Microfluidic device with bubble-based micromixer

Abstract

A kit for forming a microfluidic device with a microfluidic mixer 10 comprises a substrate with a cover-meeting surface having a relief pattern defining a mixing chamber, a gas inlet 15a proximate the

Description

Field

[0001] This application relates to bubble mixing of liquid in lite-and-portable microfluidics. Background

[0002] Bio-analytical technologies that make home based self-testing possible can reduce the burdens of centralized medical laboratory, and in many laboratories and clinical environments, lightweight, portable, simple to use, low footprint, low power consumption, low-cost devices (herein ‘lite-and-portable’) are preferred. However, the need for reliability and accuracy often makes these bioanalytical tools far less than lite-and-portable. For example, widely used lateral flow antigen / antibody tests / devices, which can be very lite-and-portable, often produce false-negative results. PCR tests are well known to give false positive results due to high sensitivity detection capability. One of the glaring differences between lateral flow testing and gold-standard lab testing is the ability to mix samples thoroughly, which is abetted by large volume sampling. Lab-On-a-Chip (LOC) technologies, and microfluidic devices in general, offer automation and rapid detection that can be useful in public health monitoring to drastically control the spread of infectious diseases, even if the tests are less sensitive and selective than more labor-intensive lab testing protocols. The use of small volumes is, in many cases, a virtue unto itself. The benefits of LOC include reduced assay reaction times, increased test sensitivities, lowered cost per test, and lower demands on trained technical staff.

[0003] Fluidic micro-mixing of microvolumes is particularly important to LOC assay protocol accuracy. A micro-mixing component facilitates mixing of relevant biological samples (solid or liquid) and reagents. Current technologies for active mixing are complicated and rely on heavy instrumentation. Furthermore, existing micromixers often require 3D structures to function and are often not mass producible. A simply designed and manufacturable micromixer is needed for LOCs. Micro-mixing remains a significant challenge within the microfluidic community and is not a simple problem to solve.

[0004] In microscale, fluidic flows are laminar because of the low Reynolds number (Re). Micro-mixing can be very slow, if solely relying on the diffusion. To mix liquids completely, a long microchannel or 3D structured channels are often required in microfluidic devices, which adds complexity and difficulty in the fabrication, integration, and assembly of miniaturized devices in high volume, and calls for substantial energy in use. These affect the portability, weight (lightness), and reliability of LOC devices. It is not trivial to design microfluidic devices that are amenable to high volume, low cost, manufacture, to produce easy to use, reliable and operable by instrument-less and lite-instrumented LOC devices.

[0005] Known microfluidic micromixers can be categorized as either active or passive mixers. Passive micromixers employ a structured-base methodology to enhance mixing of two or more liquids, which is achieved by increasing the ratio of contact surfaces or interfaces between the liquids, which in turn improves molecular diffusion between the liquids. There are several strategies used in microfluidic micromixers, including lamination, chaotic advection, injection, and the use of droplets. In one approach, a lamination micromixer splits a stream into 32 streams that are then recombined into a single stream to significantly improve mixing over basic T-mixers. In chaotic advection type mixers, special geometries or structures are manufactured in the mixing channels. A modified channel shaped for splitting, stretching, rotating, folding, and breaking of a stream is used to improve mixing. For example, a 3D serpentine micromixer to induce chaotic advection flow has been fabricated with a traditional soft lithography method where several PDMS layers were fabricated and assembled together to form the 3D channels. In another example, a tesla microvalve design was converted into an in-plane tesla micromixer that creates chaotic advection mixing. In another approach, micromixers that use injection-type mixing split the incoming liquids into multiple streams by injecting the steams at a common entry point. In yet another approach, droplet mixers rely on the shear forces generated between the droplet and carrier fluid while flowing through channels to induce rotation of the droplet causing the liquids inside to rotate in the opposite direction. The liquids inside are then sheared and rotated to mix.

[0006] Applicant’s family of patents based on WO 2013 / 120190 teaches a droplet discretization method that work very well, but requires a centrifuge, and so is decidedly not lite-and-portable. Furthermore, digital microfluidics are well-known to permit droplet mixing, but these require electronics embedded in chips, usually also requiring seals for an oil medium, or controls for avoiding evaporative losses of droplets that have not been made adequately lite-and-portable.

[0007] Active micro-mixing uses external energy sources including pressure sources, magnetic sources, electrohydrodynamic sources, electrokinetic sources, magneto hydrodynamic sources, and thermal sources to create disturbances. Time pulsing of pressure has been utilized to create disturbance for mixing. Magnetic fields and beads have been used to create disturbances in the liquids resulting in mixing. Electrodes lining both sides of a mixing channel achieved mixing by alternating voltage and frequency. Oscillating electro-osmotic flow pulsing to create disturbances has also been reported, which is a continuous flow process. An embedded microheater has been utilized to generate bubbles, which served to produce mixing disturbances. An acoustic source has been used to facilitate micro-mixing by inducing bubbles by utilizing a piezoelectric actuator positioned directly and in contact beneath the mixing reservoir. An air-liquid co-injection micromixer has been utilized in which liquids and nitrogen gas were injected at the same location to form a bubble-liquid mixture, which flows through a series of hexagonal chambers to facilitate bubble expansion and contraction inducing liquid stretching and folding to form chaotic mixing. Air-liquid co-injection micro-mixing is a continuous flow micro-mixing and requires a positive pneumatic injection pressure. Bubble mixing can be achieved in microfluidics by injecting gases into a chamber, which is like that of cylindrical tank mixing with chemicals scaled down to use in microfluidic applications. Nitrogen or air was introduced from the tank’s base to create columns of bubbles. As the bubbles ascend towards the top of the tank for evacuation, vortices are initiated around this bubble column, resulting in disturbances to surrounding liquids. This mixing tank’s aspect ratio (i.e., the ratio of height to width) is about 4:1. In centrifugal based microfluidic devices, bubble mixing has also been demonstrated in which disturbing vortices are initially generated around a bubble column where mixing efficiency was significantly enhanced by the centrifugal force. Flow control and pumping on a centrifugal based microfluidic platform has also been explored.

[0008] The above approaches have significant limitations including the following. Passive micromixers are based on diffusion mechanism requiring long mixing channels, which undesirably affects the ability to miniaturize microfluidic devices. While using 3D microchannels to reduce the microchannel length and create splitting and recombining streams improves mixing efficiency, 3D channel-based micromixers are complicated to fabricate due to the multiple-layer structure, which are not compatible with mass production methods thereby limiting commercial viability. Some active micromixers embedded with additional components such as electrodes, magnetic stirrers, microheaters and the like improve mixing efficiency, but the additional components make assembling and manufacturing devices in volume more difficult and the cost per device is relatively high. Bubble mixing in microfluidic devices has been found to offer various benefits, such as high mixing efficiencies due to turbulent flow (high Reynold's (Re) numbers) created in the process. Bubble mixing improves mixing significantly from traditional diffusion-based mixing (low Re number), but to achieve bubble mixing requires simultaneous injection of both liquids and gas into the mixing channel, and may require volumetric or throughput controls. Many devices cannot imbibe gaseous or liquid media indifferently, and air bubbles are known as severe irritants / issues in microfluidics as small volumes of gas bubbles can greatly affect fluid displacements and the level of fluidic resistances in microfluidic chips or circuits. Hexagonal chambers have been connected by a microchannel in a serial fashion to facilitate a cycle of liquid stretching (bubble expansion) and folding (bubble compression), and a specially designed comb-like structures have been used to filter out bubbles according to some prior art, however Applicant cannot speak to the comb-like structure’s efficacy in removing bubbles. To generate acoustic bubble mixing, external piezo actuators are used limiting control over bubble creation and handling of bubble removal. Furthermore, acoustic bubble formation requires additional instrumentation implementations, which increases the complexity of the device thereby limiting manufacturability. Bubble-based micromixers involving a large commercial chemical processing tank but scaled down to microfluidic levels requires the use of high aspect ratio (2 to 4) reservoirs and must be positioned vertically to function properly, limiting integration with other microfluidic components. Micromixers combining centrifugal force and bubbles are efficient but rely heavily on large instrumentation and are better suited for use in centralized laboratories.

[0009] A relatively new general microfluidic approach has developed around “open microfluidics”. While the term as currently used is quite limited, it does include “open channel microfluidics”, which concerns itself with capillary liquid two phase flows, and is principally enabled with contact angle control which is provided by selecting the liquid’s surface tension and other rheological properties, and surface treatments of the channels. Still, it is fair to say that microfluidic approaches based on three phase microfluidics: i.e. microfluidic spaces in which a solid substrate supports a liquid, separated from a gas (e.g. air) by a free surface, is a newer front in microfluidics, and it simplifies problems of air removal in 2 phase microfluidics.

[0010] There remains a need for a simple and mass-manufacturable lite-and-portable micromixer, especially for lab-on-chips (LOCs). Summary

[0011] Applicant has developed a bubble-based micromixer with surprising low costs of fabrication, and very low energy costs in terms of mixing. The micromixer may be relief patterned on a substrate to provide a chip, and may be provided with a wide array of other chip features to provide for different protocols on the chip. Described herein is a bubblebased micromixer with manufacturable three-dimensional (3D) relief features that is particularly amenable to low cost, reliable, fabrication. The micromixer is preferably in a microfluidic device that performs other microfluidic functions such as fluid displacements, and metering, as well as the microfluidic mixing, without heavy instrumentation like a centrifuge, or expensive manufacturing like the array of electronics embedded in digital microfluidics chip. The micromixer is active, in that injected gas is used to induce mixing, but needs no moving parts in the chamber to mix, and thus onerous cleaning of mixers between uses is avoided, and a single use device is particularly cost effective. Operation of the micromixer involves a pneumatic external manifold (e.g., a suction device such as a vacuum pump, a syringe, or the like) and a gas inlet to induce bubbles in a three-phase mixing chamber. By controlling low negative pressure, bubble disturbances in a mixing chamber enables mixing. Those skilled in the art will appreciate the surprising ability to create bubble mixing with a low cost, small footprint, easily mass manufactured, lite-and-portable chip, with such low energy demands. The micromixer can have a simple design, and be composed of easy to form structures, to provide an easily scalable, chip with no moving parts, and permits elimination of bubbles without the use of centrifugal forces.

[0012] In some embodiments, a microfluidic device comprises a substrate having a structural relief pattern defining a plurality of microfluidic structures, on a cover-meeting surface for sealed meeting with a cover. The microfluidic structures include at least one mixing chamber for mixing a liquid. The mixing chamber is a three-phase microfluidic space. To design the mixing chamber to enable three-phase operation, for a typical range of aqueous liquids used in microfluidics: a depth d (mean recessed depth of its floor from the covermeeting surface) should be more than about 5 mm, and to avoid excessive formation, it generally need not be deeper than 20 mm, more preferably 15 mm; and the minimum dimension or “width” of the mixing chamber’s sealing periphery at the cover-meeting surface, shouldn’t be less than half the depth. Preferably d is chosen so that a fill line, at least in the vicinity of the bubbles, is 5 mm or more below the cover-meeting surface, and accordingly when a suitable volume of liquids is loaded into the mixing chamber, the free surface of the liquids to be mixed is at least 5 mm from a cover. If either of these dimensions are too small, liquid will tend to creep up the sides of the wall, instead of pooling at the bottom, making it difficult to maintain the liquid in a shape amenable to bubble mixing. Furthermore, when bubbles burst, some amount of splatter may arise. It can be desirable to avoid splatter that adheres to the cover or sidewalls away from the liquid, to avoid lost sample, to ensure mixing ratios, or to facilitate viewing of the sample in the mixing chamber. For liquids, such as some buffers, that tend to bubble or even froth, it can be preferable to increase the depth further, and for liquids and shapes that form large bubbles, it can be preferable to increase depth and periphery to provide room to expand without the bubble coming too close to the periphery (especially at a gas output that passes through the periphery). Some “thin” liquids may permit smaller depths, and widths, but with a 6 mm, more preferably 7 mm, and most preferably 9 mm depth, and at least 5 mm width, many typical microfluidic liquids can be mixed.

[0013] For three-phase microfluidic operation, a volume of liquid that can be loaded into the mixing chamber at a time would have to be above a minimum threshold, and below a maximum threshold. The minimum threshold is typically related to a volume that can reliably be delivered to cover the gas inlet, but can be greater than this, for example to ensure a fast return of sufficient volume to cover the gas inlet to allow successive pulses to be applied at a desired pulse rate, and / or to account for any lost volume due to splatter. The maximum threshold would be a minimum clearance between the free surface and cover, as noted hereinabove, and margin for reliable operation.

[0014] The gas inlet meets the mixing chamber at or near its floor, and is coupled to a port of the chip that is open to ambience, or to a substantially ambient pressure gas filled container. Preferably the port is located at or above the cover-meeting surface, to discourage liquid discharge from the mixing chamber therethrough. The gas inlet may be hydrophobic, to further discourage imbibition.

[0015] Apart from the gas inlet, at least a second microfluidic channel opens into the at least one mixing chamber to serve as exhaust or outlet for the gas. The at least one gas outlet meets the mixing chamber proximate the cover-meeting surface, and preferably crosses a seal around the mixing chamber, as it is provided by a structural relief patterning of the substrate. In other embodiments the gas outlet can be provided through the cover, or as a relief pattern of the cover facing the cover-meeting surface. The gas outlet couples the mixing chamber with a port of the chip that is adapted to couple to the pneumatic manifold, e.g. of a suction device, adapted to draw negative relative (to ambi-ent) pressure from the mixing chamber of -0.345 kPa (-0.05 psi) to -3.5 kPa (-0.5 psi).

[0016] Each channel may be network-connected for only gas transport, or may transport liquid or gas depending on a mode or condition of the chip. Likewise the channel may be network-connected for unidirectional flow, or for half duplex flow. In some embodiments channels may be network-connected for half duplex flow with gas flowing in one direction, and liquid flowing exclusively in the other. For example the at least one gas inlet may also serve as a liquid extraction microfluidic channel, as both are preferably proximate the floor of the mixing chamber. The mixing chamber must have at least one opening to an inlet, which is located at or near a floor of the mixing chamber, and at least one gas outlet that meets the mixing chamber at or near a seal of the mixing chamber with the cover for pneumatic extraction (i.e., vacuum suction) to reduce pressure in the mixing chamber above a free surface of the liquid to be mixed, to below ambient pressure level, or that of a pressure in the gas supply. In some embodiments the gas inlet meets the mixing chamber passing through the floor near its centre, and in others it may be on one side of the floor. If it is on one side, the outlet is preferably on a side of the outlet that is substantially diametrically opposed to the side of the inlet, as this increases a path distance between the inlet and outlet, and tends to reduce risk of liquid splatter, or bubble entrainment in the outlet. If there are multiple inlets, they are preferably on opposite sides of the floor, or somewhat evenly distributed around a periphery of the floor, and if so the outlets are preferably arrayed so that each outlet paired with an inlet that is substantially diametrically opposed to the paired inlet, or somewhat evenly distributed around a periphery of the ceiling or the sealing edge therefor.

[0017] In some embodiments, fluid control is more efficient if separate liquid supply microchannels are used instead of using microfluidic channels for both liquid and gas conveyance at different chip loading states. It can generally be efficient to provide gas transfer through a distinct set of channels than those used to transfer the liquid(s) to be mixed, as separate controls can simplify operation. Branching of channels can also permit one segment of a same, branched, channel to be half duplex, and others not. If the same liquid supply is used for removal of mixed liquid, the outlet to this transfer channel is at or near the floor. It can be efficient to supply liquid at a check valve or one-way valve, to prevent liquid from being retracted along the transfer channel. The inlet is coupled to ambience, or to a port for coupling to a gas supply.

[0018] The substrate is part of a chip, which may be part of a cartridge. The chip may be formed by affixing the cover to seal around the channels and the mixing chamber (at least away from the channels); and a cartridge may be formed by further adding stiffening members to the chip, to facilitate handling, and improve alignment in a handling device. The stiffening members may be reusable while the chip may be made disposable to avoid contamination issues. The substrate may have through holes and relief patterning on both sides thereof, and may have a cover on both sides thereof, either or both of which may have relief patterning defining microfluidic channels and or chambers. As the substrate includes at least one three-phase chamber (i.e. the micromixer) the substrate may have relief patterning on a different scale than the microfluidic channels. The microfluidic channels may efficiently be made by distinct forming techniques on the cover, instead of the substrate, even if this imposes some measure of alignment requirements, or the substrate may be subject to two forming processes. The cover may be a pierceable seal.

[0019] In any embodiment where imbibition of liquid into the gas outlet is apt to damage the pump, or impair control thereover, and risk of imbibition remains, the gas outlet preferably comprises a liquid trap adapted to extract any droplet of splatter from the flow. Such traps may include: a keep for a high surface area material (e.g. fabric, fabric pile, nonwoven mat, or cellulose) with an adjoining drainage tray; an enlarged cross-sectional area for velocity drop; and / or an abrupt change in direction for inertial separation from the gas.

[0020] In some embodiments, a method of mixing in a microfluidic device comprises introducing one or more liquids into a mixing chamber in the microfluidic device, to cover a floor of the mixing chamber, filling the mixing chamber to a point where a free surface remains at least 4 mm below a cover surface that provides a ceiling of the mixing chamber. Once the mixing chamber is occupied, the method involves drawing a gas through the liquid in the mixing chamber by applying a negative pressure in the mixing chamber above the free surface, in a range of -0.345 kPa to -3.5 kPa relative to ambient pressure. A gas inlet is present in the mixing chamber, that meets the mixing chamber adjacent to the floor, or through the floor. Creating and bursting at least one bubble in the liquid mixes the liquid(s) in the mixing chamber.

[0021] Drawing the gas may comprise pulsing gas through at least one gas inlet. The microfluidic device is adapted to mix a liquid in the mixing chamber by applying the negative pressure to draw a gas through the liquid in the mixing chamber thereby creating at least one bubble in the liquid. The negative pressure is in a range of-0.345 kPa to -3.5 kPa. In some embodiments, the negative pressure is in a range of-1 kPa to -1.7 kPa. The bubble formation causes the free surface in the mixing chamber to rise, and this motion can encourage mixing, especially if the rise leads to wetting of a large surface area. Larger surface areas are wet when the free surface without gas lies below a relatively flat shelf over which the liquid spreads during bubbling. By providing channels or a slight general slope the liquid can be encouraged to return after the gas is removed from the liquid. Continued application of the negative pressure can cause the at least one bubble to burst. A burst bubble very rapidly redistributes the liquid skin, chaotically mixing the liquid. In some embodiments, a plurality of bubbles can be created and rise through the liquid in a stream during a single pulse of negative pressure, and bursting of many bubbles efficiently mixes liquid. In some embodiments, the microfluidic device has two chambers connected by a microfluidic channel. Adding fluidly connected mixing chamb-ers permits mixing of numerous liquids.

[0022] Various liquids (e.g., biologically relevant liquids, food, air or water samples, effluent, products or biproducts) can be delivered to the mixing chamber from microfluidic channels connected proximate either a top or bottom of the mixing chamber, or through a pierceable (self-healing) cover. The liquids are typically aqueous, or otherwise oil-based, and typically has, or could have, dissolved, carried or suspended particles, toxins, chemicals, or other proteins, viruses, and organic matter to be tested. The liquid has a density and viscosity less than that of tepid liquid honey and more than that of ethanol.

[0023] The opening of the mixing chamber to a mixed liquid extraction microfluidic channel can be located anywhere on a floor of the mixing chamber (e.g., at a center of the floor or at an edge of the floor anywhere around a perimeter of the mixing chamber) or can be in a wall of the mixing chamber close to the floor anywhere around the perimeter of the mixing chamber. The mixed liquid channel opens into the mixing chamber below an operational fill level of the liquid volume in the mixing chamber, which is typically substantially lower than a fill level of the liquid with bubbles. In some embodiments, a plurality of gas outlet channels are distributed around a perimeter of the mixing chamber at a cover-meeting surface of the mixing chamber. These gas outlets are preferably independently controlled, or collectively controlled to open different gas outlets at different moments, to draw the bubbles through the liquid in different directions, further increasing uniformity of mixing. Similarly, independently controlled, or collectively controlled set of gas inlets can be used to vary paths through the liquid in the mixing chamber, however typically the gas supply is less effective to this end, especially with smaller volumes, as the liquid naturally assumes a hemispherical shape, and the features that encourage the liquid to quickly return to covering the inlets typically narrow the area at the bottom of the mixing chamber, relative to the top. If pluralities of gas inlets and out-lets are used, they are preferably coordinated such that at any moment open inlets and open outlets are as separated as they can be given the options, to maximize a length of path of bubbles through the liquid.

[0024] In some embodiments, the mixing chamber comprises a sloped sidewall. The sloped sidewall promotes liquid reflowing to cover the gas inlet after bubbles burst. When bubbles burst, liquid in the mixing chamber is pushed in various directions, but in a much more abrupt manner than the bubble formation, although both processes affect a volume and shape of the liquid’s free surface. In some embodiments, the mixing chamber has a floor of minimum surface area, to reduce dead volume, and the mixing chamber grows monotonically in surface area when moving from the floor to a top, cover-meeting surface. In some embodiments, the mixing chamber has fixed protuberances to promote more thorough mixing. In some embodiments, the sloped sidewall is inclined upwardly away from a gas inlet to provide a ramp that the liquid increasingly covers when expanded by inclusion of air bubbles. In some embodiments, the sidewall is inclined upwardly in at least two different directions away from the opening, i.e., from where the bubble or bubbles are created, e.g. forming two planar or curved ramps, or a frustoconical sidewall with an axis perpendicular to the floor. The ramp or cone surface may allow for a tidal action of the liquid in the mixing chamber, when a level of the liquid rises and falls.

[0025] A bubble can be generated by drawing a gas from the external environment (or other gas supply) through the liquid in the mixing chamber via negative pressure in the mixing chamber above the free surface of the liquid. Growth of the bubble causes the liquid in the mixing chamber to expand, roll, split, fold, and recombine, thereby mixing the liquid. Timing and rate of bubble formation can be controlled with negative pressure duty cycles to encourage thorough mixing. With soapy liquids, for example, long off times in the duty cycle are advisable to permit bursting of more bubbles before introducing new bubbles. In addition, alternating between vacuum suctioning, blocking, and venting in a programable controlled manner can further enhance mixing efficiency.

[0026] Several mixing operational modes are available to the micromixer. Such operational modes include, for example, Bubble Formation and Burst (BFB) mode, Tidal Action, Bubble Formation and Deformation (BFD) mode, and Bubble Stirring. Bubble Formation and Burst (BFB) mode'.

[0027] In accordance with the BFB mode, sufficient negative pressure is applied for a length of time that causes the at least one bubble to grow from the (or at least one of) gas inlet, to rise through the liquid, and to burst at the free surface of the liquid. The bubble formation and burst mode may comprise closing each microfluidic channel meeting the mixing chamber is closed, except at least one gas outlet coupled to a negative relative pressure supply and meeting the mixing chamber above a free surface of the liquid, and at least one gas inlet coupled by a channel. Generally this can be efficiently provided by leaving open the gas inlet throughout a pulsed sequence of suction, unless there are a plurality of gas inlets, in which case one would typically coordinate control such that only one of the inlets is open at a time.

[0028] Strict temporal alignment is not required if both inlets and outlets are gated, as negative pressure can build up above the free surface while the gas inlet is closed, but bubble growth, detachment from the inlet opening, and movement through the liquid requires the negative pressure. It should be noted that the invention may work without closure of all of the other microfluidic channels that meet the mixing chamber, however closure is the preferred mode of operating, as it permits a lowest power pump to operate, reduces a hysteresis or lag between depressurization and bubble forming, and generally improves control and reliability. With the negative relative pressure applied by the gas outlet, air from the mixing chamber above the free surface is removed, and gas from the supply, or ambience is imbibed into the liquid through the gas inlet. Once a buoyancy of the grown bubble provides enough force, the bubble rises through the liquid, all the while expanding to match a density above the free surface. Once the bubble meets the free surface, a thinning film of liquid rapidly effaces offering less and less resistance until the bubble bursts. If the suction on the outlet persists, this gas rapidly disperses and is drawn into the outlet. A stream of bubbles rising through the liquid causes circulatory movements of the liquid, and some mixing, but the bursting of the bubbles provides for a very fastacting mixing effect, and can, depending on a size of the bubble relative to the volume of the liquid plug, substantially affect boundary conditions across the envelope of the liquid plug, leading to local and more distributed mixing effects, via pressure waves. Bubble bursting can also be facilitated by a sudden removal of negative pressure or venting. Pulsing the negative relative pressure, or alternating between pressure and venting with a duty cycle can increase mixing efficiency. In some embodiments, the micromixer is operated to either intermittently or continuously withdraw gas from the mixing chamber. The time period in which the negative pressure (suction) is ‘On’ controls the amount of bubbles formed while the time period in which the negative pressure is ‘Off’ can increase the rate of bursting. The parameters of the duty cycle can be tailored to properties of the liquid in the mixing chamber. Tidal Action:

[0029] The bubble formation and bursting changes a volume of the liquid slug (which is understood herein to refer to a volume of liquid that is partially constrained by the chip, and partially defines a free surface, and has a shape that may vary depending on air bubbles entrainment). This change in volume can have a disproportionate effect on a surface area to volume ratio of the slug, depending on a shape of the mixing chamber. In some embodiments, the slug can have a volume more than twice that of the liquid (without bubbles). By selecting the shape of the mixing chamber, a tidal action can be produced, with high tides associated with bubble lift-off, and low tides associated with bursting, events. As the bubble grows, the free surface rises, generally with liquid from the top middle moving radially outwards, and spilling radially outwards, and spilling onto surfaces of the mixing chamber tends to redistribute the liquid from a centre of the bubble path, towards a periphery of the mixing chamber, and also to bring liquid from the bottom of the mixing chamber, towards the top, during the bubble forming events. This leads to a larger scale circulatory flow that is mostly upward and radially outward. During the bubble bursting events gravity and surface tension cooperate to bring all of the liquid back down, but surface adhesion may tend to slow the motion of the spilled liquid on the newly wetted sidewall, and thus redistribution during collapse offers a more localized circulatory flow in the opposite direction, such that the two flows do not tend to cancel each other out, but rather enhance mixing, both locally and at a greater distance from the bubble collapse. This effect can be maximized with ramped sidewalls, and particularly with sidewalls that have a fairly shallow angle, making them arguably more like a floor than a sidewall, as the closer the ramp is to flat (parallel with floor), the greater the change in surface area is provided per unit rise in the free surface. However, the flatter the ramp, the more exact the position of the free surface must be to enable this tidal mixing to occur, and thus the more accurately the liquid would have to be metered; furthermore the flatter the ramp, the more resistance the liquid may have to return to a position overlying the gas inlet. Relying on surface tension to pull the liquid back to the overlying position may be slow and uncertain, and thus it is generally better to provide an angle in the range of 2-10° to maximize tidal action. Bubble Forming and Deforming (BFD) mode:

[0030] The BFD mode is suited for high viscosity liquids or liquids that include surfactants as an additive. If the liquid has high viscosity, it is relatively easy to control bubble growth without bursting the bubble, and growth, and shrinking of the bubble can be arranged by bursting or by releasing the negative relative pressure. If the liquid has a surfactant or otherwise tends to froth or foam up, it can be desirable to avoid BFB, although this calls for more direct supervision and control. It is expected that mixing of foamable liquids with air might be otherwise impossible. The BFD mode may comprise the following steps: a) the gas supply inlet is provided with a volume of gas and then blocked; b) a low negative pressure is applied to the gas outlet, c) the gas in the liquid extraction microfluidic channel is drawn into the mixing chamber and a single bubble is formed in the liquid in the mixing chamber. The blocking of the liquid extraction microfluidic channel limits the amount of gas in the liquid extraction microfluidic channel so the gas expands in the bubble to equalize with the pressure on both sides of the bubble’s surface, d) with a short ON period, the single bubble is formed without bursting, e) when the negative pressure is removed quickly (vented), the single bubble shrinks in size due to the increase in pressure in the mixing chamber until the bubble disappears because blocking the liquid extraction microfluidic channel fixes the total amount of gas in the liquid extraction micro-fluidic channel. The mixing is achieved as the liquid expands and folds due to the bubble expanding and contracting, and is particularly useful in combination with tidal action. Bubble stirring:

[0031] Bubble stirring uses numerous bubbles that form sequentially and burst at respective times and positions that vary in a somewhat controlled manner to stir the liquid. In some embodiments, two or more gas outlets are distributed substantially evenly (each peripheral distance between two adjacent outlets is within ± 60% of average) around a perimeter of the mixing chamber, and in some embodiments two or more gas inlets are distributed substantially evenly (each peripheral distance between two adjacent outlets is within ± 60% of average) around a perimeter of the floor of the mixing chamber. In some cases there are multiple inlets and multiple outlets. Whenever there are multiple inlets or outlets, it is preferable that at least some of the inlets or outlets are blocked at each instant of the process, and in many it is preferably that at most one of the multiplicity (or each multiplicity) be open at each instant. Whether the multiplicity is of inlets or outlets, by blocking some at some moments, and others at other moments, the directed path through the liquid changes, which results in more mixing in comparison with single fixed inlet and outlet designs. Duty cycles and arrangements for alternating closed and open inlet / outlets can be provided with exercise of the common knowledge in the art. The sequence can be in any order, for example one to another around the perimeter of the mixing chamber or diagonally across the mixing chamber to create back and forth liquid mixing motion. In some embodiments, the gas is drawn sequentially into the mixing chamber in a circulating manner around a perimeter of the mixing chamber at different locations around the perimeter of the mixing chamber to stir the liquid in the mixing chamber. If multiplicities of both inlets and outlets are provided, it can be preferable to coordinate blockage to ensure a sufficiently long distance through the liquid is provided, for example by blocking all but one outlet and one inlet that are diametrically opposed from each other.

[0032] The at least one mixing chamber has a height to width aspect ratio of less than 2:1. In some embodiments, the aspect ratio is less than 1:1. In some embodiments, the aspect ratio is less than 0.5:1.

[0033] The microfluidic device comprises any suitable substrate material. In some embodiments, the substrate material comprises a polymeric material not excluding any biocompatible low-cost polymer. In some embodiments, the substrate comprises a thermoplastic material, e.g., Injection moldable thermoplastics like Polystyrene (PS), Polycarbonates (PC), Cyclic olefin copolymer (COC), Poly(methyl methacrylate), Polyurathane, and Cyclic olefin polymer (COP). In some embodiments the substrate comprises a thermoplastic elastomer (TPE) such as Dryflex, Sariink, Monprene, Santoprene, Lapreane, Mediprene, etc. Specific embodiments of microfluidic devices were produced with COC (Zeonor), and PC (Makrolon2458 (Clear)).

[0034] The gas may be any gas suitable for the desired mixing process. In some embodiments the gas comprises air, one or more inert gases (e.g., nitrogen, argon), one or more reactive gas (e.g., oxygen, carbon dioxide) or any mixture thereof.

[0035] The micromixer can be formed into the microfluidic device by any suitable method, for example injection molding, 3D-printing, layer-by-layer assembly, machining, etc. Because the micromixer has no moving parts and requires minimal assembly procedures, the micromixer is particularly amenable to being manufactured by molding methods, especially injection molding methods. Specific examples of microfluidic devices were fabricated using 3D printing and injection molding.

[0036] Microfluidic devices with micromixers described herein are surprisingly efficient based on a combination of a simple to fabricate structure, even if a depth of forming is somewhat high, combined with active dynamic mixing by bubble formation. The devices described herein are significantly better at mixing than traditional microfluidic serpentine channels and do not require instrumented centrifugal forcing functions to mix efficiently, and furthermore require very high pressures to force liquid over long distances. The low aspect ratio mixing chamber structure makes the devices suitable for monolithic injection molding fabrication methods. The micromixer does not require many-layer structures for assembly, thereby reducing assembly costs. The micromixer is suitable for lite-instrument and instrument-less microfluidic devices as negative relative pressure is suitable for movement. Some prior art bubble-based mixers have high aspect ratio mixing zones where bubbles burst and escape at the top of a vertical mixing zone resulting in inferior mixing of liquid in regions lower in the mixing zone. In contrast, the present invention can ensure bubbles pass through most of the liquid volume, by arrangement of the gas inlet and outlet, and shape of the walls. By placement of the gas inlet almost 1 cm below the top cover of the mixing chamber, splatter from the bursting of the bubbles can be limited to the sidewalls, and may be aggregated with the slug by raising the level by increasing bubble content, and then decreasing the bubbles without bursting them after the mixing is complete. This allows for mixing in a fluid-parsimonious manner that can be important for handling expensive reagents, and for good use of products, and / or to provide reliable metering of products for subsequent processing.

[0037] The presently described micromixer can also create stirring-like liquid circulation through a succession of directed bubble formation and bursting, adding an additional capability to the micromixer that is different from previous air stream micromixer concepts, which do not involve bubbles. Unlike some prior art micromixing technologies that use a magnetic micro-stirrer embedded inside the chip to generate a circular motion of liquids, the presently described micromixer has no physical contact of liquid with a stirring device and there are no embedded moving components, thereby reducing the unit cost of the microfluidic device. The presently described micromixer can be utilized in an array of micromixers with a mixed liquid output of one micromixer serving as liquid supply to another micromixer, and a single microfluidic mixer can be used for repeated mixing processes as a protocol may require, with different parts of the liquid withdrawn, and / or different liquids added between bubble mixing procedures. The use of negative relative pressure (vacuum suction) bubble-based micromixing offers advantages (besides energy consumption) over other pressure-driven methods, such in centrifugal method), for example permitting the use of devices having lower bonding strength between layers.

[0038] By selection of geometrical shape of sidewalls (e.g., cylindrical, conical, spherical, etc.) and the geometrical parameters thereof (height, diameter, draft angle, etc.), a single micromixer can be designed to accommodate a range of liquid volumes, for example volumes of liquid in a range of 15 pL to 1 mL have been mixed with a single sloped ramp, which is useful for complex protocols requiring several different wash and treatment steps. The present device provides for a low amplitude, negative relative pressure-driven, simple, and controllable mixing process that is suitable for an automated microfluidic system for sample preparation as well as for point-of-care (POC) diagnosis in low resource environments, and can be made lite and portable.

[0039] Further features will be described or will become apparent in the course of the following detailed description. It should be understood that each feature described herein may be utilized in any combination with any one or more of the other described features, and that each feature does not necessarily rely on the presence of another feature except where evident to one of skill in the art. Brief Description of the Drawings

[0040] For clearer understanding, preferred embodiments will now be described in detail by way of example, with reference to the accompanying drawings, in which:

[0041] FIG. 1A is a schematic drawing in perspective view, of part of a structural re lief-patterned substrate featuring a first embodiment of a micromixer in accordance with the present invention;

[0042] FIG. 1B is a panel of time sequence photographs showing mixing of a liquid, chiefly in BFB, in a prototype device of Fig. 1A;

[0043] FIG. 2A is a schematic top plan view of a relief-patterned substrate comprising a second embodiment of a micromixer, with 4 inlets and 2 outlets;

[0044] FIG. 2B is a panel of time sequence photographs showing mixing of a liquid in bubble stirring and BFB mode in a prototype device of FIG. 2A;

[0045] FIG. 3A is a schematic perspective view of a relief-patterned substrate comprising a third embodiment of a micromixer, having a ramped sidewall;

[0046] FIG. 3B is a magnified partial view of the micromixer of FIG. 3A further comprising a liquid splitting feature;

[0047] FIG. 3C is a panel of time sequence of schematic cross-sectional side elevation views showing mixing in the device of FIG. 3A, showing BFB and tidal action;

[0048] FIG. 3D is a schematic cross-sectional side elevation view showing mixing in BFB mode, with bubble stirring, and bubble mixing in a fourth embodiment of a micromixer having conical or two paired ramped surfaces;

[0049] FIG. 4 is a schematic cross-sectional side elevation view showing integration of a metering chamber with micromixer in accordance with a fifth embodiment of the present invention; and

[0050] FIG. 5 is a panel of photographs of a magnetic bead mixing apparatus, comprising a partial image of a chip, and two rows, each of four enlargements, the top row showing accretion of beads, and the bottom showing resuspension of the beads. Detailed Description

[0051] FIGs. 1 (i.e. 1A and 1B) depict a part of a structural relief-patterned substrate 11 defining a micromixer 10 and (8 instances of) a prototype chip based on the design. The substrate 11 is preferably composed of biocompatible plastic capable of injection molding, and may be recyclable, or biodegradable. The micromixer 10 is centred around a mixing chamber 12 and three microfluidic channels 14, 15, 16. The mixing chamber 12 has a height (depth) of 5 to 15 mm, which might appear to be excessive to those familiar only with 2 phase microfluidics, but is effective for bubble mixing, even though an intended volume for mixing may be from 10 to 60%, more preferably 33 to 55%, most preferably 25 to 50%, of the capacity of the mixing chamber 12. Specifically, the depth of chamber 12 in the prototype of FIG. 1B was 9 mm. While a depth / height of chamber 12 may be extreme for 2 phase microfluidics, the channels 14, 15, 16 and length and width of the chamber 12 may all be quite similar to dimensions of 2 phase microfluidics. Thus, an aspect ratio (depth or height to width) of the chamber 12 is typically less than 2:1, and is about 0.5:1 as shown, and the length is 1 to 8 times the width.

[0052] The channels 14, 15, 16 are preferably designed for non-capillary flow and advantageously are designed to move under a same negative relative pressure that drives bubble mixing, and preferably uses a native contact angle of the plastic. Channel 14 is a gas outlet (pneumatic extraction channel) having a first end 14a opening into the mixing chamber 12 at a top peripheral edge of the mixing chamber 12 (i.e. where a cover-meeting surface of the substrate 11 meets the chamber 12) and a second end 14b adapted to be connected to a suction pump (not shown) via a port of the chip. Specifical-ly as shown, the port would be provided either by a through-hole in the substrate 11, or by a through-hole in the cover (not shown), the port preferably having a nipple or sealing surface for establishing a seal with the suction pump, although in some embodiments a clamp around a back side of the substrate 11 can provide sufficient sealing force to press a supple ring against the cover away from the through-hole to provide desired sealing. Applicant currently prefers using a multi-hole gasket shaped to overly a provisioned number of ports on a top or bottom side of the chip, that is applied by adhesive to the surface. While the first embodiment has all microfluidic channels and chambers relief-patterned onto the substrate 11, it will be appreciated that subject to alignment requirements, a network of the microfluidic channels can be supplied instead by relief patterning of the cover, leaving large depth, bulk-scale features formed into the substrate, and reserving all microfluidic channels and finer scale features to be provided by the cover, yielding the same chip network.

[0053] Channel 15 is a gas inlet having a first end 15a meeting the mixing chamber 12 at a floor 13 of chamber 12 and a second end (not in view). Like the gas outlet, the second end meets a port of the chip, which may be simply a microfluidic vent, or a port for coupling to a low (near ambient) positive pressure gas supply, for example. Channel 15 is shown having a serpentine path segment over the cover-meeting surface of the substr-ate 11. The path through the cover-meeting surface which terminates at a snorkel-bore in a hemicylinder 17 protruding from a wall of chamber 12. The snorkel-bore couples to a rectangular opening at the first end 15a. A safe margin above a top of the rectangular opening, is understood to be a minimum fill level of the liquid for mixing. The second end of channel 15 is vented to ambience when air is used as the bubble-generating gas.

[0054] Channel 16 is a separate liquid sample line, for introducing an aqueous liquid into the mixing chamber 12. In other embodiments, with suitable controlling topology, valves, vents and pressure supply lines, either or both the gas inlet and gas outlet could be dually used to supply the liquid. The sample line 16 has a first end 16a meeting the mixing chamber 12 at the cover-meeting surface periphery of the mixing chamber 12. The invention works just as well if the liquid is introduced into the chamber 12 from below, such as at or near the floor, e.g. with a separate snorkel, or could reuse the snorkel within the hemi-cylinder 17, by branched coupling to the channel 15. Depending on the network structure, it may be preferable to provide the liquid supply and gas inlet in the same line, without any branching, as suction applied by channel 14 can first pull liquid into the chamber 12, and then draw gas, the whole process can be performed in a single step. The opening at the first end 16a is shown with a downward flaring that enlarges the opening although this is not necessary, and it might give the impression that the channels have a particularly high aspect ratio, which is not the case. The channels have an etch depth no more than 2.5 time the mean channel width, and no less than 1 / 5‘h the mean channel width, to provide a reasonably low surface area to cross-sectional area, and to limit flow resistance. Some design suites default to a standard channel depth, such as about % mm, or some other value that is typically between 0.1 mm and 0.35 mm. Often width of channel is varied where needed, and can range from about 50 to 500 pm. A pillar 18 extending upwardly from the floor 13 is situated in front of and close to the first opening 16a to guide liquid into the mixing chamber 12 in the illustrated embodiment, however this is not necessary, and is not even desirable in some cases, as it may actually impair liquid entry, may reduce mixing efficiency, or complicate fabrication.

[0055] While micromixer 10 is illustrated without a cover, a cover would normally be placed over top of the mixing chamber 12, to prevent contamination, oxidation and / or dehydration of liquid, even if it weren’t used for applying negative pressure to above a free surface of liquid in the chamber 12 for inducing bubble mixing. The three microfluidic channels 14, 15, 16, are sealed by the cover to prevent leakage of fluids, and to enable fluid manipulations on the chip. While the substrate itself is believed patentable, the utility of the substrate is tied to sealing the substrate with a cover

[0056] In operation, a chip (substrate 11 and cover) provides the micromixer 10 with its mixing chamber 12 loaded with a liquid (including two or more liquid phases, at least one of which being aqueous, and / or an aqueous liquid with powders, cells, or beads) to be mixed. The liquid loaded more than completely covers the gas inlet, even without any gas bubbles increasing the slug volume. Conveniently, the liquid may be loaded by closing a valve to the gas inlet 15, opening sample line 16, and applying negative pressure on the gas outlet 14 to draw one or more liquids to be mixed into chamber 12. In some embodiments, beads, lyophilized product, particulate solid matter, cells or cellular matter, or other films of liquid or solids were placed on the floor of the chamber 12, prior to loading of the liquid. Sample line 16 is then closed and the gas inlet channel 15 opened so that pulses of the negative pressure applied via the gas outlet rarify gas in the mixing chamber 12, above the free surface of the liquid slug. This pulsed suction draws gas through the gas inlet, and through the slug that otherwise obstructs the gas flow. This drawing of gas starts with entry of gas into the first opening 15a at the bottom of the mixing chamber 12 just above the floor 13. This entry enlarges to form a first bubble 19 (only one labeled) in the liquid. When the bubble expands to a volume with sufficient buoyancy to overcome attachment to the floor and periphery of the first opening 15a, the first bubble detaches and rises through the slug. All the while the slug grows in volume in proportion to the added volume. While FIG. 1A schematically illustrates 5 bubbles, without showing a fill line of the liquid, and while the bubbles look to be distributed radially around the opening 15a, those skilled in the art will appreciate that the bubbles generally rise after formation, and while the bubbles do tend to spread radially around in somewhat the manner shown, it is only after several bubbles back up in a bubble stream, that they do so.

[0057] The time sequence shown in FIG. 1B includes snapshots of the micromixer before, during, and after a single pulse of about 0.1 s duration: in experiments performed on various chips with different liquids, pulse durations of between 0.1 and 0.5 s were used, and relaxation intervals between pulses varied between a duty cycle of 50% and about 10%. The images were sequential, but not uniformly spaced in time. The mixing chamber 12 is initially bubble-free (state 1: top left), then bubbles 19 start to form (state 2) and bubbles 19 keep forming to a maximum bubble density (state 6: bottom, second from left) until the negative pressure is turned off, whereupon bubble formation stops and the number of bubbles 19 diminishes due to bubble bursting (state 7) and finally the bubbles disappear altogether (state 8). State 3 shows proliferation and fanning of the bubbles as they rise, and State 4 shows bubbles dividing and shrinking in volume as they aggregate on the free surface, owing to a time taken to thin the bubble’s films and burst. State 5 shows how bubbles can tend to creep around a periphery of the mixing chamber 12. Throughout the sequence, bubbles 19 burst and new bubbles 19 are formed causing disturbances in the liquid, which causes mixing of the liquid. The negative pressure applied to the pneumatic extraction channel 14 may be cycled through on / off cycles to improve the rate of mixing. Depending on diffusion rates of the two liquids being mixed, it has been found that % to ½ s pulses of negative 0.1 Psi (0.69 kPa) pressure can mix a 250 to 500 mL volume of aqueous samples and reagents.

[0058] FIGs. 2 depict a schematic micromixer 20 (which optionally shares specifications above of micromixer 10 as a whole, or in any detail and with reference to any feature thereof) in accordance with a second embodiment of the present invention, and 6 photographs of a prototype chip based on the design in 8 respective states. Herein common names of different reference numerals in different examples indicate a same, or functionally equivalent form or structure for the feature, and descriptions are not always repeated in respect of each embodiment and feature. Unlike the first embodiment, the second embodiment is a complete chip, with no function other than to provide the micromixer 20. To this end the cover may be a peelable resealable film that permits direct loading and withdrawal of liquid from the chamber, or a self-sealing membrane with needles for injecting and withdrawing liquids into the micromixer 20 between mixings.

[0059] FIG. 2A is a top plan view of a substrate 21 bearing a relief pattern for mixing a liquid predominantly in the bubble-stirrer (BS) mode. A mixing chamber 22, meets six microfluidic channels 23, 24, 25, 26, 27, 28. The device 20 is illustrated without a cover, which, in use, is placed overtop of cover-meeting surface of the substrate 21 (i.e. the whole top plan surface, away from the relief-patterned recesses for channels, throughbo-res, the chamber 22, and 4 corner cutouts that reduce mass and plastic costs of the chip, while maintaining rigidity) to seal around the mixing chamber 22 and microfluidic channels and prevent leakage of fluids. In some embodiments, the mixing chamber 22 has dimens-ions within the ranges described for chamber 12.

[0060] Channels 23 and 24 are gas outlets having respective first ends 23a and 24a that meet the mixing chamber 22 through a peripheral rim of the cover-meeting surface. Specifically, as shown, a short channel is provided as a recess in the cover-meeting surface, which extends from a through-bore of the substrate, to the chamber 22. Each gas outlet has a respective second end (23b and 24b), provided at a port, adapted to be connected to a respective suction pump (not shown), or to a valving or switching plate for controlling suction applied to these two gas outlets. The port is defined in the cover, and thus is not in view in FIG. 2A; all that is seen is an opening to a throughbore of the substrate 21. This throughbore is aligned with a recessed channel (shown in ghost view: dashed lines) that runs along a back side of the substrate, much as the short channel is defined at the cover-meeting surface. The dashed lines in FIG. 2A all refer to channels on the back side of the substrate, and to parts of vias that are covered by substrate 21. The openings at the backside of the substrate, and the channels, requires a backing plate to enclose the structure, just as the cover does for the top side. The backing plate may be the same as the cover, and may be a polycarbonate sheet of about 0.2 mm thickness, for example, or similar, with a suitable adhesive, however in some embodiments, better imaging or viewing of transparent liquids can be provided if the backing plate is opaque, and coloured in coordination with imaging and lighting. Ultrasonic welding, solvent and thermal bonding, anodic thermal bonding can be used instead of adhesives if needed.

[0061] In some embodiments it is preferable to only apply suction to at most one of these two gas outlets at a time (which can be provided with a switch or sets of valves), and in other embodiments it can be desirable to enable both ports to be independently controlled. As shown, the first openings 23a and 24a of the channels 23 and 24, are situated at 20 opposite ends of the mixing chamber 22, which maximally separates them. While maximal separation may be optimal, it will be appreciated that less than maximal separation can be fully satisfactory. While FIG. 2 shows an embodiment having 2 gas outlets, it will be appreciated that it could alternatively have another number, such as 3 or 4 gas outlets. In generally, more uniform spacing around the rim is desirable to improve dirigibility of bubbles through the slug. As each adjacent pair of the 2 to 4 gas outlets naturally bounds a respective part of the periphery, and as the 2 to 4 gas outlets collectively partition the periphery into 2, 3 or 4 lengths, Applicant notes that the spacing should be uniform enough that the closest adjacent pairs do not span a length that is less than 1 / 3 of any other adjacent pair’s span. Preferably, if there are an even number of gas outlets, and, at the top rim, the chamber 22 is symmetric on two axes (which the second and third embodiments happen to be) each of the gas outlets is paired with another gas outlet such that their first openings lie diametrically opposed to each other (i.e. their midpoint is nominally the intersection of the two axes). The same desire for peripherally spreading the gas outlets applies to the inlets as well, and such a distribution scheme is shown with the 4 gas inlets (25a and 27a matched, and 26a and 28a matched).

[0062] The microfluidic channels 25, 26, 27 and 28 are gas inlet channels having first ends 25a, 26a, 27a and 28a, respectively, that meet the mixing chamber 22 at a floor thereof, and second ends 25b, 26b, 27b and 28b, at respective ports of the chip. It may be noted that half-moon openings are provided by partial alignment of the vias with the mixing chamber’s peripheral extent, as this is a convenient way to limit a size of the openings at 25a, 26a, 27a, and 28a, without requiring finer resolution fabrication routes. It will be appreciated by those skilled in the art that limiting the size of the openings can reduce imbibition of liquid from the chamber 22, and added resistance to bubbling as a result of liquid plugging these channels while / afterthe liquid is loaded. These may be free vents (to ambience), or adapted to be connected to a gas supply (not shown), but are preferably coupled by valves to permit selective opening and closing of the gas inlets, such that, in use, most of the time, only one inlet and one outlet is open at any time. The valving may be designed to permit at least one, and at most two, inlets to be open at a time, or at most one. Preferably inlets 25a and 28a are at least typically closed when suction is applied via outlet 23a, and inlets 26a and 27a are at least typically closed when suction is applied via outlet 24a, as this provides longer paths for bubbles through the slug. One or more of the second openings 25b, 26b, 27b and 28b of the vent channels 25, 26, 27 and 28, respectively, can be adapted for use as sample lines to introduce a liquid sample into the mixing chamber 22. Bubbles are created at each of the first openings of the inlets by opening a desired vent channel, closing the other vent channels and opening one of the pneumatic extraction channels.

[0063] In operation, the mixing chamber 22 of the micromixer 20 is provided with a liquid or liquids to be mixed by using one of the inlet channels 25, 26, 27, 28 as a sample line and using outlet channel 23 or 24, or one of the outlets to convey the sample and the other to apply suction. With reference to FIG. 2B, six images of a chip are presented. The cover is transparent, but has ports bearing nipples for coupling to tubing (only two of which in view). Bubbles 29 are created, displacing and expanding the slug, then move through the slug under buoyancy, and burst in the liquid in the mixing chamber 22. By coordinated switched openings of one of the outlets and one of the inlets, rotation of the loci of bubble bursts on the surface can be encouraged in a somewhat annular sweep around the mixing chamber 22. Not only does bubble-creation and bursting cause mixing as described above for the micromixer 10, but the rotation of the bubble trajectories through the slug, and variation in locations of the bursts also creates a stirring motion in the liquid, which enhances the mixing process. In FIG. 2B, the sequence of bubble formation follows the identified order. In preparation for the annular stirring operation, all the channels 23, 24, 25, 26, 27 and 28 are closed and there is no bubble formation (upper left image). To obtain the rotational mixing pattern: a) open channels 24 and 25; b) close channels 24 and 25 and open channels 23 and 26; c) close channel 26 and open channel 27; d) close channels 23 and 27 and open channels 24 and 28, and repeat. The outlets toggle at half the frequency of the inlets in this process. Bubble formation (and mixing) is terminated when all the channels 23, and 24 are closed (lower left image). In this example, suction can be continuously applied (via either gas outlet), or a pulse of suction can be applied with each opening of an inlet.

[0064] One tendency illustrated in the panel is that bubbles formed at a periphery of the chamber tend to rise and remain in contact with the side wall. This tendency can be helpful for ensuring mixing, as the volume in the middle tends to move and mix more readily, but liquid along the edges tends to be more stagnant, especially if the bubbles can be urged to move along the side walls at different moments in the mixing process. The bubble collapse process is most effective for mixing fluids, by chaotic mixing, even along bottom edges, especially in relatively shallow slugs.

[0065] Different sequences can create different patterns of bubble formation in the bubblestirrer mode. For example, a diagonal sequence of bubble formation can be utilized for ‘diagonal mixing’ using the following sequence of channel openings and closings: a) open channels 24 and 25; b) close channels 24 and 25 and open channels 23 and 27; c) close channel 27 and open channel 26; d) close channels 23 and 27 and open channels 24 and 22 28. Furthermore, the time taken for a bubble to rise may allow for a beneficial lag. A bubble that is first pulled towards suction A, can later be pulled toward suction B, for a longer trajectory through the slug. While the illustrated examples for controlling this micromixer 20 so far have only one opening at a time, both of ends 26a and 27a open at the same time while outlet 23 is applying suction, may offer another trajectory for bubbles.

[0066] FIG. 3A depicts, in perspective view, a micromixer 30 (optionally sharing specifications with micromixer 10 or 20 and / or its features) in accordance with a third embodiment of the present invention. The third embodiment, like the second, is a complete chip, even though integration with other chip components and functions for readout, metering, incubating, filtering, and other purposes, is expected to be more typical. The third embodiment has the advantage of being able to mix a larger range of volumes of liquids, requiring less accurate metering of the supplied liquid(s), and permitting multiple additions of liquids between mixing steps as many protocols require. Furthermore the third embodiment mixes more quickly, completely or reliably than the first embodiment, given the tidal action, and requires fewer ports and less fabrication complexity than the second.

[0067] The micromixer 30 comprises a substrate 31 bearing a top-side relief that defines most of a mixing chamber 32. Two microfluidic channels 34 and 35 include througbores (unless the chip is formed by additive manufacturing, in which case the channels can be routed through the solid material with much greater flexibility), which may or may not be coupled to other channel segments at the bottom surface. Noted that if both ports of the chip are on the bottom surface, the port to the gas inlet (channel 35) preferably has a valve (e.g a solenoid or manually releasable rubber seal) to enclose an air plug, at least during sample loading, until the suction is applied by gas outlet (channel 34). The micromixer 30 is shown without a cover, which is placed over top of the substrate 31 when in use (at least after loading). The mixing chamber 32 has a height to width aspect ratio of about 0.6:1. The microfluidic channel 34 (gas outlet) has a first end 34a opening into the mixing chamber 32 at a top of the mixing chamber 32, adjacent a top end of a ramped side-wall 33; and a second end not in view. A short channel cut into the cover-meeting surface of the substrate 31 extends between the first end 34a and a throughbo-re 34c. This througbore is adapted connected to a suction pump (not shown) either at a port on the bottom side of the chip, or a port coupled to the throughbore by another channel segment. A small, light, low-power pump may be built-in to the chip, or may snapfit into the chip for some applications, however given onerous cleaning and sterilization processes required to make the illustrated chip reusable, and the very low cost of the chip itself (which may be under 1$ US if injection molded on a large scale), a simple coupling of a disposable chip to a system comprising the pump, a controller, and a power supply, is desirable for more applications and use cases. The system may further comprise one or more of the following subsystems: a sensor for reading out data from a sample to answer chip value of one or more samples; a camera (with or without lighting) for stateful monitoring of a process on the chip, and / or for reading out data from sample to answer; a thermal regulation and control system for controlling temperature changes in respective chambers of the chip; a magnetic field generating device or mover for selectively collecting magnetic beads or particles from a sample in a chamber; one or more (e.g. mechanical, electro-mechanical, electromagnetic, pneumatic or hydraulic) actuator for a flow control element such as a valve at a port of the chip; a communications system for exchanging information between a computer and one or more of the above subsystems; or a processor for controlling one or more of the above subsystems.

[0068] The channel 35 is a gas inlet having a first end 35a open to the chamber 32 at a floor 36 of the mixing chamber 32 and a second opening (not shown) adapted to be connected to a gas supply or open vent (not shown). The floor 36 is generally a c-shaped flat surface, with a cut-out provided by a ramped side-wall 33, although branched arms of the c-shape are curved out of plane and rise more gradually away from the branching than the side-wall 33. The weakly sloped branched arms of the floor are bounded externally by a normal (typically 89°) sidewall and internally by a rising edge of the (more steeply) ramped side-wall 33. The rising edges may extend at a maximal angle of about 89°, or any lower angle, or the ramped surface may be smoothly curved, defining a single surface. The sloped branched arms define return troughs for liquid drawn up onto the ramped sidewall 33 during bubble mixing, which is believed to provide faster return of liquid to the floor, specifically encouraging liquid to flow towards the opening end 35a of the floor 36, and increases circulation in the chamber 32 away from the bubble stream. The troughs could have lowest points (in width-wise section) where the trough meets the normal sidewall, or at the interface with the rising edge, or both (in which case the trough is a planar slide), but preferably at least the trough is rounded and smoothed with respect to both the normal sidewalls and raised edges. Moreover the edge between side-wall 33 and trough can be rounded, substantially avoiding any sharp edges within the chamber 32. Rounder surfaces are typically more easily formed. Unlike the that shown in FIG. 3A, in other embodiments, the side-wall 33 extends a full width of chamber 32 (i.e. no side troughs), or there may be only one side trough. Furthermore, the side-wall could be replaced by a raised protrusion from the floor 36, and thus not meet or form any part of a side-wall, and still function to provide a broad surface for expanding contact with the liquid during mixing, and for mixing a range of volumes of liquid.

[0069] As is useful, the first ends 35a and 34a, are substantially separated, at disparate ends of the chamber 32. However, they need not be maximally separated as shown: placement of the opening 34a above the top of the ramped side-wall 33 may be avoided to limit risk of liquid or splatter being ingested by the gas outlet channel 34, especially if the chamber 32 is at risk of being overfilled (for some applications, simply-designed physical structures can be provided on the substrate to preclude overfilling). Accordingly in another embodiment, the opening to the channel 34 in an otherwise same design, is directly above one of the branched arms of the c-shaped floor 36. Note also that chan-nel 34, or any other structure between the first end 34a and suction pump can have a trap or structure permitting limited volume ingestion of liquids into the gas outlet, without risk. To prevent contamination, Applicant prefers placing traps on the single use chip.

[0070] Regarding the simply-designed physical structures that can be used to prevent overfill, the simplest is a dividing wall within the chamber (12, 32, and possibly even 22, though possibly best provided by an annular wall, in that case). Provided that the sample is exclusively fed to the side of the chamber with the gas inlet opening (mixing compartment), and the compartment behind the dividing wall has a sufficient volume for the overflow (or a (possibly passive) drainage system), a height of the dividing wall will determine a capacity of the mixing compartment. What will depend on many details of design, is whether flow or leakage into the other compartment will arise during bubble mixing, and whether that will be a problem for a given process.

[0071] In some methods, suction is applied after filling of the chamber, and prior to opening of the gas inlet, to grow a single bubble at the gas inlet opening. Given the limited air available for the bubble (volume of gas inlet between valve and opening to gas inlet), the growth will be slow, which is excellent for controlling expansion of the slug. If duration of the suction is controlled (e.g. e.g. fixed number or fraction of standard pulse), the bubble will grow to a given size, and then shrink back, but expansion of the slug is apt to ensure volume in excess of a desired volume, can be extracted, and conversely that the volume remaining after that duration, can be bubble mixed without blocking the gas outlet opening. This can be performed with only the equipment for the bubble mixing, assuming that there is a valve on the gas inlet (which is not strictly necessary if the gas inlet extends a safety margin above a height of the chamber to avoid liquid leaking out).

[0072] While a dividing wall, or topologically equivalent structure, may be a simplest structure to produce an overflow, it is by no means the only solution. Another alternative is to provide another suction port to draw excess fluid from the chamber after filling, the opening of this overflow channel being at a fixed height from the floor. Once the chamber is filled, if there is no resistance on this channel, one can assume the chamber is underfilled 25 or well filled, and if not, once the liquid plug is removed from the overflow channel, the chamber should be well filled. This requires that the gas outlet serve as a vent during the overflow channel withdrawal, or that another vent be provided, but is well sealed prior to bubble mixing. Note, all requirements for overflow prevention can be avoided with use of a metering device in a supply line of the chip. One metering structure is taught by Applicant’s co-pending US provisional application serial number 63 / 619256.

[0073] In various embodiments of the present invention, an overflow to prevent overfilling of the chamber can be provided. In a variant of the first embodiment, the overflow is provided by an opening in the pillar 18 and a snorkel embedded therein with venting provided by the gas outlet. In another variant of the first embodiment, the dividing wall can be provided to connect the pillar 18 to the sidewall at two locations to enclose the overflow compartment up to a given height of the chamber 12. In a variant of the second embodiment, the dividing wall is provided as an annular ring in a middle of the chamber 22, the interior of the overflow compartment may have passive or active suction to remove more liquid if variability of liquid supply is unconstrained. In a variant of the third embodiment, the over-flow can conveniently be located on a curved sidewall adjacent the gas inlet, as a suction line at a suitable height above the floor. In a second variant of the third embodiment, the curved sidewall is demoted to the wall, and an area behind the sidewall is the overflow compartment.

[0074] FIG. 3B depicts a variation of the micromixer 30. In this variant, the gas outlet is provided in a port of the cover, which is not in view. A liquid splitter 37 is provided at the end of mixing chamber 32, opposite the first opening 35a of the vent channel 35. The splitter 37 comprises a ramp (e.g., having a 12.5° incline with respect to the floor 33) attached to the inclined floor 33 over which and around which the liquid can flow, creating additional mixing action in the liquid in the mixing chamber 32. Liquid splitter 37 has little surface area for deforming the slug during expansion, but when bubbles burst in succession and a volume of the slug rises and falls rapidly, the surface can improve mixing in the chamber 32. Placement of the splitter 37 away from the region where bubble bursting is most consistent and dense, and away from the paths of the bubbles through the slug, is particularly preferred for fast, and complete mixing.

[0075] FIG. 3C is a schematic illustration of a single cross-sectional side elevation view through a middle of a variant micromixer 30. This variant has a lower depth to length ratio than that of FIG. 3A, and features incidentally changed locations of the channels 34, 35, which may be of far shorter length (as shown) than in previous embodiments. While the floor of this variant, in this view, is shrunken to a lip of the gas inlet’s opening (at end 35a) to the chamber 32, this floor extends in a direction into the page a distance, in the present 26 embodiment. Preferably the edge between sidewall and floor is not too sharp away from the gas inlet opening if complete withdrawal of the mixed liquid is desired, as sharp edges are far more prone to trapping liquid. Extended branched arms or slightly raised wings (not in view) are included in some variants of this chip, and thus the floor may be substantially c-shaped. The floor is defined FIG. 3C,1 schematically illustrates the angle of inclination (a) of the ramped side-wall 33. To increase a change in surface area as a function of increased volume of the slug, it is desirable to keep the surface as flat as possible (a—>0), however the closer the ramped side-wall is to flat, the more stringently the elevation of the ramped surface would have to be relative to fill level, and if flat, there is no benefit for improved mixing with different volumes of liquid. Applicant prefers 2° <a <40°, depending on how well metered the liquid is, and an expected extent of volumetric increase provided by bubble mixing. Noted that, as shown, a is just under 14°. Furthermore, the separation of the top of the ramped side-wall 33 from the cover (h) can be important in some cases, particularly if the footprint of the mixing chamber 32 is minded and the overfilling is not otherwise controlled, or if liquid delivery is desired through the gas outlet and the liquid volume is small. As explained in Applicant’s unfiled co-pending application NRC2024-004, to reliably control delivery of small volumes of liquid, a gravitationally driven check valve can be used. Conveniently the depth of the chamber 34 required for bubble mixing is slightly greater than the needs for gravitationally driven check valve. If the slug is likely to cover the ramped side-wall 33, h can be chosen to be greater than about 4 mm.

[0076] The panel of images in FIG. 3C show 3 states of bubble mixing enhanced by tidal action. Herein ‘tidal action’ merely suggests lapping of water on a shoreline as a result of wave action of a body of water. In operation, the mixing chamber 32 is provided with a liquid (or liquids) 37. As shown in FIG. 3C,1, the liquid 37 to be mixed has a free surface 38. Headspace provided by the separation h may ensure that the liquid 37 cannot fill the chamber 32, in combination with an overflow system, metering system, or other volume-limiting mechanism of the microfluidic chip, which would guarantee that chamber 32 is a three-phase chamber. FIG. 3C,1 shows the liquid after loading, which as shown, covers part, but not all, of the ramped side-wall 33. To commence bubble mixing, the gas inlet is opened, and the suction is applied, preferably in pulses, via gas outlet. FIG. 3C,2 shows a moment when a bubble bursts, and the slug is about maximally expanded. It will be noted that the slug, at this juncture, covers a substantially larger fraction of the ramped side-wall 33. The maximal extent may fail to cover the sloped surface of the ramped side-wall 33, as shown, or it might in some cases extend a bit above it. If so the value of h becomes more important for guarding against volumetric losses in the mixing chamber 32 (where this is critical), ingestion of liquid in the gas outlet (where this is critical), or splatter, streaking, or smearing of a ceiling of the chamber 32 (provided by cover 39), to the extent that imaging or visualization through the cover 39 is desired. Applicant notes that a fairly large and variable volume of liquid may be lost to dead spaces within the chamber 32 if insufficient depth and headspace h are provided. Arrows on FIG. 3C,2 show general motion of the slug as a whole, but fail to convey the fluidics. The rising of the slug directly above the gas inlet opening (end 35a), in a short period of time, tends to redistribute the liquid with some of the liquid moving generally upwards, and liquid above the contact line with the sloped surface, generally moving in the only unconstrained directions as a result: upwards of the contact line; and (if provided) on top of liquid in the branched arms of the c-shaped chamber. The liquid tends to tumble upon itself and to spread over the sloped surface during expansion, whereas during contraction, which leads to FIG. 3C,3, a funnel pipe flow of the liquid is provided with generally faster movement near the middle, at least for typical friction coefficients with the liquid at the surface. Bubble bursting (BFB) effects add more local turbulences and affect the overall flow dynamics, which favour mixing.

[0077] FIG. 3D schematically depicts a variant of the micromixer 30 in cross-sectional elevation view. Instead of a ramped side-wall 33 extending principally from the floor in a first Cartesian direction (and possibly subject to rounding or smoothing), FIG. 3D shows that a centrally located opening at end 35 permits two opposing floors to be used, especially useful with two or more gas outlets used in alternation. While FIG. 3D can be seen with prismatic or Cartesian completion, and this is one variant of the micromixer 30, the variant Applicant has tested has a frustoconical ramped side-wall 33, i.e. a ramped side-wall 33 extending radially around the minimized floor, which is essentially a rim of gas inlet opening (end 35a). Ends 34a, 36a respectively of channels 34, 36 are 2 of 3 channels that serve as gas outlets.

[0078] FIG. 3D also schematically shows how rapid switching between gas outlets can vary a path or trajectory of bubbles through the slug 37. It should be noted that the effect is greatly exaggerated, as even with an instant shift in pressure gradients, the bubbles negligeable inertia will ensure a spreading, and not a splitting, of the bubble stream.

[0079] FIG. 4 depicts a micromixer 40 on a chip edge-coupled to a process driver 50 in a side elevation cross-sectional view. The chip is composed of a substrate 41 bearing relief patterning on three sides: a top, a bottom and a coupling edge. The relief patterning from the top defines features that optionally share specifications with same-named features of other embodiments and variants. Mixing chamber 42 has three vertically stacked parts: a headspace 42a, frustoconical midsection 42c, and a bottom bowl 42b. Gas outlet microfluidic channel 44 is rather compact, allowing for low hysteresis control over the volume 28 above a liquid (in use), and has adjacent ends 44a,b. Gas inlet microfluidic channel 45 is mostly provided by relief patterning on the bottom side of the substrate, as is an overflow chamber 46. The gas inlet extends from a throughbore to the bottom of the substrate 41 to a port on the coupling-edge (not in view) as the bottom plane segment of the gas inlet is routed around the overflow chamber 46. Top (49) and bottom (49a) covers seal around channels and chambers on the top and bottom surfaces.

[0080] The transition in the mixing chamber 42 between bowl 42b and midsection 42c also serves as a separating rim for a metering cup in accordance with the teachings of Applicant’s co-pending US provisional 63 / 619256, the entire contents of which are incorporated by reference. A short channel 46a connects the mixing and overflow chambers to let excess volume into the overflow chamber 46, when suction is applied through overflow port 46b. The short channel 46a can be relief patterned from the top side, bottom side, or coupling edge, or may be drilled into the substrate after the substrate was fabricated, but some attention is made to the dimensions of the hole to ensure resistance to liquid entering the channel under gravity. Thus gravity alone does not compel liquid above the transition in chamber 42 to flow into overflow chamber 46 when the overflow port 46b is closed, despite the weak seals provided by a valve of the process driver 50 and chip seal.

[0081] The process driver 50 has numerous seals 52 for coupling to respective ports of the chip, although only 3 are in view (only two of which labelled). Each seal 52 has an associated valve 55, which is coupled by a pneumatic network to at least one of an open vent, or a plenum of a suction pump 56. Advantageously, the process driver 50 and chip are designed to ensure that only air or gas is conveyed over the pneumatic network, such that the chip is a single-use consumable, but the process driver 50 is reusable without onerous sterilization procedures. In some embodiments, the driver 50 is adapted to connect a gas cartridge to a port at the second end of gas inlet. Preferably one or more ports are provided to transfer liquids to and from the mixing chamber 42. Generally, a gravitational check-valve can be convenient for bringing liquid into the chamber 42, and bottom coupled openings are needed for efficient extraction of liquids from chambers. While each valve 55 can have different requirements for a given process, some requiring closure and opening to vent, some requiring closure and opening to suction, and others requiring open to suction or open to vent, it is generally convenient to provide valves with 3 states that open to all three, to accommodate some variations in the process for which the driver is made, as long as sealing is sufficient and the 3 state valves are cost efficient.

[0082] In use, the liquid is brought into the chamber 42, preferably from an opening along a top peripheral rim of the chamber, on the cover-meeting surface of the substrate, i.e. at the elevation of channel 44, but separated peripherally by at least 5 mm from channel 44 29 to prevent ingestion. The liquid plug is preferably delivered by closing required valves to respective ports (including the overflow valve), and drawing suction through channel 44, in which case the supply opens into the chamber 42 from a location separated peripherally from channel 44 by at least 5 mm to prevent ingestion. There may be two or more liquid supply steps, in some protocols, or the liquids to be mixed may have been precombined (but not well mixed), depending on the protocol. Once the liquid(s) are loaded, they may preferably have a fill level or mean free surface, at or above the transition between the bowl 42b and midsection 42c. A port to channel 45 is opened and the port to the supply is closed. The gas, whether from a cannister that is at most nomin-ally above ambient pressure, or ambient air, is drawn by suction through the gas inlet, and through the liquid slug, which can expand substantially such that most of the liquid can be displaced from the bowl 42b into the midsection 42c. Alternatively, instead of opening (completely) the gas inlet, in accordance with the BFD mode, where the supply of gas through the inlet is either blocked intermittently, or otherwise choked to limit supply, as this allows the bubble to be formed but not burst, which can be critical to mixing liquids that have a tendency to foam or froth when mixed with air, especially if the air is allowed to form small bubbles. While the BFD mode can be slower, it can allow mixing of liquids that would be quite difficult to mix, and likely otherwise impossible to mix with bubbles. The diameter of the opening at end 45a, and diameter of the bowl 42b can be chosen to enhance the effective displacement of liquid.

[0083] Once the sample is mixed, either by BFD, or BFB (obviously in concert with tidal action) the sample can be allowed to settle, and the mixed liquid can now be metered, by opening the valve of the gas inlet to ambience, and drawing suction on the overflow port 46b. Once resistance on this suction ends, there is no liquid (or foamy) plug covering short channel 46a, and the chamber is metered by the retraction of the overflow volume, which falls into the trap provided by chamber 46. The trap is a fluidic dead-end. Thereafter, closure of the overflow port 46b, and drawing suction on another line coupled (at least in part) to the gas inlet channel 45, permits the mixed, metered sample to be delivered to another compartment, or alternatively, the metered volume can be mixed with another liquid that is supplied as explained hereinabove.

[0084] It will be appreciated that various services can be added to the process driver 50, for various purposes. E.g.g.: a thermal regulator, heater, cooler, or thermometer, can be added to treat / monitor liquid in a chamber of the chip; a camera or sensor (preferably noncontact) can be used to statefully monitor the chip, and control operations of valves in accordance with a designed protocol, in coordination with an image analysis process, and processor, obviously with a power supply, but with or without human input and or output devices; an electrochemical, pH, or thermal sensor, or camera for imaging a sample for recording or reporting a result of an assay; a mechanical pressure sensor for determining a pressure in the chamber. Examples

[0085] FIGs. 1B and 2B show photographic evidence of chips made and used to bubble mix, in accordance with the present invention. In addition an embodiment with a conical side-wall was manufactured, somewhat as shown in FIG. 4 but without the overflow chamber 46. This embodiment was used for mixing which it did very well (quickly and with low over all energy and time). In addition, FIG. 5 shows a part of a chip, and enlar-gements of the chip in 4 timesteps, showing on the top a capture process, and on the bottom a process for bubble mixing superparamagnetic nanoparticles (MNPs) in liquid.

[0086] FIG. 5 left shows part of the chip used. It has a number of reservoirs for controlled dispensing of liquids, such as reagents, reactants, buffer, lysing agent, developer agent, for preparation and performance of a sample to answer protocol. A recess in the back-side of the chip is provided for receiving a magnet, just below the mixing chamber. Chips featuring the micromixer with a wide variety of other features, have also been manufactured.

[0087] The top row of enlargements show how within in 2.28 s the MNPs are collected on a bottom curved crevice at the bottom of the chamber. An arrow is shown at the 5.58 s points to the densified MNPs. Two other black lines in the image are image artifacts. A difference in darkness of the liquid in the chamber before and after the magnetization, with sporadic bubbling, is more obvious when viewing the colour photographs, where the liquids greenish colour contrasts strongly with the brown colour when the MNPs are well mixed. Sporadic bubble mixing, despite applying pressures that tend to disrupt the accretion of MNPs, was found to generally accelerate the accretion process. While it is unclear what fraction of MNPs were accreted by the 1.44 s timestep, by 2.28 s the density of accreted MNPs and colour of the liquid, that substantially all MNPs have been pulled out of the liquid. This is confirmed by the same contrast between the colours within the T = 5.58 s timestep and the 2.28 s timestep. No bubble mixing is shown at these timesteps.

[0088] The bottom row of enlargements show that, within about 2.58 s, a dense packing of the MNPs can be distributed throughout a liquid. Except for the absence of the metal (reflective) magnet, the first enlargement in the bottom panel is substantially identical to the last timestep of the top row. Operation of the vacuum was manual, each pulse having a duration of 0.2 to 0.5 s, and separated by substantially equal durations. The first few pulses distributed most of the MNPs. By the T = 0.45 s timestep, a little bit of the left side of the liquid, above its dominant bubble, still shows some green colouration but a bottom bubble and cloud of MNPs overlying the crevice preclude clear assessment of the density of the accreted MNPs. By the 2.58 s timestep, the colour is about uniformly brown. A density of the uniformly loaded MNPs in the deepest part of the chamber defies asses-sment of the 5 MNPs, but very little change is perceived in the density or colouration between the 2.58 and 6.15 s timesteps. Uniform mixing is believed to be provided very shortly after the 2.58 s timestep. The depth gradients within the chamber, as a result of the sloped side-wall, induce some measure of confounding features, that cooperate with the brown vs. green colouration, but the images show successful mixing, and collection of MNPs, which 10 Applicant submits is remarkable with such a low power, low cost, system.

[0089] The novel features will become apparent to those of skill in the art upon examination of the description. It should be understood, however, that the scope of the claims should not be limited by the embodiments but should be given the broadest interpretation consistent with the wording of the claims and the specification as a whole. 15

Claims

1. A kit for forming a microfluidic device with a microfluidic mixer, the kit comprising: a substrate with a cover-meeting surface for sealed meeting with a cover to form a chip, the surface having a relief-pattern defining:a three-phase mixing chamber for mixing a liquid, the mixing chamber having: a floor that is a recessed depth (d) below the surface, and a periphery around the surface for sealing with the cover;at least one microfluidic gas inlet meeting to the mixing chamber proximate the floor, in fluid communication with a port of the substrate;at least one microfluidic gas outlet in fluid communication with the external environment of the substrate the gas outlet meeting the mixing chamber proximate the cover meeting surface; andat least one liquid microfluidic channel for supplying at least one liquid to be mixed into the microfluidic mixer, and / or for drawing at least some of mixed liquid out of the microfluidic mixer; and,a suction device adapted to apply a negative relative pressure in a range of -0.345 kPa to -3.5 kPa to the gas outlet when the substrate is assembled to form a microfluidic chip and the micromixer is loaded with a volume of liquid.

2. The kit of claim 1, wherein the mixing chamber’s periphery has a length and a width, where d is 5 to 15 mm, and a depth-to width aspect ratio is less than 2:1, and wherein each of the plurality of microfluidic channels: has a mean hydraulic diameter of 100 to 500 pm; or is 150 to 300 pm wide, and 50 to 150 pm deep.

3. The kit of claim 2, wherein: the depth is 6 to 12 mm; the aspect ratio is less than 1:1; and width is less than, or equal to the length.

4. The kit of claim 2, wherein: the depth is 7 to 9 mm; the aspect ratio is less than 0.5:1; and width is at most half the length.

5. The kit of any one of claims 1 to 4 wherein the substrate comprises a metal such as an aluminum, titanium, steel, or magnesium alloy, or a polymeric material, such as a thermoset, thermoplastic, or elastomer.

6. The kit of claim 5 with the substrate made of a biocompatible polymeric material, such as a thermoset, or thermoplastic.

7. The kit of any one of claims 1 to 6, wherein at least one of the plurality of microfluidic channels is network coupled to: convey either a liquid, or a gas depending on a liquid fill condition of the microfluidic device; or convey fluid to, or remove fluid from, the mixing chamber, depending on operation of one or more valves of the microfluidic device; expose a sample to: a sensor; a camera for imaging; or a magnetic field.

8. The kit of any one of claims 1 to 7 wherein at least one of the plurality of microfluidic channels is a sample supply for introducing a liquid sample into the mixing chamber in one mode of operation, the sample supply meeting the mixing chamber proximate, or passing through, the periphery, and coupled to a metering chamber or supply reservoir.

9. The kit of any one of claims 1 to 8, wherein the periphery encloses an area greater than twice an area of the floor, and the mixing chamber monotonically enlarges with elevation from the floor to the periphery.

10. The kit of any one of claims 1 to 9, wherein the plurality of microfluidic channels comprise 2 to 4 gas outlets spaced around the periphery such that each adjacent pair of the 2 to 4 gas outlets delimits a respective part of the periphery having of a respective length, and the spacing is uniform enough that no adjacent pair’s length is more than three times that of another adjacent pair.

11. The kit of claim 10 wherein each of the 2 to 4 gas outlets has a coordinated corresponding gas inlet and pulses of relative vacuum pressure is applied by the gas outlet while its paired inlet is open.

12. The kit of claim 7, where the mixing chamber comprises at least one ramped sidewall extending from the floor, or from above the floor, to at least 1 / 5th d, and a mean angle of the ramped sidewall with a mean plane of the floor is from 2° to 33°.

13. The kit of claim 12, wherein the ramped sidewall extends principally from the floor in a first Cartesian direction, providing the periphery with a floor side and a shallow side, and each of the at least one gas outlet meets the periphery closer to the shallow side than the floor side; or the ramped sidewall is a partial conic surface.

14. The kit of claim 1 to 13 further comprising one or more cover sheets for sealing against the cover-meeting surface to enclose: the mixing chamber other than at respective openings to the plurality of microfluidic channels; and part of the at least one gas outlet, away from a port thereof, wherein the seal between the part of the gas outlet and one or more cover sheets permits the at least one pneumatic extraction channel to communicatea negative pressure in a range of -0.345 kPa to -3.5 kPa, relative to ambient pressure, with the mixing chamber, and the port is configured to be connected to the suction device.

15. The kit of claim 14, dependent directly or indirectly from claim 8, wherein the one or more cover sheets further enclose the metering chamber or the supply reservoir, and a volume of the enclosed metering chamber, or supply reservoir, is between 10% and 60%, more preferably 25% to 50%, of a volume of the mixing chamber.

16. The kit of claim 14 or 15 further assembled to form a microfluidic chip.

17. The kit of claim 14 or 15 further comprising the suction device adapted to couple to a port of the chip draw a negative relative pressure of -0.345 kPa to -3.5 kPa at the gas outlet.

18. The kit of claim 17 assembled to form a microfluidic chip, with the suction device mounted to the port of the chip to draw negative relative pressure from at the gas outlet.

19. A method of mixing in a microfluidic device, the method comprising:introducing a liquid into a three-phase mixing chamber in the microfluidic device, until between 10% and 60%, more preferably 25% to 50%, of a volume thereof is filled with liquid, whereby a free surface of the liquid is at least 4 mm below a cover of the three-phase mixing chamber; anddrawing a gas through the liquid in the mixing chamber by applying a negative relative pressure in the mixing chamber above the free surface, in a range of -0.345 kPa to -3.5 kPa, while at least one gas inlet connecting a floor of the mixing chamber to a gas supply is open, to create at least one bubble of the gas in the liquid to move the liquid within the mixing chamber.

20. The method of claims 19, wherein drawing the gas comprises:pulsing gas through the at least one gas inlet, by intermittently valving one of the at least one gas inlet, or a valve to the negative pressure supply, or by intermittently stopping and starting a pump for creating the negative pressure supply;sequentially opening one of a plurality of valves of one of a plurality of gas outlets that meet the mixing chamber at different points on the periphery of the mixing chamber above the free surface, while the negative pressure is applied to draw bubbles through the liquid in different directions as a function of time;sequentially opening one of a plurality of valves of one of a plurality of gas inlets that meet at different points around the floor of the mixing chamber, while the negativepressure is applied, to draw bubbles through the liquid in different directions as a function of time; orbubbling gas through the at least one gas inlet, by creating a steady draw of the negative relative pressure to direct bubbles along a trajectory through the liquid, displacing and raising a free surface of the liquid by injection of the bubbles, to move the liquid into contact with a ramped sidewall of the mixing chamber away from the bubbles’ trajectory, and then releasing the pressure to allow the liquid to slide back to a starting location, in a better mixed arrangement.

21. A microfluidic chip comprising:a three-phase mixing chamber for mixing a liquid, the mixing chamber having a floor that is a recessed depth (d) below a ceiling;at least one microfluidic gas inlet meeting the mixing chamber proximate the floor, in fluid communication with an inlet port of the substrate;at least one microfluidic gas outlet in fluid communication with a negative relative pressure pneumatic chamber of the chip,where a stiffness of the negative relative pressure pneumatic chamber, seals of the microfluidic chip, and materials of the microfluidic chip, permit the negative relative pressure pneumatic chamber to enclose a negative relative pressure in a range of -0.345 kPa to -3.5 kPa when the mixing chamber is filled with a liquid.AMENDMENTS TO THE CLAIMS FILED AS FOLLOWS06 06 25Claims:

1. A kit for forming a microfluidic device with a bubble mixer, the kit comprising: a substrate with a cover-meeting surface for sealed meeting with a cover to form a chip, the cover-meeting surface having a relief-pattern defining:a three-phase mixing chamber for mixing a liquid, the mixing chamber having: a floor that is a recessed depth (d) below the cover-meeting surface, and a periphery around the cover-meeting surface for sealing with the cover;at least one microfluidic gas inlet meeting to the mixing chamber proximate the floor, in fluid communication with a port of the substrate;at least one microfluidic gas outlet in fluid communication with the external environment of the substrate, the microfluidic gas outlet meeting the mixing chamber proximate the cover-meeting surface; andat least one microfluidic channel for supplying the bubble mixer with at least one liquid to be mixed in the mixing chamber, and / or for drawing at least some of the mixed liquid out of the bubble mixer; and,a suction device adapted to apply a negative relative pressure in a range of-0.345 kPa to -3.5 kPa to the microfluidic gas outlet when the substrate is assembled to form a microfluidic chip and the mixing chamber is loaded with a volume of liquid.

2. The kit of claim 1, wherein the mixing chamber’s periphery has a length and a width, where d is 5 to 15 mm, and a depth-to width aspect ratio is less than 2:1, and wherein each of the at least one microfluidic channel: has a mean hydraulic diameter of 100 to 500 pm; or is 150 to 300 pm wide, and 50 to 150 pm deep.

3. The kit of claim 2, wherein: the depth is 6 to 12 mm; the aspect ratio is less than 1:1; and width is less than, or equal to the length.

4. The kit of claim 2, wherein: the depth is 7 to 9 mm; the aspect ratio is less than 0.5:1; and width is at most half the length.

5. The kit of any one of claims 1 to 4 wherein the substrate comprises a metal such as an aluminum, titanium, steel, or magnesium alloy, or a polymeric material, such as a thermoset, thermoplastic, or elastomer.

6. The kit of claim 5 with the substrate made of a biocompatible polymeric material, such as a thermoset, or thermoplastic.06 06 257. The kit of any one of claims 1 to 6, wherein at least one first of the at least one microfluidic channel is network coupled to:convey either a liquid, or a gas depending on a liquid fill condition of the microfluidic device;convey fluid to, or remove fluid from, the mixing chamber, depending on operation of one or more valves of the microfluidic device; orexpose a sample to: a sensor; a camera for imaging; or a magnetic field.

8. The kit of any one of claims 1 to 6 wherein at least one first of the at least one microfluidic channel is a sample supply for introducing a liquid sample into the mixing chamber in one mode of operation, the sample supply meeting the mixing chamber proximate, or passing through, the periphery, and coupled to a metering chamber or supply reservoir.

9. The kit of any one of claims 1 to 8, wherein the periphery encloses an area greater than twice an area of the floor, and the mixing chamber monotonically enlarges with elevation from the floor to the periphery.

10. The kit of any one of claims 1 to 9, wherein the at least one microfluidic channel comprise 2 to 4 gas outlets spaced around the periphery such that each adjacent pair of the 2 to 4 gas outlets delimits a respective part of the periphery having a respective length, and the spacing is uniform enough that no adjacent pair’s length is more than three times that of another adjacent pair.

11. The kit of claim 10 wherein each of the 2 to 4 gas outlets has a coordinated corresponding gas inlet and pulses of relative vacuum pressure is applied by the gas outlet while its paired inlet is open.

12. The kit of claim 7, where the mixing chamber comprises at least one ramped sidewall extending from the floor, or from above the floor, to at least 1 / 5th d, and a mean angle of the ramped sidewall with a mean plane of the floor is from 2° to 33°.

13. The kit of claim 12, wherein the ramped sidewall extends principally from the floor in a first Cartesian direction, providing the periphery with a floor side and a shallow side, and each of the at least one gas outlet meets the periphery closer to the shallow side than the floor side; or the ramped sidewall is a partial conic surface.06 06 2514. The kit of claim 1 to 13 further comprising one or more cover sheets for sealing against the cover-meeting surface to enclose: the mixing chamber other than at respective openings to the at least one microfluidic channel; and part of the at least one microfluidic gas outlet, away from a port thereof, wherein the seal between the part of the microfluidic gas outlet and one or more cover sheets permits at least one pneumatic extraction channel to communicate a negative pressure in a range of -0.345 kPa to -3.5 kPa, relative to ambient pressure, with the mixing chamber, and the port is configured to be connected to the suction device.

15. The kit of claim 14, dependent directly or indirectly from claim 8, wherein the one or more cover sheets further enclose the metering chamber or the supply reservoir, and a volume of the enclosed metering chamber, or supply reservoir, is between 10% and 60%, more preferably 25% to 50%, of a volume of the mixing chamber.

16. The kit of claim 14 or 15 further assembled to form a microfluidic chip.

17. The kit of claim 14 or 15 further comprising the suction device adapted to couple to a port of the chip draw a negative relative pressure of -0.345 kPa to -3.5 kPa at the gas outlet.

18. The kit of claim 17 assembled to form a microfluidic chip, with the suction device mounted to the port of the chip to draw negative relative pressure from the microfluidic gas outlet.

19. A method of mixing in a microfluidic device, the method comprising:introducing a liquid into a three-phase mixing chamber in the microfluidic device, until between 10% and 60% of a volume thereof is filled with liquid, whereby a free surface of the liquid is at least 4 mm below a cover of the mixing chamber; anddrawing a gas through the liquid in the mixing chamber by applying a negative relative pressure in the mixing chamber above the free surface via a gas outlet proximate a cover-meeting surface opposite a floor of the mixing chamber that is a recessed depth (d) below the cover-meeting surface, in a range of -0.345 kPa to -3.5 kPa, while at least one gas inlet connected to the mixing chamber proximate the floor, with the gas inlet open to a gas supply , to create at least one bubble of the gas in the liquid to move the liquid within the mixing chamber.

20. The method of claims 19, wherein drawing the gas comprises:06 06 25pulsing gas through the at least one gas inlet, by intermittently valving one of the at least one gas inlet, or a valve to the negative pressure supply, or by intermittently stopping and starting a pump for creating the negative pressure supply;sequentially opening one of a plurality of valves of one of a plurality of gas outlets that meet the mixing chamber at different points on the periphery of the mixing chamber above the free surface, while the negative pressure is applied to draw bubbles through the liquid in different directions as a function of time;sequentially opening one of a plurality of valves of one of a plurality of gas inlets that meet at different points around the floor of the mixing chamber, while the negative pressure is applied, to draw bubbles through the liquid in different directions as a function of time; orbubbling gas through the at least one gas inlet, by creating a steady draw of the negative relative pressure to direct bubbles along a trajectory through the liquid, displacing and raising a free surface of the liquid by injection of the bubbles, to move the liquid into contact with a ramped sidewall of the mixing chamber away from the bubbles’ trajectory, and then releasing the pressure to allow the liquid to slide back to a starting location, in a better mixed arrangement.

21. A microfluidic chip comprising a cover for sealing a substrate, and the substrate that is relief-patterned to define:a three-phase mixing chamber for mixing a liquid, the mixing chamber having a floor that is a recessed depth (d) below a ceiling;at least one microfluidic gas inlet meeting the mixing chamber proximate the floor, in fluid communication with an inlet port of the substrate;at least one microfluidic gas outlet in fluid communication with a negative relative pressure pneumatic chamber of the chip, the microfluidic gas outlet meeting the mixing chamber proximate a cover-meeting surface sealing the mixing chamber, where a stiffness of the substrate surrounding the negative relative pressure pneumatic chamber, seals of the substrate with the cover, and materials of the substrate and cover, permit the negative relative pressure pneumatic chamber to enclose a negative relative pressure in a range of -0.345 kPa to -3.5 kPa when the mixing chamber is filled with a liquid.41

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