shown in Figures 25A and 25B, but operation 2600 may be performed by any other suitable system or device.

[0308] Operation 2600 begins in block 2602, where the magnet is deactivated. The magnet 2556 may be deactivated, for example, by moving the magnet 2556 away from the tip of the stylus 2550, or by removing power from the electromagnet comprising the magnet 2556.

[0309] Next, in block 2604, a compressive force is applied to the microfluidic droplets containing iron and / or non-ferrous particles to be suspended by mechanical actuation. For example, the compressive force may be provided by a mechanical actuation force, which may be applied by the 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.

[0310] Next, in block 2606, the compressive force may be released or reduced in order to suspend the iron particles and / or non-ferrous particles in the droplet. For example, the 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 iron particles or non-ferrous particles within the droplet 2540.

[0311] In some cases, additional agitation of the microfluidic droplets 2540 may be desirable to enhance the particle distribution within the microfluidic droplets 2540. To provide this additional agitation, a compressive force may be repeatedly applied and removed (or reduced) over a predetermined number of cycles. Thus, in block 2608, the number of completed compression cycles is determined. A completed compression cycle may include the application and removal or reduction of a compressive force. If the number of compression cycles is less than the predetermined number, 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, operation 2600 may terminate.

[0312] Next, in block 2604, a compressive force is applied to the microfluidic droplets containing the iron and / or non-ferrous particles 2541 to be suspended by mechanical actuation. The compressive force may be provided by a mechanical actuation force, which may be applied to the first sheet 2510 and / or the second sheet 2520 through the stylus 2550. The compressive force may deform or stretch the droplets 2540.

[0313] Next, in block 2604, the compressive force may be released or reduced in order to suspend the iron particles and / or non-ferrous particles 2541 in the droplet 2540. The removal or reduction of the compressive force may cause the droplet 2540 to return to a spherical or semi-spherical shape, which at least partially suspends the iron particles or non-ferrous particles within the droplet 2540.

[0314] In some examples, the stylus may include or be connected to a reservoir or other liquid container that can be used to hold the liquid drawn from the gap of the microfluidic device. Exemplary devices are described later in conjunction with Figures 27–38.

[0315] Figure 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 in Figure 19A. The first sheet 2710 may include a slit septum or other configurable opening (not shown) that may allow droplets 2740 to be drawn in (removed) from the gap 2730.

[0316] Figure 27B shows another diagram of the microfluidic device 2700. A septum 2711 may be contained on one of the sheets of the microfluidic device 2700. As shown, the septum 2711 is located on (contained within) 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 the liquid from "leaking" from the first sheet 2710. The droplet 2740 may move beneath the septum 2711.

[0317] Figure 27C shows another diagram of the microfluidic device 2700. A stylus 2750 may be positioned substantially on the septum 2711. The stylus 2750 may include a lumen 2751 which may be connected to the septum 2711 and configured to receive a droplet 2740. In some examples, negative pressure may be provided to and / or through the stylus 2750 to draw the droplet 2740 into the stylus 2750 through the septum 2711. The stylus 2750 may then move the droplet 2740 to another location within the microfluidic device 2700.

[0318] Figure 28 is a flowchart illustrating an exemplary operation 2800 for aspirating liquid from a microfluidic device. Operation 2800 is described below with respect to the microfluidic device 2700 shown in Figures 27A-27C, although operation 2800 may be performed by any other suitable system or device.

[0319] Operation 2800 begins in block 2802, where the droplet is positioned close to the septum. For example, in any executable operation provided herein, the droplet 2740 may be moved substantially below the septum 2711.

[0320] Next, in block 2804, the stylus is moved onto the septum. For example, the stylus 2750 may be moved onto the septum 2711. The stylus 2750 may include a lumen 2751 that can be connected to the septum 2711.

[0321] Next, in block 2806, the droplet is drawn into the stylus. For example, negative pressure may be applied by or through the stylus 2750. This negative pressure may draw the droplet 2740 into the lumen 2751.

[0322] In some examples, liquid may be supplied to or returned to the microfluidic device through the septum. Several examples are described below in conjunction with Figures 29 and 30.

[0323] Figure 29A shows 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 in Figure 27A. Thus, the first sheet 2910 may include a septum 2911.

[0324] In some examples, a liquid which may be contained within the stylus 2950 may be injected into the gap 2930 through the septum 2911. The stylus 2950 may be positioned on the septum 2911 before the liquid is injected. In some examples, the lumen 2951 of the stylus 2950 may be connected to the septum 2911. Positive pressure may be applied to the lumen 2951 to push the liquid out of the stylus 2950 and form a droplet 2940 in the gap 2930. Therefore, the applied pressure may exceed the closing force of the septum 2911.

[0325] Figure 29B shows another diagram of the microfluidic device 2900. The stylus 2950 may be moved away from the droplet 2940. Furthermore, to reduce the gap 2930, the stylus 2950 may provide a compressive force to the first sheet 2910 (and / or the second sheet 2920). The reduced gap 2930 may move the droplet 2940 relative to the first sheet 2910 and the second sheet 2920.

[0326] Figure 29C shows another diagram of the microfluidic device 2900. The stylus 2950 may be moved to move the droplet 2940. Once the movement of the droplet 2940 is complete, the stylus 2950 may then be positioned to remove the compression source to the first sheet 2910 and / or the second sheet 2920. In this way, the stylus 2950 can act as a pipette for moving the liquid (e.g., droplet 2940) within the microfluidic device 2900.

[0327] Figure 30 is a flowchart illustrating an exemplary operation 3000 for manipulating a liquid in a microfluidic device. Operation 3000 is described below with respect to the microfluidic device 2900 shown in Figures 29A-29C, but operation 3000 may be performed by any other suitable system or device.

[0328] Operation 3000 begins in block 3002, where a droplet is dispensed from the stylus of the microfluidic device through the septum into the gap. For example, the stylus 2950 may be positioned on the septum 2911. Positive pressure may be applied to the stylus 2950 and / or the lumen 2951 to inject the liquid into the gap 2930 through the septum 2911.

[0329] Next, in block 3004, mechanical actuation selectively reduces the distance between the two sheets 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 the stylus 2950 in close proximity to (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.

[0330] Next, the distance between the two sheets in block 3006 is restored. For example, the compressive force applied in block 3004 may be removed, which allows 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.

[0331] In some examples, the stylus may include additional devices or systems that can be used to process droplets within the stylus. Some examples are described below in conjunction with Figures 31-38.

[0332] Figure 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 in Figure 18A. The stylus 3150 may include a temperature control element 3155. Any liquid drawn from the gap 3130 through the temperature control element 3155 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 positioned below a septum 3111 which may be contained within the first sheet 3110. The stylus 3150 may be positioned on top of the septum 3111. In some examples, the temperature control element 3155 may provide heating or cooling to the surrounding area based on a control voltage or signal, etc.

[0333] Figure 31B shows another diagram of the microfluidic device 3100. The droplet 3140 may be drawn into the stylus 3150 through the septum 3111 from the gap 3130. In this configuration, while the droplet 3140 is inside the stylus 3150, it may undergo temperature processing provided by the temperature control element 3155. In some examples, the stylus 3150 and / or the temperature control element 3155 may include a temperature sensor (not shown). A controller (not shown) may monitor or control the temperature of the droplet 3140 as part of the temperature processing. For example, the controller may monitor the temperature of the droplet 3140 and control the temperature provided by the temperature control element 3155.

[0334] Figure 31C shows another diagram of the microfluidic device 3100. After temperature treatment, droplets 3140 may be injected into the gap 3130 through the septum 3111 in the first sheet 3110.

[0335] Figure 32 is a flowchart illustrating an exemplary operation 3200 for heat-treating a liquid in a microfluidic device. Operation 3200 is described below with respect to the microfluidic device 3100 shown in Figures 31A-31C, but operation 3200 may be performed by any other suitable system or device.

[0336] The operation begins in block 3202, where the stylus is positioned on the septum and droplet. For example, the stylus 3150 may be positioned on the septum 3111, which is on the droplet 3140.

[0337] Next, in block 3204, the droplet is drawn into the stylus through the septum. For example, negative pressure may be provided to the stylus 3150. Accordingly, the droplet 3140 may be drawn into the stylus 3150 through the septum 3111.

[0338] Next, the temperature of the droplet is 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.

[0339] Next, in block 3208, a droplet is injected into the gap through the septum. For example, a temperature treatment may be completed. Positive pressure may be applied to the stylus 3150, which causes the droplet 3140 to be injected into the gap 3130 through the septum 3111. In some examples, the stylus 3150 may be repositioned relative to the first sheet 3110 and / or the second sheet 3120 before the droplet 3140 is returned to the gap 3130.

[0340] Figure 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 in Figure 18A. The stylus 3350 may include a temperature control element 3355. The temperature control element 3355 may be positioned toward a portion of the stylus 3350 that can contact the first sheet 3310. The stylus 3350 and the temperature control element 3355 are positioned on the first sheet 3310 on the droplet 3340. In this configuration, the temperature control element 3355 may provide heat to the droplet 3340 through the first sheet 3310 for any temperature-controlled processing.

[0341] In some examples, the temperature control element 3355 may include a temperature sensor (not shown). In this configuration, a controller (also not shown) may control the temperature of the droplet 3340 to be within a desired temperature range.

[0342] Figure 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 in Figure 18A. The stylus 3450 may include a light sensor 3455 and a light source 3456. In some examples, a reaction occurring or occurring in a droplet 3440 may be monitored by sensing light transmitted or reflected through the droplet 3440. Thus, the droplet 3440 may be drawn into a stylus 3450 that can detect and / or measure transmitted and / or reflected light. The amount of detected light may be associated with the progress or completion of the reaction.

[0343] In some examples, a stylus 3450 may be positioned on a septum 3411 contained within a first sheet 3410 to measure the reaction. In addition, a droplet 3440 may be positioned below the septum 3411.

[0344] Figure 34B shows another diagram of the microfluidic device 3400. As shown, the droplet 3440 may be drawn into the stylus 3450 through the septum 3411. 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 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 detected light may be associated with the progress or completion of the reaction. In this manner, the detected light may indicate the progress or completion of the reaction.

[0345] Figure 34C shows another diagram 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 configuration, after determining the transmission or refraction of light through the droplet 3440, the droplet 3440 may be returned to the gap 3430 for further processing.

[0346] Figure 35 is a flowchart illustrating an exemplary operation 3500 for performing a reaction measurement. Operation 3500 will be described below with respect to the microfluidic device 3400 shown in Figures 34A-34C, but operation 3500 may be performed by any other suitable system or device.

[0347] The operation begins in block 3502, where the stylus is positioned on the septum and droplet. For example, the stylus 3450 may be positioned on the septum 3411, which is on the droplet 3440.

[0348] Next, in block 3504, the droplet is drawn into the stylus through the septum. For example, negative pressure may be provided to the stylus 3450. Accordingly, the droplet 3440 may be drawn into the stylus 3450 through the septum 3411.

[0349] Next, the droplet reaction is monitored 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 the light transmitted and / or reflected from the droplet 3440. In some cases, the transmitted and / or reflected light may be associated with the rate of progress of the reaction occurring within the droplet 3440.

[0350] Next, in block 3508, a droplet is injected into the gap through the septum. For example, the reaction may have been completed. Positive pressure may be applied to the stylus 3450, which causes the droplet 3440 to be injected into the gap 3430 through the septum 3411. In some examples, the stylus 3450 may be repositioned relative to the first sheet 3410 and / or the second sheet 3420 before the droplet 3440 is returned to the gap 3430.

[0351] Figure 36 shows 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 in Figure 18A. The stylus 3650 may include a light source 3655. The light source 3655 may be positioned toward a portion of the stylus 3650 that can contact the first sheet 3610.

[0352] The photodetector 3621 may be facing the light source 3655. As shown, the photodetector 3621 may be positioned on the second sheet 3620. When the droplet 3640 is positioned between the light source 3655 and the photodetector 3621, the photodetector 3621 may detect transmitted and / or reflected light. Thus, the light source 3655 and the photodetector 3621 may monitor the progress of the reaction as described with respect to Figures 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 positioned on the second sheet 3620.

[0353] Figure 37A shows 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 in Figure 18A. The stylus 3750 may include a sonication probe 3755. In some examples, the sonication probe 3755 may deliver acoustic and / or ultrasonic stimuli to the droplet 3740. The sonication probe 3755 may be any viable piezoelectric device.

[0354] In some examples, a stylus 3750 may be positioned on a septum 3711 contained within a first sheet 3710 to deliver sound wave or ultrasonic stimulation. In addition, a droplet 3740 may be positioned below the septum 3711.

[0355] Figure 37B shows another diagram of the microfluidic device 3700. As shown, the droplet 3740 may be drawn into the stylus 3750 through the septum 3711. Once the droplet 3740 is inside the stylus 3750, the sonication probe 3755 may be activated or enabled, which allows sound waves and / or ultrasound to be delivered to the droplet 3740. The delivery of the sound wave stimulus may be controlled by a controller (not shown).

[0356] Figure 37C shows another diagram 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 the septum 3711 in the first sheet 3710. In this configuration, after delivering an acoustic stimulus to the droplet 3740, the droplet 3740 may be returned to the gap 3730 for further processing.

[0357] Figure 38 is a flowchart illustrating an exemplary operation 3800 for performing sound wave processing. Operation 3800 will be described below with respect to the microfluidic device 3700 shown in Figures 37A-37C, but operation 3800 may be performed by any other suitable system or device.

[0358] The operation begins in block 3802, where the stylus is positioned on the septum and droplet. For example, the stylus 3750 may be positioned on the septum 3711, which is on the droplet 3740.

[0359] Next, in block 3804, the droplet is drawn into the stylus through the septum. For example, negative pressure may be provided to the stylus 3750. Accordingly, the droplet 3740 may be drawn into the stylus 3750 through the septum 3711.

[0360] Next, in block 3806, sonic treatment may be delivered to the droplet. For example, a sonication probe 3755 may apply sonic and / or ultrasonic treatment to the droplet 3740.

[0361] Next, in block 3808, a droplet is injected into the gap through the septum. For example, sonic processing may be completed. Positive pressure may be applied to the stylus 3750, which causes the droplet 3740 to be injected into the gap 3730 through the septum 3711. In some examples, the stylus 3750 may be repositioned relative to the first sheet 3710 and / or the second sheet 3720 before the droplet 3740 is returned to the gap 3730.

[0362] Figure 39 shows a block diagram of 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 device 3929, a processor 3930, and a memory 3940.

[0363] In some examples, a pressure actuator 3920 connected to the processor 3930 may be used to provide forces, including compressive and actuation forces, to one or more sheets of a microfluidic cartridge. In some examples, the pressure actuator 3920 may use mechanical, pneumatic, and / or electric actuators to provide the compressive and / or actuation forces. The compressive 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 way, droplets may be drawn out of the gap in the microfluidic device and into the stylus.

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

[0365] One or more heaters 3924, also connected to the processor 3930, may be used to provide heat to one or more microfluidic droplets. The heat may be used during the analysis or assay of the microfluidic droplets. In some examples, the heater (e.g., heating element) may be positioned close to the side of a hydrophobic and oleophobic sheet. In some other examples, the heater may be positioned within a stylus.

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

[0367] The optical sensor 3927, also connected to the processor 3930, may detect the presence and / or location of any droplet, such as any microfluidic droplet placed between two or more hydrophobic and oleophobic sheets.

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

[0369] The sonic device 3929, also connected to the processor 3930, may provide sonic or ultrasonic processing to 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 connected to the memory 3940, may be one or more suitable processors capable of executing scripts or instructions of one or more software programs stored in 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 that may be located on a hydrophobic and oleophobic sheet. Thus, the processor 3930 may use the septum position data 3941 to position the stylus on any particular septum.

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

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

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

[0375] The processor 3930 may run a magnet control module 3944 to selectively control, enable, and / or disable one or more magnets 3922. In some examples, the execution of the magnet control module 3944 may cause power to be applied to electromagnets contained within the magnets 3922. In some examples, the 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 an electrode control module 3948 to selectively provide an electric field to one or more electrodes. For example, the execution of the electrode control module 3948 may provide one or more large electric fields to electrode 3926 to cause electroporation on the cell membrane in the microfluidic droplet.

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

[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 together with the pressure actuator control module 3942, thereby guiding the application of a compressive force to one or more hydrophobic and oleophobic sheets.

[0379] The processor 3930 may run an acoustic control module 3950 to control the acoustic device 3929. For example, the 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 acoustic stimulation and / or ultrasonic stimulation or processing to the droplet.

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

[0381] cartridge As described above, the cartridges described herein may generally include an elastically deformable first (e.g., upper) sheet, a second (e.g., lower) sheet, and a frame separating the two to form an air gap. The first sheet may be an elastomer material such as polyester (e.g., TPE), natural rubber, synthetic rubber, nitrile rubber, silicone rubber, urethane rubber, chloroprene rubber, or ethylene vinyl acetate (EVA). The second sheet may be the same material or a different material. The sheets of the cartridge are generally planar in structure and may be membranes, layers, etc. The first and second sheets may be held taut on or pressed 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] Figures 40A–40D illustrate examples of cartridges 4006, 4006' as described herein. The cartridges include a frame 4017, a first sheet 4007, and a second sheet 4009. In the examples shown in Figures 40A, 40C, and 40D, the cartridges are divided into three lanes 4005, 4005', and 4005''. In this example, the frame is configured as a partition with three distinct lanes. The exemplary cartridge shown in Figure 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-sections of the cartridges shown in Figures 40C–40D illustrate an example with three lanes formed by the frame / partition 4017. The first sheet 4007 is held taut on the top of the frame, for example by welding and / or adhesive 4013. The second sheet 4009 in this example is also held taut on the frame and welded and / or bonded thereto. An air gap 4011 is formed between the first and second sheets. In Figures 40C and 40D, a portion of the cartridge is shown mounted on a mounting section of a mechanical microfluidic actuator, which has a concave area and multiple vacuum ports for sealing the second (lower) sheet into the mounting area to create a tight thermal connection between the second sheet and the mounting area. Sealing the second sheet 4009 to the base, as shown in Figure 40D, also extends the air gap 4011 to a greater height compared to the unmounted configuration of the cartridge.

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

[0384] Figure 41 shows an exploded view of an example of cartridge 4100. In this example, the cartridge includes a first (e.g., upper) frame 4117 on which a first sheet 4107 is mounted, taut and stretched flat. All four sides of the sheet may be held taut. The sheet may be fixed to the frame by pinning, clamping, welding, gluing, riveting, or other means. In this example, the first sheet 4107 also includes a pair of openings 4133, 4133' configured as input / output windows ("windows") into which a fluid substance (e.g., droplets, drop gloss) can be applied by manual or automatic means (e.g., pipetting). The fluid substance may be added to the air gap through the windows, close to the sides of the windows, so that a mechanical microfluidic actuator can be used to apply force close to the windows to manipulate the droplet (or multiple droplets) within the cartridge by further pulling the droplet into the air gap. The windows 4133, 4133' may be of 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 of any size or any ratio of the surface size of the first sheet. For example, the window may be 50% to 100% of the width of the surface of the first sheet and about 0.1% to 10% of the length of the surface of the first sheet. In some examples, the window is 1 mm to 10 cm in length and 1 mm to 5 cm in width. Larger windows may be used. As described 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 Figure 41 also includes a second frame 4121 to which a second sheet 4109 is attached. The second sheet may be under tension, or it may be configured to be fixed to the base of a mechanical microfluidic actuator, and therefore may be attached more loosely. 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 film, FEP film, etc. The first sheet may be adhesively attached to the first frame by an adhesive, for example, a double-sided adhesive film (e.g., 3M 300LSE, 2 mm 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 fixed to the spacer frame. In some examples, the spacer frame is made of a hydrophobic and oleophobic substance (material), such as PTFE.

[0387] Alternatively, in some examples, only a single frame may be used, which may not include spacers, and the first seat may be attached to the first side of the frame, while the second seat may be attached to the 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] Figures 42A and 42B illustrate another example of a part of the cartridge. In this example, the cartridge is shown 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 made of any suitable material (e.g., polyester, ABS, or a polymer material such as POM (glass fiber reinforced)). Figure 42B shows an enlarged side cross-sectional 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 mm thick, double-sided adhesive). In Figures 40 and 41A-41B, the first and second sheets have a thickness of approximately 20-60 μm (for example, 25-50 μm) and may be formed from an elastomer material.

[0389] Any of these cassettes may include a hydrophobic frame (backbone), such as polypropylene, and may include one or more internal structures, not limited to spacer frames. 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, barcodes, etc.) that can uniquely identify the cassette. The cassettes may be oriented, for example, to be positioned within the base of a mechanical microfluidic actuator in a preferred or exclusive orientation, or they may not be oriented (to be applicable in any orientation). In some cases, the cartridges may include specific "top" and "bottom" parts, and may be marked or coded (including color coding) and / or keyed so that the upper surface is "up" to fit within the base of a mechanical microfluidic actuator.

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

[0391] In Figure 43B, the cartridge is shown mounted on a base of a mechanical microfluidic actuator 4322. In this example, the mechanical microfluidic actuator includes a concave 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. Figure 43B shows the state with an applied suction force; this 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 greater height 4319' (compared to the neutral height 4319).

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

[0393] Figures 44A–44B schematically illustrate two alternative examples of a cartridge installed and fixed in the installation area of ​​a mechanical microfluidic actuator. In Figure 44A, the cartridge includes a frame 4406, and the first sheet 4407 and the second sheet 4409 are mounted on the frame 4406 and separated from each other to form an air gap 4421. In Figure 44A, as in Figures 43B–43C, the installation area 4419 of the mechanical microfluidic actuator includes a vacuum port that fixes the lower (second) sheet 4409 to the surface of the installation area (in contact) so that there is no gap between the installation area and the second sheet; the second sheet is held fixed to the installation area as shown. In Figure 44A, the droplet 4412 may be heated / cooled through the lower (second) sheet by applying thermal energy through a specific sub-region of the installation area of ​​the mechanical microfluidic actuator, or otherwise operated. In this example, the entire installation area is shown to be concave; in other examples, only a portion of the installation area is concave, forming a well (described later) that can hold droplets.

[0394] Figure 44B shows another example of a cartridge held within the mounting area of ​​a mechanical microfluidic actuator. In this example, the cartridge includes a frame 4406, as well as an upper sheet 4407 and a lower sheet 4409; the cartridge is fixed to the mounting area by a fixture such as a clamp 4420 that applies a fixing force to the frame to hold the cartridge immobilely in place. In this example, the mechanical microfluidic actuator 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 Figure 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 actuators described herein may include a fixture (e.g., a clamp, a lock, etc.) that holds the cartridge relative to the mounting area of ​​the mechanical microfluidic actuator.

[0395] As will be described in more detail later, any of these mechanical microfluidic actuators may include a controller and one or more thermal regions; the thermal regions may locally heat / cool the installation region and, therefore, the droplets in the air gap of the cartridge on this portion of the installation region.

[0396] In general, any of the mounting areas of a mechanical microfluidic actuator may include a shape to which the lower sheet of the cartridge can conform, which can have numerous advantages, such as fixing droplets in a specific area (e.g., pinning droplets) and / or enhancing thermal energy transfer. Figures 44C-44E schematically illustrate an example in which the mounting area of ​​a mechanical microfluidic actuator includes a protrusion. In Figure 44C, the cartridge is shown mounted on the mounting area of ​​the mechanical microfluidic actuator 4419'', which includes a rail 4411 (e.g., a rail area). The cartridge may be held downward by a fixture (not shown; clamp, magnet, etc.). Thus, the droplet 4412 between the first sheet 4407 and the second sheet 4409 is held at the center of the air gap 4421 area (e.g., by capillary force) and may be moved within the channel (e.g., in and out of the cross section shown in the figure) by a force applicator that applies a force to reduce the height of the air gap. Figure 44D shows an example in which the mechanical microfluidic actuator 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 Figure 44C. In Figure 44E, the mechanical microfluidic actuator 4419'''' is similar to that shown in Figure 44D but includes a dome-shaped rail 4411''. Any of these examples may, but are not required, include a vacuum manifold with vacuum ports for securing the second sheets 4409, 4409', 4409'' to the outer surface of the installation area. Alternatively or additionally, these devices may include fasteners (such as clamps, locks, or magnetic fasteners) for holding the cartridge in place.

[0397] In general, the methods and apparatus described herein may include the use of rail regions within an air gap. The rail regions may generally have a gap height on either side of the rail region that is smaller than the gap height of the surrounding region in the air gap, such as around the outer perimeter of the air gap. The rail regions may form a raised bed. As described above, the rail regions may be formed by deflecting a second (e.g., lower) sheet of the air gap; in some examples, the second sheet is more rigid than the first sheet (e.g., formed of a relatively hard material), and the rail regions may be formed of the more rigid second sheet. Surprisingly, droplets may avoid the region on the outer perimeter (closer to the rail regions) which has a larger gap height, which may prevent loss of droplet volume, especially for droplets with smaller volumes (e.g., less than 15 μL, less than 10 μL or less, less than 5 μL or less, less than 1 μL or less, less than 1 μL or less, etc.).

[0398] In general, the apparatus described herein may be configured to keep a film (e.g., a membrane, sheet, etc.) taut. The methods and apparatus described herein are configured to provide surface mechanical actuation ("MAOS"), which may include a MAOS cartridge. The surface may be the surface of a film within the cartridge, and as described above, a thin elastic film may be included on the upper and / or lower surfaces of the active area of ​​the cartridge. In general, a droplet may be dispensed between two films (or a film and a second surface) and moved to a target area through compression of the upper and / or lower film to reduce the gap between the two (e.g., the height of the air gap). To allow for repeatable droplet movement, the upper film of the MAOS cartridge may be sufficiently elastic to return to its original shape after compression. In addition, the upper film (and / or, in some examples, the lower film) may be kept taut so as to be planar during use, including after the force on the film has been removed. Tension may be applied by the cartridge itself or by a tensioner engaged with the cartridge. In some cases, the cartridge may be integrated with the tensioner.

[0399] Therefore, the cartridge may include a tensioner, which may be configured as a film tensioning subsystem that stretches the film so that the film is taut, with a force applied to move the droplets so that the film is taut, with a force applied to move the droplets so that the film is taut, with a force applied to move the droplets so that the film is taut, with a force applied to move the droplets. In some cases, the tensioner (e.g., the tensioning subsystem) applies tension to the film by including one or more (e.g., multiple) clamp rib features that fit into recesses located at the top of the cartridge. The film is stretched and taut across the entire surface of the cartridge as it is pushed into the cartridge recesses. The film can then return to its original plane, even after maximum compression has been applied by the MAOS system.

[0400] This is illustrated in Figures 72A-72B, 73A-73C, and 74A-74B. For example, Figure 72A shows an example of the underside of a tensioner 7282 for use with (or to be integrated with) a cartridge 7288. The tensioner may be configured to clamp onto the cartridge such that the lower part of the tensioner (shown in Figure 72A) fits with the upper part of the cartridge (or a portion of the cartridge) to secure the elastic film between them and apply tension. As described above, the film may be transparent or translucent to allow for the visualization of droplets. In Figures 72A-72B, the cartridge includes a plurality of (e.g., eight) lanes 7287. The tensioner includes a plurality of tensioning projections 7884 around the outer periphery of the active region that forms the lanes in which droplets move. In Figure 72A, the projection comprises elongated projections (e.g., ribs) 7284 located on all four sides, where contact with the cartridge is made together with the upper part of the cartridge 7288. Thus, tension is applied to the film in both the x and y directions. The cartridge 7282 (or a portion of the cartridge) shown in Figure 72A may also include a second film on the back surface (not visible in Figure 72A) which may be mounted around the active region (e.g., lane 7287). In some cases, this lower film may be made of the same material as the elastic film described above. The lower film forms the lower surface of the air gap. The lower film may also be driven relative to the device receiving the cartridge in order to drive droplets within the air gap of the cartridge.

[0401] Figure 72B shows an example of the upper surface 7288 of a MAOS cartridge (or a portion of a cartridge). The exemplary cartridge shown includes an elastic membrane 7290 on lanes that is elastically deformable to move droplets. In this example, the membrane is attached to the cartridge 7288 but may be fully tensioned by a tensioner. In Figure 72B, the shown cartridge portion also includes a recessed region 7286 located beneath the upper film, which may receive tensioning projections from a tensioner around the active region.

[0402] In the exemplary cartridge shown in Figure 72B, the film includes an opening 7291 at the first end of the cartridge for loading material into the lanes of the air gap; and a second opening 7292 at the opposite end of each lane from which the material can be removed after being driven through the air gap (by MAOS).

[0403] In some cases, the tensioner may be permanently connected to the cartridge. In some cases, the tensioner may be temporarily connected to the cartridge. When connected to the cartridge, the tensioner may be considered part of the cartridge, for example, as a subsystem of the cartridge. In some cases, the tensioner may be integrated into a device that holds the cartridge and drives the movement of droplets within the cartridge. As described herein, any of these devices may also include a droplet dispenser (e.g., a dispensing robot) which is part of the device.

[0404] Figures 73A–73C illustrate examples of cross-sectional views through exploded assembly diagrams of a cartridge similar to those shown in Figures 72A–72B, including a tensioner 7382. For example, Figure 73A shows the tensioner 7382 above a cartridge portion 7388 that includes an air gap formed between an upper (e.g., elastic) film 7392 and a lower film 7393. The film, configured to receive force to move droplets within the air gap, may be fixed around the end or edge 7398 of the cartridge, for example, by being sealed, as shown in Figure 73A.

[0405] As shown in the enlarged views of Figure 73B (showing the tensioner) and Figure 73C (showing the cartridge side), the cartridge 7388 includes a receiving channel 7396 into which a tensioning projection 7384 from the tensioner can be inserted to apply tension (in both the x and y directions in this example). The portion of the film extending over the receiving channel 7396 is pushed down into the receiving channel 7396. Since the film is fixed around the periphery of the active area (e.g., around the cartridge), this may reduce the risk of slack in the film over the active area.

[0406] In general, tensioners may help define the lanes. In the examples shown in Figures 72A–72B, 73A–73C, and 74A–74B, the tensioners include channels that align with the lanes. The walls forming these channels may also help press and hold the film against the cartridge; this may further help to apply tension to the film, and may also help to isolate the channels from one another by limiting the variation in air gap height to specific channels.

[0407] In some cases, the cartridge may be fixed to a device that drives the droplet movement by a tensioner. For example, the tensioner may act as a cover on the cartridge while clamping the cartridge to the driver device. As shown in Figures 74A-74B, the tensioner may be pressed against and held against the cartridge, for example, by being pressed against and clamped against the cartridge, and locked in place. Alternatively, as described above, the tensioner may be integrated into the cartridge.

[0408] For example, in Figures 74A and 74B, the cartridge 7493 is clamped by the tensioner 7491 such that the tensioning projection (e.g., one or more ridges or elongated tensioners) extends into the tensioner receiver (e.g., a tensioner receiver channel) 7496 on the cartridge. The elastic film 7492 is fixed to the edge of the cartridge (e.g., at the outer circumference 7498) (e.g., by adhesive); when the tensioning projection from the tensioner engages into the tensioner receiver 7496, the film is pulled taut.

[0409] Mechanical microfluidic actuators As mentioned, any of these devices may include a mechanical microfluidic actuator. Figures 45A–45E illustrate an example of a portion of a mechanical microfluidic actuator. In Figure 45A, the mechanical microfluidic actuator 4500 includes a mounting area (seat) 4531 on which a cartridge can be fixed. In the illustrated example, the mounting area includes several parallel lanes 4532 (eight are shown) running along the length of the mounting area. In this example, the seat includes several vacuum ports connected to a vacuum manifold 4538 for applying an attractive force to fix the lower (second) seat of the cartridge in a mated position within the mounting area. In addition, each lane of the mounting area includes several different zones: a zone 4533 for thermal control, a zone 4535 for magnetic field application, or a zone 4542 for magnetic field and thermal control. The thermal control zones may be thermally connected to a heater / cooler (e.g., a Peltier device), and each of the magnetic control zones may include a local electromagnet. This is illustrated in Figure 45B, where the mounting area 4531 is made transparent to show the thermal control zone 4533, the electromagnet zone 4539, and the thermal control / magnetic combination zone 4542. In this example, the base of the mechanical microfluidic actuator may be a heat sink 4536 to allow for localized application of heating / cooling. Figure 45C shows an example of a mounting area for a mechanical microfluidic actuator with a cartridge 4506 mounted in the mounting area 4531.

[0410] This example illustrates three types of zones arranged in an alternating pattern along the length of each lane in the installation area (which may correspond to the lanes in the cartridge 4506 as shown in Figure 45C), but other patterns of zones and / or other types of zones (e.g., heating / cooling, magnetic, electrical energy, sensing / imaging, UV application, sonication application, etc.) may be included. Examples of installation area topologies are shown in more detail in Figures 45D and 45E. For example, in Figures 45D and 45E, the installation area may include a plurality of wells 4541 formed therein, which may be located below the thermal control area. For example, in Figure 45D, the installation area of ​​the mechanical microfluidic actuator includes a plurality of thermal control areas configured as wells 4541 having shallow bowl-shaped recesses formed of a thermally conductive material 4533. The bowls also include a suction port 4555 which is in contact with a suction manifold 4538 to hold a second sheet 4509 downward. Methods of driving droplets into a well (e.g., with a force applicator) can pin the droplets within the well and can significantly reduce or limit evaporation, especially when heating the droplets (e.g., for thermal cycling).

[0411] Figure 46 schematically illustrates an example of a portion of an apparatus similar to those shown in Figures 45A-45E (including a cartridge and a mechanical microfluidic actuator), including a vacuum port 4638 that secures the second sheet 4609 of the cartridge to the mounting area 4631 of the mechanical microfluidic actuator. In Figure 46, a droplet 4612 is shown within an air gap area and is coated with a drop gloss 4652 material. The drop gloss coating may be formed of a material that restricts evaporation and is immiscible with the droplet.

[0412] In general, any droplet of appropriate size, including microliters and submicroliters, may be used. However, in some cases, smaller droplets (e.g., less than 2 μL) may be difficult to transfer with high reliability. It may also be beneficial to use fluid transfer of droplets of any size that does not require negative pressure (e.g., aspiration), such as without pipetting. Figure 47 illustrates an example of a method for reliably transferring very small droplets, including (non-limiting) 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. This concave region / void may be configured to hold a specific droplet volume, for example, less than a few microliters (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.). A larger volume may be used as a well (e.g., 1–50 μL, 0.1–50 μL, etc.). The droplet void region 4673 may be inserted into a solution of the fluid 4675 to be transported and removed, so that droplets 4677 of the expected size and volume are trapped within the end of the solid transport member 4671, as shown. This droplet may be released, for example, within an air gap by immersion in a solution with lower surface tension (e.g., another droplet) 4679 (e.g., drop gloss); as illustrated, this immersion results in the displacement and release of droplet 4677.

[0413] Generally, these devices can handle smaller volume droplets by increasing the volume / amount of immiscible fluid (drop gross) so that the final volume is large enough for displacement within the air gap using mechanical actuators as described herein.

[0414] Figure 48 shows another schematic diagram of the mechanical microfluidic actuator 4800. In this example, the mechanical microfluidic actuator includes a force dispenser 4878 (e.g., a stylus, bearing, roller, etc.) and a force dispenser driver subassembly 4874 (e.g., a force dispenser subassembly). The force dispenser subassembly may include one or more drivers (e.g., x and / or y motion drivers, z motion driver 4873, etc.), as well as / or a frame or gantry, on which the force dispenser is driven to change position and / or apply force to the cartridge 4877 when the cartridge is placed on the device's cartridge base 4878. The force dispenser subassembly may also include one or more stepper motors, motion rails (e.g., a gantry / frame), and / or a home switch.

[0415] The mechanical microfluidic actuator in Figure 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 installation area which may be in thermal communication with a heating and / or cooling element (e.g., a Peltier device); it may include multiple heating / cooling elements. Any of these mechanical microfluidic actuators 4800 may also include a magnetic control subassembly 4885, 4885' for controllably applying a magnetic field into the air gap. Figure 49 illustrates an example of a row of magnetic elements (electromagnets) 4985 within a base 4819 of a mechanical microfluidic actuator.

[0416] In some examples, the mechanical microfluidic actuator device may include cartridge fasteners 4876, 4876' (e.g., holders, clamps, locks, etc.) for securing the cartridge to the cartridge base or mounting area 4878 of the mechanical microfluidic actuator. In Figure 48, the device includes a vacuum / suction subassembly (not shown) for applying suction force to secure the cartridge within the mounting area. In some examples, the mechanical microfluidic actuator device may include a fluid handling (e.g., pipetting) subassembly 4883 for adding and / or removing fluid from the air gap. Other subassemblies forming part of the mechanical microfluidic actuator 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 the mechanical microfluidic actuator). The mechanical microfluidic actuators described herein may also include one or more control inputs (e.g., keyboards, touchscreens, buttons, switches, etc.) and / or one or more outputs (e.g., displays, LEDs, wireless communication outputs / inputs, etc.), as well as hardware, software, and / or firmware for controlling them. In some cases, the same features may be used for the control inputs and outputs. Generally, the mechanical microfluidic actuators described herein may include one or more controllers 4899 for controlling and coordinating the operation of various subassemblies.

[0417] Any of these devices may include leveling. For example, in Figure 48, the device includes an adjustable leveling foot 4881.

[0418] In general, the mechanical microfluidic actuators described herein may be single-cartridge use (e.g., use with a single cartridge at a time) or may be configured to use multiple cartridges. Figure 50 illustrates an example of a mechanical microfluidic actuator device 5001 configured to be used with a single cartridge 5077, at least partially enclosed within a housing 5095. The device includes a force applicator subsystem 5097 (e.g., a three-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., a controller, power distribution, user interface board, touchscreen, etc. (and in some examples, for applying power to one or more electrodes, e.g., for electroporation and / or electrochemical procedures on the cartridge, etc.). The device also includes a thermal subsystem 5092 (e.g., a Peltier, high-power TEC driver, heat spreader, heat sink, etc.), a controller 5091, and input / output 5093 (e.g., a display / touchscreen). Figure 51 illustrates an example of a multiplex device 5101 that enables the handling of multiple cartridges in parallel. As shown in Figure 51, either a single cartridge or a multiplex configuration may also include or be configured to be used with a fluid handling system (e.g., a liquid handler 5163).

[0419] Figures 52A and 52B schematically illustrate an example of a mechanical microfluidic actuator as described herein, showing one possible arrangement of the subassemblies described herein. For example, in Figure 52A, the apparatus includes a liquid staging subassembly for adding / removing liquid from a cartridge (e.g., temperature control, input, chip housing, chip disposal, etc.), as well as a chassis subassembly (e.g., chassis, fan, leveling feet, switches / buttons, touchscreen, etc.), and a power distribution subassembly (main power supply, power distribution network, 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 handling subsystem including a motion controller (driver, position sensor, etc.), and may also control submodules comprising a temperature subassembly (Peltier subassembly), a magnetic subassembly, a linear motion subassembly, and a cartridge receptacle (e.g., cartridge data), each of which can provide input to the controller. [Examples]

[0420] Figures 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 so that they can move 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 5393 (also called the inner surface facing the air gap) of the first and second sheets may be hydrophobic and oleophobic. The sheets may be formed from a hydrophobic and oleophobic material or coated with a hydrophobic and oleophobic material.

[0421] A watery 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 and second sheets may be fixed facing each other at a predetermined distance and generally parallel, with an air gap between them. The first and / or second sheets may be held taut. At least the first sheet is formed of an elastomer material so as to be deformable when a force (e.g., a mechanical stylus as shown in Figures 53B and 53C) is applied to it, and return to a generally parallel configuration when the force is released. For example, in Figure 53A, a mechanical force applicator (stylus 5375) is positioned above the top of the first (e.g., upper) elastomer sheet 5307. As described above, one or more moving drive units (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 that includes one or more tensioners (e.g., a tensioning frame) that keep the first and / or second sheets taut.

[0422] As shown in Figure 53B, the mechanical force actuator 5375 may be driven downward relative to the first sheet with respect to the region adjacent to the droplet. Locally reducing the height of the air gap (with a continuous gradient as shown) may drive the droplet into that region of lower height by the resulting increased capillary force. Thus, a mechanical force actuator driven across (and relative to) the top of the sheet, as shown in Figures 53B and 53C, moves the droplet within the air gap.

[0423] In any of these examples, the height of the air gap may be reduced by a gradient, and the distance between the upper and lower (first and second) sheets may be reduced but they will not come into contact with each other. For example, the height may be reduced by approximately 5% to 90% (e.g., approximately 10% to 80%, approximately 20% to 60%, approximately 10% to 50%, etc.). In some cases, it may be advantageous to reduce the height by approximately 5% to 60%, but not exceeding 60% (e.g., not exceeding 55%, not exceeding 50%, not exceeding 45%, not exceeding 40%, not exceeding 35%, not exceeding 30%, not exceeding 25%, etc.). This may allow the gradient to drive movement, but the area may be limited to a localized area on 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 Figure 53C). As shown in Figures 53B-53C, applying force (by a mechanical force applicator / stylet) to elastically deform the first sheet reduces the distance of the air gap between the first and second sheets in a local region within the air gap adjacent to the fluid droplet, and causes the droplet to move within the air gap, following the reduced height region formed by the stylet.

[0424] In any of the methods and apparatus described herein, the sheet forming the air gap is made of a hydrophobic and oleophobic material; non-hydrophobic and oleophobic materials did not function in many of the examples shown. In addition, the material forming the inner surface of the air gap may be substantially non-porous.

[0425] As mentioned, any droplet may be coated with a layer of drop gloss, which may be a substance with low surface tension (e.g., oil), such as a gloss coat, and which may be immiscible with the droplet; being immiscible with the droplet may also prevent or limit evaporation.

[0426] In general, the methods and apparatus described herein may include the use of at least 0.01% of a surfactant in or around the moving droplet. Surprisingly, the inventors have found that the use of a surfactant in the droplet (e.g., 0.01% or more, 0.02% or more, 0.025% or more, 0.01% to 1%, 0.01% to 0.7%, 0.01% to 0.5%, 0.01% to 0.25%, 0.01% to 0.1%, etc.) or in the gloss layer surrounding the droplet may make the movement of the droplet within the air gap, when pulled by the reduced gap height as described herein, more predictable. While not bound by theory, this may be due to the effective surface tension of the droplet; therefore, the use of a surfactant in either or both the drop gloss and / or the droplet may make the droplet move more predictably and firmly. Without surfactants, droplet movement may be less predictable and, in some cases, may not be able to follow a mechanical actuator as it moves across the surface. Any suitable surfactant may be used. For example, the drop gloss used may contain a nonionic surfactant (e.g., Brij-35) or other hydrophobic polymer. In some examples, the droplets may contain surfactants such as Pluronic, Tween-20, or Tetronic. Thus, in any of these methods and apparatuses, either or both of the drop gloss and / or droplets may contain a surfactant (e.g., 0.01% or more of the surfactant). In some cases, the surfactant may be added before initiating any step that moves the droplet by locally reducing the gap height in the region adjacent to the droplet.

[0427] In examples where a mechanical force applicator (e.g., a stylus) is used, the contact surface of the stylus may be sized in proportion to the air gap and / or the size / volume of the droplet. Specifically, the aspect ratio of the stylus, e.g., the size of the stylus tip relative to the droplet size, and / or the size of the stylus tip relative to the height of the air gap, may be selected to be 1:0.5 to 1:20 (tip:droplet).

[0428] DNA sequencing and DNA synthesis The methods and apparatus described herein may be particularly used for enzymatic processing of polynucleotides, including (non-limited) sequencing and / or synthesis.

[0429] For example, these methods and apparatus may be used to perform synthetic DNA sequencing (SBS). SBS offers many significant benefits to the scientific research community and has enabled many new diagnostic applications; these include increased output from sequencing instruments, faster turnaround times for results, and cost reductions of several orders of magnitude compared to the preceding primary sequencing method, Sanger sequencing. Sanger sequencing relies on electrophoretic separation of DNA fragments created by specially modified termination nucleotides. SBS eliminates the need for separation and enables the implementation of large-scale 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) to detect aneuploidies such as Down syndrome in the blood of pregnant women, genetic carrier testing to inform parents about potential genetic risks, stratification of tumor patients, and profiling and early detection of tumors through sequencing of nucleic acids in blood (known as cell-free sequencing).

[0430] SBS is a flow-based sequencing technique in which a series of liquid formulations are introduced into a flow cell containing a DNA template that has been isolated, purified, and processed to create a sequencing "library." The template DNA is loaded into the flow cell and placed on a random or structured array, which creates separate "clusters" for each DNA library fragment. Reagents are delivered sequentially, thereby enzymatically adding a single fluorescently labeled nucleotide in each cycle. The flow cell is imaged each time a fluorescent nucleotide is added, with each unique fluorescent label representing a specific base (A, C, G, T); the flow cell is processed through software that assigns the next base in the sequence. After imaging, the fluorescent dye and nucleotide block groups are chemically cleaved and washed away in preparation for the next cycle. Typically, this process is repeated for 75–600 cycles, with another base added each cycle, which is recorded (captured) through imaging and software analysis. Many protocols include a process (usually during the run) to create a complementary strand of DNA, which is also sequenced to improve coverage and accuracy. The method for creating this complementary strand uses the same reagents as those used to create the newly synthesized DNA to be sequenced in SBS.

[0431] Current methods typically employ a delivery method to sequentially provide a continuous flow of each reagent. These reagents are delivered by pumping or pressure mechanisms; each reagent overflows the flow cell, completely filling it, and then flows in to replace the next reagent required to drive the cycle. Although these volumes are fairly small, carryover from step to step or from cycle to cycle can impair the resulting sequencing, so extra volume is needed to avoid carryover. Figure 54 schematically illustrates the SBS sequencing cycle and overall processing map. The microfluidic methods and apparatus described herein, including the washing step, may be used to flow various components within a “flow cell” configured as described herein.

[0432] Conventional digital microfluidics (such as those using electrowetting) have been proposed for nucleic acid sample preparation as a front-end for sequencing processes and as an alternative to manual benchtop library preparation or conventional robotic pipetting systems, but they have several drawbacks. Electrowetting on dielectrics (EWOD) has been successfully applied to front-end processes such as DNA isolation from patient samples and sequencing library preparation for loading into sequencing instruments. While electrowetting is reasonably suitable for automating upfront processes, the complexity of the sequencing process itself presents several technical and practical economic challenges. Of particular note is the requirement for flow cell imaging after each nucleotide addition cycle. In an ideal embodiment, a fully integrated sequencing process would allow the sample to be introduced into the system and provide DNA sequencing results as output. EWOD does not seem likely to succeed as a fluidic solution for such a fully integrated process.

[0433] Methods and apparatus described herein, also known as surface mechanical actuation, in which a mechanical force (e.g., compressive force) is applied to drive one or more droplets, may offer significant advantages over multiple microfluidic techniques, including electrowetting, and may allow for the use of a single fluid engineering technique throughout the entire sequencing process, including SBS. As described above, the use of mechanical compression to alter capillary forces to move droplets in two dimensions may offer many advantages, particularly with respect to sample preparation, and may be used in virtually all necessary steps, including nucleic acid isolation, library generation, cluster generation, primer loading and hybridization, and multi-cycle sequencing reactions, including the steps illustrated in Figure 54.

[0434] Figures 55A-55B illustrate an example of the use of mechanical compression to alter capillary force to move droplets in flow cell 5501 in order to introduce sequencing primers into clusters of a DNA template (for example, as part of the synthesis sequencing process described above). Figure 55A shows the loading and washing steps related to the introduction of primers into the sequencing template in flow cell 5501. As with any method described herein, multiple mechanical force inductors (stylus 5575) may be used simultaneously on the same flow cell (e.g., cartridge). These mechanical force inductors may be controlled / actuated independently or collectively. In Figure 55A, the cartridge / flow cell contains regions in which the primers, with cluster formation, are arranged either unpatterned or patterned (e.g., as nanowells). The steps for generating clusters may also be performed using mechanical force inductors as described herein, or these steps may be performed by pipetting and washing.

[0435] The first droplet 5502 may first be attracted onto the cluster using the first stylet and then incubated on the cluster as shown in Figure 55B (this allows for hybridization); then it may be drawn away from there and, in some cases, removed from the flow cell (e.g., to a waste depot). Next, a second droplet 5504 (wash buffer) may be attracted onto the cluster to wash the cluster.

[0436] Figures 56A–56E illustrate the steps associated with each cycle of SBS performed within the cartridge (e.g., flow cell) 5501. As described above, multiple mechanical force applicators (styluses 5575), or the same stylus, may be used sequentially. In this example, the figure shows the unidirectional movement of the droplet, for example, from left to right. However, the methods and apparatus described herein may allow movement in two dimensions (for example, movement across the entire plane of the air gap, and movement in any direction within this plane). Thus, in some examples, reagent droplets may be moved to the side and reintroduced or reused (and furthermore, depleted components, such as nucleotides, enzymes, or other chemicals, may be refilled).

[0437] In Figure 56A, an SBS cycle may be performed, consisting of primer hybridization and washing (see Figures 55A-55B), followed by the application of a nucleotide-polymerase reaction mixture, washing, and extension and subsequent imaging for multiplex sequencing. Figure 56A shows the start of the first cycle; in this cycle, droplets containing nucleotides and polymerase are added to a cluster in the air gap of the cartridge / flow cell 5501 by pulling them using a mechanical actuator. After a suitable time, the droplets may be pulled away from the cluster, and one or more washing droplets may be moved onto the cluster during imaging (to identify additional nucleotides) (Figure 56B). The washing droplet may be moved again to detach it from the cluster, and a droplet containing the dye / terminator cleavage component may be moved onto the cluster (Figure 56C); this droplet may then be moved and detached using a mechanical actuator, and the same or a different mechanical actuator may be used to move another droplet of nucleotide and polymerase to initiate a second cycle (Figure 56E).

[0438] The microfluidic methods and apparatus described herein may also be used for applications other than SBS sequencing. For example, these methods and apparatus may be used for the enzymatic synthesis of DNA oligos (which is very similar to SBS processing), such as the cyclic enzymatic addition of nucleotides with reversible terminators. Other non-limiting examples may include DNA oligo synthesis.

[0439] For example, the methods and apparatus 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 schematically illustrates the overall workflow from nucleic acid extraction to sequencing using a system for surface mechanical actuation (e.g., mechanical compression to alter capillary forces).

[0440] In Figure 57, top and side views of an 8-lane cartridge similar to those described above may be integrated into a system containing an array of cooling areas (e.g., Peltier) and magnetic and / or resistive heating zones, which may enable the cartridge and system to perform DNA / RNA extraction, library preparation, and sequencing in designated areas. In Figure 57, the cartridge includes an air gap with multiple different reaction wells (including thermal control / cooling). The cartridge is divided into lanes (eight lanes are illustrated) and contains areas for DNA / RNA sequencing, library preparation, and sequencing (e.g., SBS). Lane dividers may separate the lanes.

[0441] Figure 58 illustrates the operation of the cartridge and system shown in Figure 57 for extracting polynucleotides (e.g., DNA, RNA, etc.) from a clinical sample. Figure 58A shows a schematic side view of DNA / RNA extraction from a clinical sample on the cartridge. In this example, a 250 μl droplet of a clinical sample (e.g., blood, saliva, and tissue homogenate) containing 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 method described herein, and incubated at 70°C for 10 minutes. The reaction (Rxn) droplet is then actuated to the magnet / resistance 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 engaged until a pellet is formed, the supernatant is discarded, and the pellet is washed twice with 600 μL of VHB washing buffer. The pellet may be resuspended in 600 μL of SPM washing buffer and driven by surface mechanical actuation to clean the magnet / resistance heater zone; the magnet may be activated and the washing buffer may be moved for disposal. Finally, the eluted nucleic acids are eluted into 30 μL of eluent.

[0442] In Figure 58, droplets of clinical samples (e.g., blood, saliva, and tissue homogenates) are fused with lysis buffer 581, mixed in the reaction well for 5 minutes by the technique described herein (e.g., surface mechanical actuation), and incubated at 70°C for 10 minutes. The lysate droplets are then actuated to the magnet / resistance heater zone 582, fused with the binding buffer and magnetic / binding bead particles; and may be mixed for 10 minutes. The magnet may then be activated until a pellet is formed 583, the supernatant may be discarded, and the pellet may be washed twice with wash buffer. The pellet is resuspended in wash buffer and driven by surface mechanical actuation to clean the magnet / resistance heater zone; the magnet may be activated and the wash buffer may be moved for discard. Finally, the elution buffer is actuated to the pelletized beads to elute the nucleic acids and separate them from the beads.

[0443] Figure 59 illustrates the RNA sequencing workflow. In Figure 59, the side view through the cartridge shows a schematic diagram of the RNAseq workflow on the cartridge. In step 591, 2 μl droplets of fragmented RNA and the first-strand synthesis master mix are actuated to the PCR reaction well with a 45 μL dropgloss droplet by mechanical actuation on the surface (e.g., using a stylus) as described herein, and then incubated (10 minutes at 25°C, 15 minutes at 42°C, and 15 minutes at 70°C). Subsequently, a 6 μl droplet of the second-strand synthesis master mix is ​​added to the reaction (Rxn) droplet and incubated at 16°C for 60 minutes. Actuate the Rxn droplet to the magnet / resistance heater zone 592, fuse it with the 11.2 μl bead droplet, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet is formed, discard the supernatant for disposal, wash the pellet twice with 25 μl of 80% EtOH (not shown schematic diagram), and elute the cDNA sample in 6 μl of elution buffer to remove it from the beads. Then actuate the 5 μl droplet of cDNA sample to the PCR reaction well as described herein, fuse it with 1 μl of Endoprep Master Mix (with 10 μl Drop Gross), and incubate at 20°C for 30 minutes, followed by 65°C for 30 minutes. Next, actuate 3.1 μl of Adapter Ligation Master Mix and 0.25 μl of Adapter Drop as described herein, mix with the Rxn droplet, and incubate at 20°C for 15 minutes 593. Actuate the adapter ligation Rxn droplet to the magnet / resistance heater zone, fuse it with 7.28 μl of bead droplets, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet forms, then discard the supernatant for disposal, wash the pellet twice with 25 μl of 80% EtOH (not shown schematic), and elute the DNA library in 6 μl of nuclease-free water containing 5 μM TRUESEQ BARCODES 594.

[0444] Next, actuate a 5 μl droplet of the purified DNA library sample to the PCR reaction well by mechanical actuation (e.g., using a stylus), fuse it with 10.9 μl of USER / PCR master mix (with 45 μl drop gloss), incubate at 37°C for 15 minutes, then 98°C for 30 minutes, then 98°C for 10 seconds, then 65°C for 75 seconds, or 19X cycles.595 Actuate the amplified DNA droplet to the magnet / resistance heater zone, fuse it with a 12.72 μl bead droplet, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet is formed, discard the supernatant for disposal, wash the pellet twice with 25 μl of 80% EtOH (not shown in schematic diagram), and elute the DNA library in 25 μl of eluate.596

[0445] In Figure 59, droplets of fragmented RNA and the first-strand synthesis master mix are actuated to the reaction well with the drop gloss by mechanical actuation (e.g., stylus) and incubated (10 minutes at 25°C, 15 minutes at 42°C, and 15 minutes at 70°C)591. Next, droplets of the second-strand synthesis master mix are added to the reaction (Rxn) droplet and incubated at 16°C for 60 minutes. The Rxn droplet is actuated to the magnet / resistance heater zone, fused with the SPRI or Ampure bead droplet, mixed, and incubated at room temperature (RT) for 5 minutes; the magnet is activated until a pellet is formed, the supernatant is discarded for disposal, the pellet is washed twice with 80% EtOH (not shown in schematic diagram), and the cDNA sample is eluted into elution buffer and removed from the beads592. Next, actute the cDNA droplets into the reaction well (e.g., using a stylus), fuse them with the Endoprep master mix, and incubate at 20°C for 30 minutes, followed by 65°C for 30 minutes.593 Next, actute the adapter ligation master mix and adapter droplets by mechanical actuation (e.g., using a stylus), mix with the Rxn droplets, and incubate at 20°C for 15 minutes.Actute the adapter ligation Rxn droplets into the magnet / resistance heater zone, fuse them with the SPRI or Ampure bead droplets, mix, and incubate at RT for 5 minutes; activate the magnets until a pellet is formed, discard the supernatant for disposal, wash the pellet twice with 80% EtOH (not shown in schematic diagram), and elute the DNA library in nuclease-free water containing primers.594 The purified DNA library and primer mixture is mechanically actuated (by stylus) to the PCR reaction well as described herein, fused with the USER / PCR master mix with drop gloss, incubated at 37°C for 15 minutes, then at 98°C for 30 seconds, then at 98°C for 10 seconds, then at 65°C for 75 seconds, repeating up to 19 cycles.Actuate the amplified DNA droplet to the magnet / resistance heater zone, fuse it with the SPRI / Ampure bead droplet, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet forms, discard the supernatant for disposal, wash the pellet twice with 80% EtOH (not shown in schematic diagram), and elute the RNA-seq library in 25 μl of elution buffer.596

[0446] Figure 61 is a schematic side view 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 to the PCR reaction well with a 45 μl dropgloss droplet as described herein and incubated at 95°C for 5 minutes and at 60°C for 2 hours 601. The Rxn droplet is actuated to the magnet / resistance heater zone and fused with 33.3 μl of streptavidin beads and mixed at RT for 30 minutes; the magnet is operated until a pellet is formed, the supernatant is discarded for disposal, the pellet is washed (not shown in the schematic) first with a 50 μl buffer droplet preheated to 70°C and then with a 50 μl buffer droplet preheated to 48°C, and the purified DNA library sample is eluted from the beads in 7.5 μl of elution buffer 602. Subsequently, actuate a 7.5 μl droplet of the purified DNA library sample to the PCR reaction well as described herein, fuse it with 0.83 μl of primer and 8.3 μl of master mix (with 10 μl of drop gloss), incubate at 97°C for 45 seconds, followed by 8 cycles of 15 seconds at 97°C, 30 seconds at 60°C, 30 seconds at 72°C, and finally 1 minute at 72°C 603. Actuate the amplified DNA droplet to the magnet / resistance heater zone, fuse it with a 30 μl droplet of magnetic beads, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet is formed, discard the supernatant for disposal, wash the pellet twice with 25 μl of 80% EtOH (not shown in schematic diagram), and elute the DNA library in 30 μl of eluate 604.

[0447] Therefore, in Figure 60, the method for enriching the Twist exome target is performed on a cartridge as described herein, using mechanical actuation of the cartridge surface. In Figure 60, droplets 601 of DNA and hybridization mix with drop gloss are actuated to the reaction well as described herein and incubated at 95°C for 5 minutes and at 60°C for up to 4 hours. The Rxn droplets are actuated to the magnet / resistance heater zone, fused with streptavidin beads, and mixed at RT for 30 minutes; the magnet is operated until a pellet is formed, the supernatant is discarded, the pellet is washed (not shown schematic) first with a buffer droplet preheated to 70°C and then with a 50 μl buffer droplet preheated to 48°C, and the purified DNA library sample is eluted from the beads in elution buffer 602. Next, droplets of the purified DNA library sample are actuated to the PCR reaction well by mechanical actuation of the surface as described herein, fused with primers and drop gloss, and incubated at 97°C for 45 seconds, followed by 15 seconds at 97°C, 30 seconds at 60°C, 30 seconds at 72°C for up to 18 cycles, and finally at 72°C for 1 minute 603. Actuate the amplified DNA droplets to the magnet / resistance heater zone, fused with the SPRI / Ampure magnetic bead droplets, mixed, and incubated at RT for 5 minutes; activate the magnet until a pellet is formed, discard the supernatant for disposal, wash the pellet twice with 80% EtOH (not shown schematic diagram), and elute the DNA library in elution buffer 604.

[0448] Figure 61 illustrates the workflow for Aplicon-seq. For example, Figure 61 shows a schematic side view 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 on the surface (e.g., using a stylus), mixed for 5 seconds at RT, and split into two equal droplets using a liquid handler (not shown in the schematic diagram)611. Second, each droplet is fused with a 5 μl unique primer droplet accompanied by a 45 μl dropgloss droplet, and the Rxn droplets (1 & 2) are actuated to the PCR reaction well zone by mechanical actuation on the surface (e.g., using a stylus) and incubated (2 minutes at 99°C, then 17 cycles of 15 seconds at 99°C and 4 minutes at 60°C)611'. Next, Rxn droplets 1 and 2 are fused by mechanical actuation on the surface (e.g., using a stylus), actuated to the magnet / resistance heater zone, fused with 4 μl of FuPa reagent droplet, mixed, and incubated at 50°C for 10 minutes, 55°C for 10 minutes, and 60°C for 20 minutes. Secondly, droplets of 8 μl of switch solution, 4 μl of barcode adapter mix, and DNA ligase are added to the Rxn droplet by mechanical actuation on the surface (e.g., using a stylus), and incubated at 22°C for 30 minutes, 68°C for 5 minutes, and 72°C for 5 minutes.612 Subsequently, a 90 μl droplet of beads is added to the Rxn droplet, mixed, and incubated at RT for 5 minutes; the magnet is activated until a pellet forms, the supernatant is discarded, the pellet is washed twice with 150 μl of 80% EtOH droplets (not shown in schematic diagram), and the library is eluted from the beads in droplets of 50 μL of Platinum® PCR SuperMix HiFi and 2 μL of Equalizer® Primers.

[0449] In step 613, actuate a 50 μl droplet of the purified library to the PCR reaction well by mechanical actuation on the surface (e.g., using a stylus), incubate at 98°C for 2 minutes, and repeat 9 cycles of 15 seconds at 98°C followed by 1 minute at 64°C. Secondly, add a 10 μl Equalizer Capture droplet to the Rxn droplet and mix at RT for 5 minutes. In step 614, actuate the Rxn droplet to the magnet / resistance heater zone, fuse it with 6 μl of washed Equalizer® beads, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet is formed, discard the supernatant for disposal, wash the pellet twice with 150 μl of 80% EtOH (not shown in schematic diagram), and elute the DNA library in a 100 μl elution droplet.

[0450] Therefore, as shown in the Ampliseeq (2 primer pool) workflow in Figure 61, in 611, three droplets of DNA, PCR master mix, and water are fused by surface mechanical actuation (e.g., using a stylus), mixed for 5 seconds at RT, and then split into two equal droplets using a liquid handler (not shown in the schematic diagram). Secondly, in 611', each droplet is fused with a unique primer droplet with drop gloss, and the Rxn droplets (1 & 2) are actuated to the PCR reaction well zone by surface mechanical actuation (e.g., using a stylus) and incubated (2 minutes at 99°C, then 17 cycles of 15 seconds at 99°C and 4 minutes at 60°C). In step 612, firstly, Rxn droplets 1 and 2 are fused by mechanical actuation on the surface (e.g., using a stylus), actuated to the magnet / resistance heater zone, fused with the FuPa reagent droplet, mixed, and incubated at 50°C for 10 minutes, 55°C for 10 minutes, and 60°C for 20 minutes. Secondly, droplets of switch solution, barcode adapter mix, and DNA ligase are added to the Rxn droplet by mechanical actuation on the surface (e.g., using a stylus), and incubated at 22°C for 30 minutes, 68°C for 5 minutes, and 72°C for 5 minutes. Thirdly, the bead droplet is added to the Rxn droplet, mixed, and incubated at RT for 5 minutes; the magnet is activated until a pellet is formed, the supernatant is discarded, the pellet is washed twice with an 80% EtOH droplet (not shown in the schematic diagram), and the library is eluted from the beads into droplets of Platinum® PCR SuperMix HiFi and Equalizer® Primers. In 613, firstly, a 50 ul droplet of the purified library is actuated to the PCR reaction well by mechanical actuation on the surface (e.g., using a stylus), incubated at 98°C for 2 minutes, and repeated 9 cycles of 15 seconds at 98°C and 1 minute at 64°C. Secondly, the Equalizer Capture droplet is added to the Rxn droplet and mixed at RT for 5 minutes.In 614, actuate the Rxn droplet to the magnet / resistance heater zone, fuse it with the washed Equalizer™ beads, mix, and incubate at RT for 5 minutes; activate the magnet until a pellet forms, discard the supernatant for disposal, wash the pellet twice with 80% EtOH (not shown in schematic diagram), and elute the DNA library into the elution droplet.

[0451] Liquid introduction and removal Figures 62A to 62J illustrate an example of a method for applying a liquid (droplet) into a cartridge, such as the cartridge described herein. For example, in Figure 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 substance (as described above) may be pipetted into the air gap of the cartridge. For example, about 10 to 45 μL of drop gloss may be pipetted into the air gap, and then the pipette tip may be removed (Figure 62B). Next, as shown in Figure 62C, a droplet of the aqueous reactant may be inserted into the air gap in the same manner. The droplet may be pipetted on or in close proximity to the drop gloss (which is added first). In some examples, the drop gloss may be combined with the droplet before being pipetted together with it. Alternatively, the drop gloss may be added after the aqueous (reaction) droplet has been added. In general, liquid substances may be introduced by pipette tips, and either a unique application (dedicated single tip / sample) or a universal application (shared tip for multi-dispensing) can be used to introduce reagents across one or more lanes of a cartridge. In Figure 62C, any volume of aqueous reaction mixture, such as approximately 250 nL to 80 μL, may be used.

[0452] In Figures 62C-62D, pre-dispensed drop gloss encapsulates the aqueous reagents, protecting them from surface contamination and evaporation during the workflow steps. Ethanol and wash buffer do not require drop gloss. The volume of drop gloss may be greater than, less than, or equal to the volume of the aqueous droplets. In some examples, as shown in Figures 62A–62D, 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), so even reaction droplets of very small volumes (e.g., as small as 250 nL) can be manipulated; the reaction droplet may be bound with excess drop gloss, which encapsulates it and allows for the manipulations described herein (movement, binding, splitting, heating, mixing, etc.) even within a channel having a relatively large channel height (e.g., 1.5 mm or more). Thus, in some examples, the systems described herein can also dispense reagents / master mixes / samples in small volumes of 250 nL and large volumes of 80 uL. In some examples, this system can dispense volumes of 10–45 μL for drop gloss. In some of these examples, the lanes shown can accommodate a total volume of up to 150 μL (drop gloss + reagent) or a reagent volume of up to 80 μL. Larger or smaller lane widths and / or heights may be used.

[0453] As shown above, during the introduction of liquid into the inlet of each lane, the dispensing tip is lowered (straight down) relative to the lower film surface to ensure that a portion of the droplet is inside the channel (due to the intrinsic wetting properties of the liquid) upon dispensing. Capillary pressure may be used to pull the droplet into the air gap and away from the opening (or towards the edge of the opening) so that the droplet can be manipulated as shown in Figures 62E-62G. Thus, for small volumes 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 the small volume. For example, as shown in Figures 62E-62F, a mechanical manipulator (stylus) may be lowered to an elastically deformable upper sheet to reduce the height (on or, more preferably, in close proximity to the droplet), and the mechanical manipulator (stylus) may be drawn across the surface of the upper sheet as shown in Figure 62G to move the droplet surrounded by the drop gloss. As illustrated, the reagent is protected inside the drop gloss droplet. In Figure 62F, the stylus compresses the film surface in close proximity to the inlet hole (at a safe distance to avoid stylus contamination). The drop gloss / reagent droplet is then drawn into a narrower gap (by capillary action, including increased capillary force), 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., drop gloss and aqueous droplet) across heating / cooling zones and / or magnetic / isothermal heater zones.

[0454] Figures 62H-62J illustrate the removal of droplets from the cartridge. The drop gloss substance may first be removed from the droplet, for example, by contact with a lipophilic substance that can wick the drop gloss substance, or by mechanical separation. Alternatively, the droplet may contain the drop gloss substance together with an aqueous substance. In Figure 62H, the stylus drives the reaction droplet (e.g., including a product library or other reaction products, as shown and described above) close to an opening (e.g., an inlet hole) that leads through the upper sheet into the air gap, at a safe distance to avoid stylus contamination. This is illustrated in Figure 62I. The droplet to be removed is near the opening leading into the air gap, but not in the opening itself. However, since the upper sheet is made of an elastic material, it may be deformed by the pipette tip for access, as shown in Figure 62J. In this example, the pipette tip is inserted into the inlet hole (Figure 62I) and reaches a position above the lower film. Next, the pipette tip is moved toward the droplet (Figure 62J), which temporarily deforms the upper sheet (film) until the pipette tip reaches the sample position (at this point, the droplet is only partially sandwiched between the upper and lower sheets); and the droplet is then aspirated into the pipette until it is completely removed or a specific volume is removed. The pipette tip is then returned to the inlet opening, then rises, and moves toward the destination of the product (e.g., a tube / plate) so that the operator can collect the substance at the end of the run.

[0455] Evaporation control In general, these methods and apparatus may be configured to prevent or reduce evaporation. Generally, a drop gloss coating of an aqueous substance may, alone or in combination with applying a force (e.g., mechanical force) to the droplet, result in both enhanced heating uniformity and prevention of evaporation. For example, in some variations of the methods and apparatus described herein, aqueous droplets may evaporate less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%) when heated to 95°C or above for at least 30 minutes. In one example, droplets of an aqueous reaction mixture (20 μL in 45 μL of drop gloss) heated to 95°C for 30 minutes evaporated a total of approximately 5.8%.

[0456] Figure 63 illustrates an example in which a droplet (e.g., drop gloss + aqueous droplet) is held in a reaction well formed in the lower layer (which is elastically deformable, like the upper layer) by applying a suction force to conform the lower layer to form a well in the air gap. 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 for controlling the temperature of the droplet in the air gap. In Figure 63, the droplet was heated to 95°C for 40 minutes (20 μL of 45 μL of drop gloss). A stylet (illustrated in this example as a roller stylus) may be held above the droplet, above the top of the sheet. The applied mechanical force may hold or pin the droplet within its position relative to the heating area. This may also help to insulate the droplet. In some examples, the portion of the stylet above the droplet may be insulating. In some examples, the lower layer (sheet) may be more thermally permissible than the upper layer (sheet). Therefore, the methods and apparatus described herein have the potential to prevent evaporation in the air gap remarkably well compared to other microfluidic systems.

[0457] Air gap height As described above, the air gap formed between the upper and lower sheets when installed, for example, as part of a cartridge within the cartridge mounting surface of a mechanical microfluidic actuator device, or when integrated within a mechanical microfluidic actuator device, may generally be any suitable height (e.g., air gap height or thickness). For example, the air gap may have a height / thickness 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, about 0.2 mm to 3 mm, about 0.3 mm to 2 mm, about 0.1 mm to 1.5 mm, etc.). Generally, the devices described herein (including, but not limited to, cartridges) may include an air gap formed between a first (e.g., upper) sheet and a second (e.g., lower) sheet. As described above, its height may be adjusted and / or set by the mechanical microfluidic actuator device, such as the shape of the cartridge mounting surface. In particular, the cartridge and / or mechanical microfluidic actuator device may include, or be configured to form, a gutter region having greater height; this greater height, and the resulting reduction in capillary forces, may prevent fluid droplets from moving to the edges of the cartridge (or lanes within the cartridge), otherwise the droplets may be attracted to the sides. This is illustrated in Figures 44B-44E.

[0458] In some examples, the lower surface of the cartridge may be formed of a deformable material and may conform to the base of the cartridge mounting surface of the mechanical microfluidic actuator device. The upper and lower sheets (surfaces, layers, etc.) may be formed of the same material or different materials. As mentioned, the upper surface may be made of an elastically deformable material so that it can be actuated repeatedly (hundreds or thousands of times) using a stylus as described above. The lower sheet may also be conformable to a similar or lesser extent and may be held, for example by vacuum, to conform to the cartridge mounting surface of the mechanical microfluidic actuator device. Thus, the cartridge mounting surface of the mechanical microfluidic actuator device may include a manifold for diverting and applying suction force to the back surface of the second (lower) sheet; in some cases, this may result in the lower sheet being conformed and held so that it adjusts the height of an air gap region to form, for example, a gutter, well, etc., which may help control or guide the movement of droplets by mechanical actuation as described herein.

[0459] For example, Figure 64A shows an example of a portion of a cartridge, including an upper sheet 6410 and a lower sheet 6410 having hydrophobic and oleophobic surfaces facing an air gap region open to the atmosphere. The height of the air gap 6488 may be 0.5 mm to 1.5 mm in this example. In Figure 64A, the cartridge mounting region 6419 of the mechanical microfluidic actuator device is shown, and the cartridge is lowered onto the mounting region 6469. Figure 64B illustrates the cartridge mounted on the mounting region 6419. The lower sheet 6420 may be attached to the mounting region. For example, a suction force may be applied between the mounting region and the lower sheet to hold the lower sheet in the mounting region in a releaseable manner (so that the lower sheet conforms to the mounting region). In some examples, the mounting region may include a manifold of suction openings for applying the suction force. In this example, the air gap may have an initial height 6488 (height before being placed on the mounting region of the device) of about 0.5 mm to about 5 mm. Once the cartridge is installed on the installation area 6419, the height of some areas may be greater or smaller depending on the shape of the installation area to which the lower sheet fits.

[0460] In any of these devices, the air gap may be configured to include a well region by either forming a lower sheet to create a well, or by having a deformable lower sheet that can be conformed to the shape of a well. For example, the installation region 6419 may include a well 6456, and a conformable lower sheet 6420 of the cartridge may be attached to the shape of the installation region, as shown in Figure 64B. Figures 13A-13E show examples of wells formed in the lower part (e.g., through a gap). The well may be configured as a thermal control region (as described above) and may include a heater and / or cooler 6445 (heating / cooling element), and / or a thermal conduit in thermal contact with a heater (e.g., a resistive heater) or a cooler (e.g., a Peltier). In any of the devices and methods described herein, the well may extend at least slightly above the installation region, as shown in Figures 64A-64C. For example, the well may extend to 1% to 90% of the height 6488' of the narrow passage of the air gap (e.g., approximately 10-90%, 20-85%, 30-80%, 40-90%, 50-95%, 60-90%). In Figure 64C, the well is shown with a droplet 6440 in well 6456. Surprisingly, having a well wall that rises and protrudes from the installation area 6419 may prevent the droplet from moving out of the well area even if the droplet temperature (and therefore the surface tension and capillary forces acting on the droplet) changes, while still allowing the droplet to move in and out of the well by deforming the surface of the upper sheet 6410 and changing the capillary action as described above. In this example, the well is formed in a ring or donut shape within the installation area. The installation area may include a plurality of wells formed in this manner. The lower part of the well may include one or more suction openings. As shown in Figure 64B, one or more openings may be present around the baes of the well to press against the nearly vertical side walls of the well and pull the lower sheet.

[0461] During operation, a force (e.g., from a stylus or otherwise) may be applied to the upper sheet to locally deform it and locally reduce capillary action, thereby driving droplet movement, which, as described above, allows the droplet to follow the movement of the deformed region (e.g., the stylus). The height of the deformation may be controlled and optimized by the device. Generally, the upper sheet may be locally deformed to a height that does not touch the lower sheet during operation; however, the methods and devices described herein may still function even if the upper sheet touches the lower sheet, but preventing contact between the two sheets may reduce wear on the upper and lower sheets and potentially allow for longer cartridge operation. For example, the height of the air gap in the deformed region may be maintained within approximately 5% to 90% of the height of the air gap when the upper sheet is not deformed. In any of these examples, the height of the air gap in the deformed state (e.g., when the movement is actuated by pressing the stylus against the upper sheet and lowering it) may be adjusted based on the volume of the droplet. For example, when the fluid volume is larger (the droplet is larger), the deformation of the upper sheet may be less than when the fluid volume is smaller (the droplet is smaller). In some examples, the apparatus may detect the droplet size (e.g., optically, as described above) and adjust the depth of deformation accordingly. The droplet size may refer to the width (e.g., diameter) of the droplet (when viewing the droplet through the first and / or second sheet), or it may refer to a metric value that includes both the width and height of the droplet. The apparatus described herein may adjust the depth of the deflection of the upper sheet based on the resulting droplet size by scaling between a lower limit of deflection of the upper sheet with respect to the height of the air gap (e.g., 5%, 10%, 15%, 20%) and an upper limit of deflection of the upper sheet with respect to the height of the air gap (e.g., 95%, 90%, 85%, 80%); the depth may be scaled between the upper and lower deflection limits for a range of droplet sizes between an upper threshold of size (e.g., 2 cm in diameter) and a lower threshold of size (e.g., 2 mm).For example, by limiting the height change of the stylus (in z), the deflection of the upper sheet may be limited to a lower deflection limit (e.g., 10% deflection) for droplets at or above the upper size threshold. For example, by limiting the height change of the stylus (in z), the deflection of the upper sheet may be limited to an upper deflection limit (e.g., 90% deflection) for droplets at or above the lower size threshold.

[0462] It should be noted that while many of the examples described herein refer to applying a stylus by pressing it against the upper sheet to reduce the height of the air gap, in any of these apparatuses and methods, the cartridge (e.g., the upper sheet) may be pressed against and driven by the stylus, or the two may move relative to each other, in order to achieve an equivalent effect.

[0463] Drop Gloss As described above, these methods and apparatus may generally involve the use of a drop gloss material, which is combined with or contained in aqueous droplets. A drop gloss generally refers to a surfactant that coats droplets and reduces the effective surface tension of the droplets compared to pure water. Generally, droplet movement within an air gap between the upper and lower hydrophobic and oleophobic surfaces that form the air gap can be greatly enhanced by the use of a drop gloss, especially drop glosses that reduce the surface tension of aqueous droplets to less than pure water (e.g., 72 dyn / cm at 20°C), to the level of ethanol (e.g., 22 dyn / cm at 20°C), or less. Therefore, DropGloss may be configured to reduce the surface tension of aqueous droplets to at least 10% (or more than 10%) less than the surface tension of pure water, such as 65 dyn / cm or less, 60 dyn / cm or less, 55 dyn / cm or less, 50 dyn / cm or less, 45 dyn / cm or less, 40 dyn / cm or less, 35 dyn / cm or less, 30 dyn / cm or less, 25 dyn / cm or less, or 20 dyn / cm or less (at 20°C).

[0464] In some examples described herein, the drop gloss may also reduce or limit evaporation. For example, the drop gloss may coat the droplet to prevent or limit (or limit) evaporation. However, limiting evaporation is not essential to the function of the drop gloss. Any suitable drop gloss may be used as long as it meets the specified criteria of reducing surface tension and remaining relatively inert (e.g., non-reactive) with the aqueous material and / or the material in the droplet.

[0465] waste pad Any apparatus (e.g., a cartridge) and method described herein may include an absorbent for sequestering or removing material (liquid material) from an air gap. For example, the absorbent may be a waste pad at least partially contained within the air gap. Generally, the waste pad is configured not to expand within the air gap; such expansion could undesirably alter the height of the air gap. Thus, the absorbent may be a non-expandable absorbent material, such as a non-expandable material or a material made from non-expandable fibers. Alternatively or additionally, the absorbent may be constrained not to expand, at least in the height (z) direction. For example, the absorbent may be held within the frame of the device and constrained by the frame not to expand in the z direction.

[0466] In general, the rate of droplet movement within an air gap may be determined by the movement of deformation applied in the transverse plane (x, y plane), such as the movement of the stylus in this plane. In some examples, the device may limit the rate of movement to maintain smooth droplet movement. To prevent overfilling of the air gap, the addition of fluid into the air gap may be limited. To prevent the air gap from overflowing, the absorption rate may also limit the rate of fluid addition into the air gap. For example, the rate of fluid addition may be limited to 1 L / min or less (e.g., 900 ml / min or less, 800 ml / min or less, 700 ml / min or less, 600 ml / min or less, 500 ml / min or less, 400 ml / min or less, etc.).

[0467] Integration with liquid handling systems The mechanical microfluidic actuators described herein may be configured as standalone devices or / or integrated into a liquid handling system or subsystem (e.g., modules). Examples of liquid handling subsystems include, but are not limited to, the Integra Assist Plus (INTEGRA) Pipetting Robot or other liquid handling systems.

[0468] Figure 65 schematically illustrates an example of a modularly configured mechanical microfluidic actuator shown connected to a liquid handling system. In Figure 65, the mechanical microfluidic actuator device 6505 includes a force-applying subsystem having a stylus 6509 (e.g., a bearing, roller, etc.) and a force-applying driver subassembly 6574 (e.g., a force-applying subassembly). In this example, the force-applying subassembly includes one or more drivers (e.g., x and / or y motion drivers, z motion driver, etc.) and / or a frame or gantry for driving the force-applying actuator to change position and / or apply force to the cartridge 6577 when the cartridge is installed in the cartridge seat of the device. The force-applying subassembly may also include one or more stepper motors, motion rails (e.g., a gantry / frame), and / or a home switch. The device may coordinate the position of the force-applying subsystem with that of the liquid handling subsystem so that the components of the force-applying system do not interfere with the components of the liquid handling subsystem (e.g., pipette adapter, pipette, etc.), or vice versa. The mechanical microfluidic actuator module may include communication hardware, software, and / or firmware for communicating with the liquid handling subsystem. Communication may be wireless and / or via direct connection. The controller of the mechanical microfluidic actuator module may communicate with and / or coordinate with the controller 6525 of the liquid handling subsystem. Either the liquid handling subsystem or the mechanical microfluidic actuator module may coordinate the operation of the entire system (mechanical microfluidic actuator module and liquid handling subsystem) to perform automated or semi-automated workflows, including operations such as adding / removing fluid from a cartridge and moving fluid within the cartridge (as described herein).

[0469] In Figure 65, the liquid handling subsystem 6501 (also equivalently referred to herein as a liquid handler or liquid handling robot) may also include one or more chip exchange areas 6518. Mechanical microfluidic actuator modules may be coupled to and / or docked with the liquid handling subsystem. In the example shown in Figure 65, mechanical microfluidic actuator module 6505 docks to the side of the liquid handling subsystem 6501. In some examples, mechanical microfluidic actuator modules may be mounted on the front, rear, or opposite side, or they may be plugged into the liquid handling subsystem. The base of the mechanical microfluidic actuator module may be configured to couple with the liquid handling subsystem.

[0470] As mentioned, the mechanical microfluidic actuator module may also include one or more sensors (e.g., optical sensors) for detecting / tracking / monitoring the position of one or more droplets. In some examples, the optical sensors may be positioned above the base of the mechanical microfluidic actuator module 6505. The mechanical microfluidic actuator module may also include a thermal subassembly for controlling the temperature of one or more regions of the air gap (including within one or more wells, as described above). The thermal subassembly may include a thermally conductive zone or region of the installation area that is in thermal contact with heating and / or cooling elements (e.g., Peltier devices); it may include multiple heating / cooling elements. Any of these mechanical microfluidic actuator modules may also include a magnetic control subassembly for controllably applying a magnetic field into the air gap.

[0471] Generally, the microfluidic actuator device 6505 may be connected to the liquid handling robot 6501 by a coupling 6530 configured to link the microfluidic actuator device to the liquid handling robot. The coupling may be mechanical, electrical, electromechanical, magnetic, optical, or the like. The coupling may be a mechanical coupler that stably links the microfluidic dispenser device to the liquid handler and may be fixed in place by a lock or the like. In some cases, the coupling may include electrical and / or optical connections for transmitting and / or receiving data and / or control information to the microfluidic actuator device. The coupling may be keyed so that the microfluidic actuator device fits and aligns with the liquid handling subsystem 6501. This may allow a dispenser 6515 (e.g., a robotic dispenser) to dispense liquid into / from a cartridge in the microfluidic actuator device. In general, the microfluidic actuator and the liquid handling subsystem 6501 may communicate to coordinate both the operation of the microfluidic actuator (e.g., driving droplets in a cartridge for movement, mixing, heating, splitting, etc.) and the operation of the liquid handling subsystem 6501 (e.g., adding / removing droplets, including reagents, samples, products, etc.). When coupled together, the microfluidic actuator and the liquid handler may share a processor and / or coordinate separate processors. In some cases, the processor of the microfluidic actuator may drive or control the liquid handling subsystem 6501; alternatively, in some cases, the liquid handling subsystem 6501 may drive or control the microfluidic actuator.

[0472] The coupler may engage with a track or rail on the liquid handling subsystem 6501, and / or the track or rail may extend for the movement of a portion of the liquid handling subsystem 6501 (e.g., a dispenser, a dispenser arm, etc.).

[0473] The mechanical microfluidic actuator module may include cartridge fasteners (e.g., holders, clamps, locks, etc.) for securing a cartridge to the cartridge seat or mounting area of ​​the mechanical microfluidic actuator module. The mechanical microfluidic actuator module may also include a vacuum / suction subassembly (not shown) for applying suction force to secure the cartridge within the mounting area. The mechanical microfluidic actuator module may also include one or more control inputs (e.g., keyboard, touchscreen, buttons, switches, etc.) and / or one or more outputs (e.g., display, LED, wireless communication output / input, etc.), as well as hardware, software, and / or firmware for controlling them. Alternatively or additionally, the mechanical microfluidic actuator module may receive input from a fluid handling subassembly; the fluid handling subassembly may include a separate user interface which may also be used to interface with the mechanical microfluidic actuator module. The mechanical microfluidic actuator module may include its own user interface. The mechanical microfluidic actuator module may include leveling. For example, the mechanical microfluidic actuator module may include an adjustable leveling foot.

[0474] The same mechanical microfluidic actuator module may be configured for use with a liquid handling subsystem (e.g., a liquid handling robot) and / or for use as a standalone mechanical microfluidic actuator device (where the fluid may be manually added / removed).

[0475] Additional uses The mechanical microfluidic actuators and methods using them described herein can be adapted to perform a variety of different workflows in addition to those described above. For example, the methods and apparatus described herein may be used for nucleic acid amplification. For example, Figures 66A-66B illustrate an example of a method for nucleic acid amplification using mechanical droplet actuation. In this example, the cartridge may be configured to perform amplification by thermal cycling within the cartridge; it should be noted that other amplification techniques may be used, including non-thermal amplification such as loop-mediated isothermal amplification (LAMP).

[0476] In Figure 66A, the cartridge includes an upper sheet (e.g., film) 6610 that is elastically deformable and transparent in this example, and a lower sheet that may also be elastically deformable or rigid. The upper and lower sheets may form an air gap between them and may have hydrophobic and oleophobic surfaces. In some examples, the cartridge includes a heating region or well 6656 as described above, which may be thermally coupled to a heater / cooler (e.g., Peltier) 6645.

[0477] During operation, the polynucleotide materials to be mixed may be combined with amplification components (e.g., primers, buffers, enzymes, etc.) and drop gloss 6618 to form reaction droplets that may be held within a heating zone (e.g., well) as shown in Figure 66B. For example, as shown in Figure 66A, droplets of master mix 6611 (e.g., buffers, enzymes, etc.) may be combined in the air gap with droplets containing primer 6610 and droplets of template polynucleotide (e.g., cDNA) 6609; this may be done by pipetting each droplet from the inlet into the air gap, either automatically (using a liquid handling subsystem) or manually, as shown in Figures 62A-62J. Droplets may be added through openings in the upper sheet. Droplets may be moved within the air gap region to combine and / or mix them; this may be done by mechanically deforming the upper sheet to reduce the height of the air gap and thus increase capillary force, in order to pull the droplets behind a sagging region of the upper sheet. The droplets, once mixed, may be held in well 6656 as shown in Figure 66B and reacted by cycling the temperature of the well (and therefore the temperature of the droplets) as described herein. In some examples, the reaction may be monitored by one or more sensors, including a detection (e.g., optical detection) sensor 6640, as shown. Thus, Figure 66A illustrates one example in which a droplet of cDNA (or gDNA) is mixed with gene-specific primer 6610 and PCR master mix 6611, followed by real-time PCR amplification and optional detection for, for example, up to 40 cycles. In some examples, the method may include a step of removing the reaction droplets from the well after the amplification is complete; this may be done, for example, by pulling the droplets out of the well and locally deforming the upper sheet to pull the droplets into another area of ​​the air gap in which the droplets may be washed and / or eluted. For example, the reaction droplets may be combined with a solid-phase substrate, such as beads, such as magnetic beads, and then washed, rinsed, and eluted.These steps may all be performed within the air gap using the mechanical actuation techniques described herein. The droplets with amplified material may be removed as described above by pipetting from the opening into the air gap (see, for example, Figures 62H–62J).

[0478] The methods and apparatus described herein may be used, for example, as a proteomics platform for synthesizing and / or analyzing proteins. For example, Figure 67 illustrates an example workflow for a shotgun proteomics method using mechanical actuation to move droplets as described herein. In Figure 67, the cartridge (including the air gap region) may be modified for use with a mass spectrometer 6780. In this example, a biological sample may be collected and processed, for example, by cell lysis or sedimentation, to extract proteins. In some examples, the sample may be prepared in cartridge 6701. For example, as shown in Figure 67, a sample 6712 may be added to the air gap of the cartridge, and proteins may be extracted from the sample 6771 (by known methods, such as sonication, pressure, or enzymes) to form a droplet 6713 containing proteins. The droplets may be relatively small and may be moved within the cartridge, for example, by deforming the upper sheet to pull the droplets within the air gap. The extracted proteins may be enzymatically digested 6773 with trypsin at 70°C, for example (using heater 6745), to form peptides. The resulting peptide mixture 6714 may then be subjected to pre-fractionation 6775 using a C18 solid-phase extraction monolith 6777, which may be incorporated (e.g., embedded) in the cartridge, and subsequently separated using a liquid chromatography (LC) capillary column 6779, which may be integrated between an upper sheet and a lower sheet, for example, in or connected to an air gap. Finally, the separated peptides may be analyzed using tandem mass spectrometry 6780 (MS / MS) for ionization and fragmentation, which generates fragment spectra for amino acid sequencing. Thus, in any of these instruments, the cartridge described herein may include a solid-phase extraction region, and the method of using this cartridge may include pre-fractionation. Pressure (positive pressure) may be used to spray the droplet material into the spectrometer.Therefore, the cartridge may include an inlet to the spray area that can be closed or sealed and connected to a positive pressure source. Alternatively, in some examples, the cartridge does not include an HPLC area (e.g., a capillary column), but the peptide-containing sample may be transferred to another device or system (e.g., a subsystem).

[0479] Assay In general, the methods and apparatus described herein may be used to perform one or more assays, including immuno-based assays. In some examples, the cartridges and / or mechanical microfluidic actuators described herein may be configured to perform bead-based ELISA assays. The cartridges and controllers may be configured to perform ELISA-like assays as illustrated in Figures 68A–68F. For example, a droplet containing the sample to be assayed (e.g., a biological sample, an environmental sample, etc.) may be loaded into the cartridge (including, as described above, an air gap, as well as an upper elastically deformable sheet 6810 and a lower sheet 6820), the sample droplet 6870 may be pre-treated if necessary (including lysis / destruction of one or more cells, etc.), and may be combined with a substrate 6871, such as magnetic beads coated with an antibody specific to a target protein. This is illustrated in Figure 68A. The droplets may be mixed as described above, for example, by periodically bending the upper sheet of the cartridge above the droplets. The droplets may then be merged and incubated; in the meantime, the target analyte may adhere to the magnetic beads 6871, while undesirable proteins may be removed through washing facilitated by the magnet. This is illustrated in Figure 68B, which shows a step that allows the beads to be washed as described above using a magnet at the bottom of the cartridge, which can be selectively engaged to pull the beads 6873 with the analyte downwards (pelletize); as well as a step that uses mechanical actuation to deform the upper sheet to draw the washing solution onto and away from the magnetic beads. Following incubation, a secondary antibody may be bound to the captured antigen 6875 as shown in Figure 68C, after which any excess antibody may be washed away 6877 (by selectively engaging the magnet again to pull the beads downwards, allowing rinsing and resuspension).In some examples, the secondary antibody may be linked to an enzyme, and after removing excess enzyme by washing (Figure 68D), the substrate may be used to quantitatively measure the concentration of the target protein using an embedded detection mechanism, as shown in Figures 68E-68F. In Figure 68E, magnetic beads with the antibody, the bound protein, and the secondary antibody are washed and combined (e.g., suspended) with a solution of enzyme substrate material. The substrate may react in the substrate droplet, and the resulting reaction may be detected, for example, optically, as shown in Figure 68F. In this example, the substrate droplet may be monitored for colorimetric changes over time.

[0480] Therefore, any of these devices may include software, hardware, and / or firmware for monitoring and tracking enzyme reactions, which allows assays to be performed directly within the cartridge. These assays may be performed in parallel across multiple regions (e.g., lanes) of the cartridge.

[0481] cell culture Methods and apparatus for performing cell culture and / or analyzing cultured cells are also described herein. In some examples, these methods may be used for screening, for example, to determine one or more effects of molecules (e.g., drugs, small molecules, immunotherapeutic agents, etc.) or combinations of molecules. The cartridges described herein may be adapted to allow cell growth and adhesion (e.g., transient adhesion) within an air gap. The air gap may be controlled to control culture conditions (e.g., temperature, CO2 / O2 / N2 mixture, etc.). One or more surfaces of the cartridge may be configured to allow cell binding / adhesion in order to allow cell growth within the air gap. Growth medium may be added by using mechanical control of droplet positioning as described herein.

[0482] Figures 69A–69D illustrate an example of a device (e.g., a cross-sectional view through a cartridge) and method configured for culturing cells using these devices. The cartridge may include an elastically deformable upper cartridge sheet 6910 and a lower cartridge sheet 6920. The lower cartridge sheet may include one or more adhesion regions ("adhesion pads") having hydrophilic regions (e.g., a first hydrophilic region 6935 and a second hydrophilic region 6931). Figure 69A illustrates an automated medium exchange 6903 for long-term cell culture, showing a mechanism for automated medium exchange using these devices. In this example, a fresh medium source droplet displaces the old medium from the culture site while simultaneously supplying fresh medium to the cells. This process may be automated dispensing, because a larger droplet displaces the old medium as it crosses the adhesion pad 6935, leaving a portion of the new medium behind, which occurs spontaneously due to the hydrophilic contrast along the lower sheet 6920. The cells may first be suspended and then pipetted or otherwise delivered into the air gap; the droplet containing the cells may be mechanically actuated by moving the flex area of ​​the upper sheet 6910 to pull the droplet into position on the hydrophilic adhesive pad 6935; and the cells can then be fixed and adhered to the adhesive pad. The cells may be cultured for any appropriate time, and the medium may be automatically replaced as described above by locally flexing the upper sheet with a stylus to pull a droplet of medium onto the hydrophilic adhesive pad and replace the medium.

[0483] The cells may be examined while being cultured on the adhesion pad or removed for further processing (see, for example, Figure 67 above). In some examples, the cells may be removed by applying trypsin, as illustrated in Figure 69B. For example, the cells may be subcultured using the methods and apparatus described herein. As shown in the figure, in some cases, the first generation of cells may form a monolayer on the primary adhesion spot 6935. After delivering the trypsin-containing droplet using mechanical actuation as described herein, the cells may be detached from the surface as shown in the figure 6939. The trypsin-treated cells may then be collected in a droplet of serum-containing medium and then transferred to a secondary spot for further culture. The droplet containing the trypsin-treated cells may be harvested and mechanically transported by locally bending the upper sheet with a stylus so that the droplet follows a local flexure 6941. As shown in Figure 69C, all or part of the droplet may be transported in this manner. Subsequently, the cells may be subcultured as a monolayer on the same or other adhesion pad, as shown in Figure 69D. In this example, the subcultured cells form a monolayer 6945 on the secondary adhesion pad 6931 after 72 hours.

[0484] As illustrated in Figures 70 and 71, cultured cells may be used as part of an assay. For example, Figure 70 shows an example of a cartridge (top view and a portion of a cross-sectional view). In Figure 70, the cartridge contains eight lanes and is conceptually divided into three regions: a cell culture region on the lower part 7020 (with multiple hydrophilic adhesion pads 7043, 7055), a first cell-based assay region (with one or more hydrophilic adhesion pads), and a second cell-based assay region. Although eight lanes are shown, more or fewer lanes may be used. The elastically deformable upper sheet 7010 of the cartridge (forming the upper part of the air gap) may contain multiple openings (inlet / outlet openings 7038, 7038', 7038'', 7038'''') along the sheet, particularly within the lanes and between the adhesion pads. These inlets / outlets may allow a pipette to be inserted into the air gap to add or remove material, including culture medium, trypsin, etc.

[0485] Generally, a cartridge (such as the one shown in Figure 70) may contain one or more cell culture regions, each containing one or more hydrophilic adhesion pads. These cartridges may be configured to enable the aforementioned subculturing, allowing multiple repeatable assays to be performed in parallel, and thus enabling screening assays, including toxicity assays.

[0486] Figure 71 schematically illustrates another example of a cartridge configured for use in a mechanical actuation device and also configured for an integrated setup of cell culture / cell assays. These cartridges may be used for assays in "omics" (genomics, proteomics, etc.). Similar to the example shown in Figure 70, the cartridge shown in Figure 71 includes multiple conceptual regions (e.g., cell culture region, cell death assay region, cell health assay region, and cytotoxicity region). In this example, some of the lanes (e.g., lanes 1-4) may be configured for cell manipulation. Some of the lanes (lanes 5-8) may be configured for sample preparation (or assays).

[0487] In the top / side view of the 8-lane cartridge shown in Figure 41, the cartridge may be used with a system containing an array of Peltier heating / cooling elements and / or magnetic / resistive heating zones, which can perform cell culture; cell-based assays; DNA / RNA / protein / metabolite extraction; sample preparation for genomics, transcriptomics, proteomics, metabolomics; and sequencing of specified regions.

[0488] In any of these examples, cells may be introduced into a lane, cultured under controlled experimental conditions, and assayed for cell health, cell death, toxicity, and pharmacological responses. Live cell imaging may be used by the instrument to report cell assays, and therefore the deformable upper sheet may be optically clear. The cell culture area may include a hydrophilic pad for culturing cells; cells may be dispensed into assay zones for specific assays; assays may or may not require further cell culture. As mentioned, cells may also be dispensed into a sample preparation lane for omics; in that lane, nucleic acids, proteins, and metabolites may be extracted and used as input for downstream sample preparation steps required for comprehensive analysis of the genome, transcriptome, proteome, and metabolome.

[0489] For example, a device (including a cartridge) as shown in Figure 71 may be used for assays such as (but not limited to) CRISPR-mediated knock-in and genetic and functional characterization. In this example, three edited cell clones with desired SNV knock-in and one control (wild-type cell) are introduced into cell culture areas in separate lanes and cultured until the desired number of cells is reached, using the mechanical actuation techniques described herein to change the growth medium. Aliquots of each cell clone are dispensed into the assay zones of different lanes to perform three cell assays: annexin V apoptosis, LDH cell viability, and beta-galactosidase senescence measurement. (In the case of adherent cells, the methods described above may be used to trypsinize and resuspend the cells.) The optical clarity of the deformable top film allows for live cell imaging to report the cell assays, and colorimetric and / or fluorescence signals are recorded. Aliquots of each cell clone are dispensed into different omics sample preparation lanes, where the cells are lysed and proteins are digested and prepared for proteomics mass spectrometry analysis.

[0490] It should be recognized that all combinations of the aforementioned concepts, and additional concepts described in more detail below (provided that such concepts are not contradictory), are intended to be part of the invention disclosed herein and may be used to realize the benefits described herein.

[0491] The processing parameters and sequences of steps described and / or illustrated herein are given only as examples and may vary 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 they are 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 the disclosed steps.

[0492] Any of the methods described herein (including user interfaces) may be implemented as software, hardware, or firmware, and may be described as a non-temporary computer-readable storage medium storing a set of instructions; the set of instructions may be executed by a processor (e.g., a computer, tablet, smartphone, etc.), and when executed by the processor, it causes the processor to control or perform any of the following stages, not limited to, stages of displaying, communicating with a user, analyzing, modifying, determining, or changing parameters (including timing, frequency, intensity, etc.). For example, any of the methods described herein may be at least partially implemented by a device including one or more processors having memory storing a non-temporary computer-readable storage medium storing a set of instructions for the process of that method.

[0493] Various embodiments have been described and / or illustrated herein in the context of a fully functional computing system, and one or more of these exemplary embodiments may be distributed as a program product in various forms, regardless of the specific type of computer-readable medium used for actual distribution. The embodiments 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 computer-readable storage media or within a computing system. In some embodiments, these software modules may constitute a computing system for performing one or more of the exemplary embodiments disclosed herein.

[0494] 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 being contained within a module described herein. These computing devices may, in their most basic configuration, each comprise at least one memory device and at least one physical processor.

[0495] As used herein, the terms “memory” or “memory device” generally refer to any type or form of volatile or non-volatile storage device or medium 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 drives (HDDs), solid-state drives (SSDs), optical disk drives, caches, variations or combinations of one or more of these, or any other suitable storage memory.

[0496] In addition, as used herein, the terms “processor” or “physical processor” generally refer 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, but are not limited to, microprocessors, microcontrollers, central processing units (CPUs), field-programmable gate arrays (FPGAs) implementing soft-core processors, application-specific integrated circuits (ASICs), parts of one or more of these, variations or combinations of one or more of these, or any other suitable physical processor.

[0497] The steps of the method described and / or illustrated herein, even if illustrated as separate elements, may represent a part of a single application. In addition, in some embodiments, 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, cause the computing device to perform one or more tasks, such as the steps of the method.

[0498] In addition, one or more of the devices described herein may transform data, physical devices, and / or representations of physical devices from one form to another. Additionally or alternatively, one or more of the modules described herein may transform a processor, volatile memory, non-volatile memory, and / or any other part of a physical computing device from one form of computing device to another form of computing device by the steps of running on a computing device, storing data on a computing device, and / or otherwise interacting with a computing device.

[0499] As used herein, the term “computer-readable medium” generally refers to any form of device, carrier, or medium capable of storing or carrying computer-readable instructions. Examples of computer-readable mediums include, but are not limited to, transmissive media such as carrier waves; as well as non-transient media such as magnetic storage media (e.g., hard disk drives, tape drives, and floppy disks), optical storage media (e.g., compact discs (CDs), digital video discs (DVDs), and Blu-ray discs), electronic storage media (e.g., solid-state drives and flash media), and other distribution systems.

[0500] Those skilled in the art will recognize that any processing or method disclosed herein can be modified in many ways. The processing parameters and sequences of steps described and / or illustrated herein are given only as examples and may vary 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 they are illustrated or discussed.

[0501] 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 herein. Furthermore, one step of any method disclosed herein may be combined with one or more steps of any other method disclosed herein.

[0502] 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 of the methods disclosed herein.

[0503] In this specification, when a feature or element is said to be "on" another feature or element, it may be directly on the other feature or element, or there may also be intervening features and / or elements. In contrast, when a feature or element is said to be "directly on" another feature or element, there may be no intervening features or elements. Also to be understood, when a feature or element is said to be "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. In contrast, when a feature or element is said to be "directly connected," "directly attached," or "directly coupled" to another feature or element, there may be no intervening features or elements. Features and elements described or illustrated in relation to one aspect may also apply to other aspects. Also, as will be apparent to those skilled in the art, a reference to a structure or feature placed "adjacent" to another feature may have a portion that overlaps with or is beneath that adjacent feature.

[0504] The technical terms used herein are for the sole purpose of describing specific aspects and are not intended to limit the invention. For example, the singular forms “a,” “an,” and “the” used herein are intended to also include the plural form unless otherwise explicitly stated in the context. It should be understood that the terms “include” and / or “compris” used herein specify the presence of the described features, stages, actions, elements, and / or components, but do not exclude the presence or addition of one or more other features, stages, actions, elements, components, and / or groups thereof. The terms “and / or” used herein include any and all combinations of one or more of the related enumerated items, and may be abbreviated as “ / ”.

[0505] Spatial relationship terms such as “under,” “below,” “lower,” “over,” and “upper” may be used herein to facilitate explanation when describing the relationship of one element or feature to another, as illustrated in drawings. It is understood that spatial relationship terms are intended to encompass different orientations of a device in use or operation, in addition to the orientation depicted in the drawing. For example, if a device in a drawing is inverted, an element described as being “under” or “beneath” another element or feature will be oriented “over” that other element or feature. Thus, the illustrative term “under” may encompass both upper and lower orientations. A device may also be oriented in other ways (rotated 90 degrees or to other orientations), and the spatial relationship terms herein will be interpreted accordingly. Similarly, terms such as “upward,” “downward,” “vertical,” and “horizontal” are used herein for descriptive purposes only, unless otherwise specifically indicated.

[0506] The terms “first” and “second” may be used herein to describe various features / elements (including stages), but unless otherwise indicated in the context, these features / elements should not be limited by these terms. These terms may be used to distinguish one feature / element from another. Thus, without departing from the teachings of the invention, the first feature / element described below may be called the second feature / element, and similarly, the second feature / element described below may be called the first feature / element.

[0507] Throughout this specification and the accompanying claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” mean that various components may be used together in the method and thing (e.g., devices and methods, components and apparatus). For example, the term “comprise” is understood to imply the inclusion of any element or step described, but not the exclusion of any other element or step.

[0508] In general, any apparatus and method described herein should be understood as comprehensive; however, alternatively, all or a subset of components and / or stages may be exclusive and may be expressed as "consisting of" or alternatively "essentially consisting of" various components, stages, subcomponents, or substages.

[0509] All numbers used herein, including those used in examples, may be interpreted as being preceded by the terms “about” or “approximately,” even if those terms are not explicitly stated, unless otherwise indicated. The phrases “about” or “approximately” may be used when describing size and / or location to indicate that the described value and / or location falls within a reasonable expected range of the value and / or location. For example, a number may have values ​​that are + / - 0.1%, + / - 1%, + / - 2%, + / - 5%, + / - 10%, etc., of the stated value (or range). Any number given herein should be understood to include its value approximately or approximate unless otherwise indicated in the context. For example, if the value “10” is disclosed, “about 10” is also disclosed. Any numerical range disclosed herein is intended to include all subranges that are encompassed therein. Also to be understood, where a value is disclosed, “less than or equal to that value,” “greater than or equal to that value,” and possible ranges between values ​​are also disclosed in a manner that can be appropriately understood by those skilled in the art. For example, if a value “X” is disclosed, then “less than or equal to X” and “greater than or equal to X” (for example, if X is a number) are also disclosed. Also to be understood, throughout this application, data is provided in numerous different forms; and this data represents the end point, the start point, and the range for any combination of data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, then, as with the range between 10 and 15, greater than or equal to 10 and 15, greater than or equal to 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are also deemed to be disclosed. Also to be understood, each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0510] While various exemplary embodiments have been described above, any of the numerous modifications can be made to these embodiments without departing from the scope of the invention as described in the claims. For example, the order in which the various steps of the described method are performed may often be changed in alternative embodiments, and in other alternative embodiments, one or more steps of the method may be skipped together. Optional features of various embodiments of the device and system may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as stated in the claims.

[0511] The examples and illustrations included herein illustrate, not as limitations, specific ways in which the subject matter may be put into practice. As noted, other embodiments may be used and derived therefrom, so as to be structural and logical substitutions and modifications may be made without departing from the scope of this disclosure. Such embodiments of the subject matter of the invention may be referred to herein individually or collectively by the term “invention,” but this is for convenience only and is not intended to spontaneously limit the scope of this application to any single invention or inventive concept if more than one invention or inventive concept is actually disclosed. Thus, while specific embodiments are illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for those specific embodiments. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the embodime...

Claims

1. A mechanical microfluidic actuator device configured to be connected to a liquid handling robot, An installation area configured to connect to a cartridge; A stylus configured to deform the upper surface of the cartridge when the cartridge is installed within the installation area; An inductor driver subassembly connected to the stylus and configured to control the movement of the stylus laterally along the upper surface of the cartridge while deforming the upper surface of the cartridge vertically; and A coupling is configured to connect the microfluidic actuator device to the liquid handling robot. A mechanical microfluidic actuator device comprising the following:

2. The apparatus according to claim 1, further comprising an injector drive assembly and a controller configured to control the operation of the liquid handling robot.

3. The apparatus according to claim 1, further comprising the liquid handling robot.

4. The apparatus according to claim 1, wherein the installation area comprises one or more suction ports configured to fix the cartridge therein.

5. The apparatus according to claim 1, wherein the injector driver subassembly comprises one or more drivers.

6. The apparatus according to claim 5, wherein the one or more drivers comprises x and / or y motion drivers and / or z motion drivers.

7. The apparatus according to claim 1, wherein the inductor driver subassembly comprises a frame or gantry on which the stylus may be driven to change position and / or to apply force to the cartridge.

8. The apparatus according to claim 1, wherein the applicator driver subassembly is configured to move the stylus to apply a pinning compressive force to divide a first fluid droplet in the cartridge, and to apply an actuation compressive force near the pinning compressive force to extend and form a second fluid droplet from the first fluid droplet, the pinning compressive force being greater than the actuation compressive force.

9. The apparatus according to claim 1, wherein the applicator driver subassembly is configured to move the stylus to apply a compressive force to the cartridge between two or more separate fluid droplets, and to release the compressive force to combine the two or more separate fluid droplets into a single fluid droplet.

10. The apparatus according to claim 1, wherein the applicator driver subassembly is configured to alternately move the stylus in order to apply a first compressive force and a second compressive force different from the first compressive force to the cartridge in order to mix two or more separate fluid droplets together.

11. The apparatus according to claim 1, wherein the applicator driver subassembly is configured to move the stylus to mix two or more fluid droplets together by repeatedly applying and releasing a compressive force to the cartridge in close proximity to the two or more fluid droplets.

12. The apparatus according to claim 1, wherein the applicator driver subassembly is configured to control a magnet to attract iron particles suspended in a fluid droplet within the cartridge.

13. The apparatus according to claim 1, wherein the applicator driver subassembly is configured to resuspend one or more iron particles in the fluid droplets within the cartridge by applying and releasing compressive force to the fluid droplets and by disabling the magnet.

14. The apparatus according to claim 1, wherein the installation area comprises one or more protrusions configured to deform a film on the cartridge in order to form one or more channels.

15. The apparatus according to claim 1, further comprising a thermal control area within the aforementioned installation area.

16. An installation area configured to connect to a cartridge; A stylus configured to apply mechanical pressure to deform the upper surface of the cartridge when the cartridge is installed within the installation area; An injector driver subassembly, connected to the stylus and configured to control the lateral movement of the stylus along the upper surface of the cartridge while deforming the upper surface of the cartridge vertically; One or more well-forming surfaces that extend upward from the upper installation surface of the installation area; and One or more suction ports are configured to pull the lower surface of the cartridge toward the upper searing surface so that the lower surface of the cartridge confirms with the well-forming surface. A mechanical microfluidic actuator device equipped with the following:

17. The apparatus according to claim 16, wherein the well-forming surface is configured to form a heating / cooling well that has a thermal contract with the thermal control region of the installation region.

18. The apparatus according to claim 16, further comprising an injector drive assembly and a controller configured to control the operation of the thermal control region.

19. The apparatus according to claim 16, further comprising a liquid handling robot.

20. The apparatus according to claim 16, wherein the injector driver subassembly comprises one or more drivers.

21. The apparatus according to claim 20, wherein the one or more drivers comprises x and / or y motion drivers and / or z motion drivers.

22. The apparatus according to claim 16, wherein the injector driver subassembly comprises a frame or gantry on which the stylus may be driven to change position and / or to apply force to the cartridge.

23. The apparatus according to claim 16, wherein the applicator driver subassembly is configured to move the stylus to apply a pinning compressive force to divide a first fluid droplet in the cartridge, and to apply an actuation compressive force near the pinning compressive force to extend and form a second fluid droplet from the first fluid droplet, wherein the pinning compressive force is greater than the actuation compressive force.

24. The apparatus according to claim 16, wherein the applicator driver subassembly is configured to move the stylus to apply a compressive force to the cartridge between two or more separate fluid droplets, and to release the compressive force to combine the two or more separate fluid droplets into a single fluid droplet.

25. The apparatus according to claim 16, wherein the applicator driver subassembly is configured to alternately move the stylus to apply a first compressive force and a second compressive force different from the first compressive force to the cartridge in order to mix two or more separate fluid droplets together.

26. The apparatus according to claim 16, wherein the applicator driver subassembly is configured to move the stylus to mix two or more fluid droplets together by repeatedly applying and releasing a compressive force to the cartridge in close proximity to the two or more fluid droplets.

27. The apparatus according to claim 16, wherein the applicator driver subassembly is configured to control a magnet to attract iron particles suspended in a fluid droplet within the cartridge.

28. The apparatus according to claim 16, wherein the applicator driver subassembly is configured to resuspend one or more iron particles in the fluid droplets within the cartridge by applying and releasing compressive force to the fluid droplets and by disabling the magnet.

29. A method for forming a well within the air gap of a cartridge, The step of placing the lower sheet of the cartridge on the installation area in order to connect the installation area to the cartridge; A step of applying suction force to bring the lower sheet to the mounting surface so that a well-forming surface, which is raised and extends from the upper surface of the mounting surface, forms a heating / cooling well within the air gap; and The step of driving a stylus to apply mechanical pressure to deform the upper surface of the cartridge when the cartridge is installed within the installation area. The method, including the method.

30. An upper surface comprising an elastically deformable sheet having a hydrophobic and oleophobic surface; Lower surface; An air gap between an upper surface and a lower surface, wherein the distance between the upper surface and the lower surface changes in order to allow a droplet to move through the air gap; The frame between the upper surface and the lower surface to which the sheet is attached; and A tensioner receiver on the frame covered by the sheet, the tensioner receiver being configured to receive the tensioning projection of the tensioner in order to pull the sheet into the tensioner receiver in order to keep the sheet taut when engaged with the tensioner. A cartridge for surface mechanical actuation (MAOS) that is equipped with the following features.

31. The cartridge according to claim 30, wherein the lower surface comprises a second elastically deformable sheet that is primarily hydrophobic and oleophobic.

32. The cartridge according to claim 31, wherein the second elastically deformable sheet comprises a plurality of hydrophilic cell adhesion pad regions configured to enable cell adhesion for culture within the air gap.

33. The cartridge according to claim 30, wherein the air gap is divided into multiple lanes.

34. The cartridge according to claim 30, wherein the sheet is attached to the outer edge of the frame.

35. The cartridge according to claim 30, wherein the tensioner receiver comprises a tensioner receiver channel.

36. The cartridge according to claim 30, further comprising the tensioner.

37. The cartridge according to claim 30, further comprising a plurality of openings in the upper sheet configured to provide access to the air gap.

38. An upper surface comprising a first elastically deformable sheet having a hydrophobic and oleophobic surface; The lower surface comprises a second elastically deformable sheet that is primarily hydrophobic and oleophobic; The air gap between the upper surface and the lower surface; A frame between the upper surface and the lower surface, wherein the sheet is attached to the frame on the outer edge of the frame; and A tensioner receiving channel on the frame covered by the sheet, the tensioner receiving channel configured to receive the tensioning projection of the tensioner in order to pull the sheet into the tensioner receiving channel in order to keep the sheet taut when engaged with the tensioner. A cartridge for surface mechanical actuation (MAOS) that is equipped with the following features.

39. An installation area configured to connect to a cartridge; A tensioner comprising a plurality of tensioning projections extending from the inner surface, configured to engage with one or more tensioner receivers on the upper surface of the cartridge, in order to pull the upper elastic sheet of the cartridge into the tensioner receiver and thereby keep the sheet taut; A stylus configured to deform the upper elastic sheet of the cartridge when the cartridge is installed within the installation area; and An applicator driver subassembly is connected to the stylus and configured to control the lateral movement of the stylus along the upper elastic sheet of the cartridge while deforming the upper elastic sheet of the cartridge vertically. A mechanical microfluidic actuator for surface mechanical actuation (MAOS) comprising the following features.

40. The apparatus according to claim 39, further comprising a controller configured to control the operation of an injector drive assembly.

41. The apparatus according to claim 39, wherein the installation area comprises one or more suction ports configured to fix the cartridge therein.

42. The apparatus according to claim 39, wherein the injector driver subassembly comprises one or more drivers.

43. The apparatus according to claim 42, wherein the one or more drivers comprises x and / or y motion drivers and / or z motion drivers.

44. The apparatus according to claim 39, wherein the inductor driver subassembly comprises a frame or gantry on which the stylus may be driven to change position and / or to apply force to the cartridge.

45. The apparatus according to claim 39, wherein the applicator driver subassembly is configured to move the stylus to apply a pinning compressive force to divide a first fluid droplet in the cartridge, and to apply an actuation compressive force near the pinning compressive force to extend and form a second fluid droplet from the first fluid droplet, wherein the pinning compressive force is greater than the actuation compressive force.

46. The apparatus according to claim 39, wherein the applicator driver subassembly is configured to move the stylus to apply a compressive force to the cartridge between two or more separate fluid droplets, and to release the compressive force to combine the two or more separate fluid droplets into a single fluid droplet.

47. The apparatus according to claim 39, wherein the applicator driver subassembly is configured to alternately move the stylus in order to apply a first compressive force and a second compressive force different from the first compressive force to the cartridge in order to mix two or more separate fluid droplets together.

48. The apparatus according to claim 39, wherein the applicator driver subassembly is configured to move the stylus to mix two or more fluid droplets together by repeatedly applying and releasing a compressive force to the cartridge in close proximity to the two or more fluid droplets.

49. The apparatus according to claim 39, wherein the applicator driver subassembly is configured to control a magnet to attract iron particles suspended in a fluid droplet within the cartridge.

50. The apparatus according to claim 39, wherein the applicator driver subassembly is configured to resuspend one or more iron particles in the fluid droplets within the cartridge by applying and releasing compressive force to the fluid droplets and by disabling the magnet.

51. The apparatus according to claim 39, wherein the installation area comprises one or more protrusions configured to deform a film on the cartridge to form one or more channels.

52. The apparatus according to claim 39, further comprising a thermal control area within the aforementioned installation area.

Images