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WO2024176188A1 - Micro-aliquotage centrifuge - Google Patents

Micro-aliquotage centrifuge Download PDF

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Publication number
WO2024176188A1
WO2024176188A1 PCT/IB2024/051767 IB2024051767W WO2024176188A1 WO 2024176188 A1 WO2024176188 A1 WO 2024176188A1 IB 2024051767 W IB2024051767 W IB 2024051767W WO 2024176188 A1 WO2024176188 A1 WO 2024176188A1
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WO
WIPO (PCT)
Prior art keywords
chip
membrane
array
sample
microwells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2024/051767
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English (en)
Inventor
Tae-Hyeong Kim
Daniel Brassard
Javier HERNANDEZ-CASTRO
Teodor Veres
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National Research Council of Canada
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National Research Council of Canada
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Publication date
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Publication of WO2024176188A1 publication Critical patent/WO2024176188A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B01L2300/123—Flexible; Elastomeric
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces

Definitions

  • the present invention relates in general to micro-aliquoting, i.e. dividing a liquid into many, tiny volume, microwells on a microfluidic chip, and in particular to a centrifugal chip, method and system for micro-aliquoting that avoids trapping air bubbles, for complete filling.
  • Microfluidics as a whole is an endeavour to control small volumes of fluids, generally to: test compositions for chemical or physical properties or responses; to purify samples; or to coerce behaviour of samples. While larger volume reactors may be desired for favouring some (e.g. high-throughput) reactions, there are many reasons to desire micro-aliquoting: division of sample volumes into independent reaction volumes (IRVs) of respective microwells. For example, to quantify presence or absence of an analyte, it can be efficient to divide a sample and reagent mixture into many (e.g. 10 3 -10 6 ) IRVs, and observe a number of the IRVs reporting the analyte.
  • IIRVs independent reaction volumes
  • IRVs can be valuable for ensuring exposure of a sample to a complex or inhibited reactant.
  • Cells can be exposed to biochemical species in short periods of time, with high reliability, if the cell and species are suitably confined within a definite IRV, whereas a single chamber with cells and the species: may take too long to provide equal assurances of exposure, uptake, dissolution, or other interaction; might be subject to too many additional interactions that may confound measurements; and might make constrained dosing impossible, for example.
  • microfluidic devices to partition fluids into IRVs. While there are several ways of accomplishing this, devices and equipment for reliably filling microwells with (already small volume) samples is challenging, as demonstrated by the large footprint specialized devices required for the separation, e.g. in dd-PCR systems.
  • FIGs. 3,5 of US 8481901 to Bedingham et al. suggest, but don’t seem to provide, a device for sealing a liquid in a chamber 150 of a centrifugal microfluidic CD. It is a bit of an odd disclosure in that: 1) there is no rationale for closing off the liquid into this chamber; 2) there is no process leveraging this function; and 3) the process for filling requires centrifugation, but there is no process suggested for deforming metal foil 134 on a CD while under centrifugation.
  • C18.L18-22 states that isolation can be useful to prevent crosscontamination, however, a well metered volume of sample will not provide bridging between separate chambers 150/50. Typically the volume in the chambers will be far greater than those of the channels, as clearly illustrated, and so the metered fluid is unlikely sufficient to interconnect two or more adjacent chambers. Once the liquid is air-plugged into the ends, it cannot easily escape even if centrifugation stops. Further comments at C18,L32-42 are equally unclear. Once the metal foil is plastically deformed, one wonders what use the sample will be, as it is closed off from the whole microfluidic network. One can hardly imagine a mechanism for reopening, and so the sample can be examined, but cannot interact with any other fluids or reagents, and cannot be useful in any further microfluidic process according to this system.
  • thermomechanical means is inferably an off-line process requiring the careful deformation of select regions of the CD. Why would one bother with this risky process that could crack the metal substrate, or break the seal provided by the passivation layer 132, or just fracture the substrate, when clearly “sufficient isolation may be achieved by continuously rotating the device during processing” (C18,L28-31 )? Gravity alone does not pull a small droplet out of the niche, unless the volume is large enough with respect to the surface area of the niche to permit different scale-order effects than the present invention.
  • US 2021/069709 to Chou et al. discloses a microfluidic device, created with the objective to “quickly” isolate part of a fluidic sample from the rest, to do analysis. Chou et al. seeks equally to limit motion of liquid during exposure to thermal cycling. To this end clamps are provided for their non-centrifugal device, for pressing and releasing in sequence to confine fluid in a chamber. The chamber may have spacers away from the clamp regions (ring or box). Chou et al. manage to produce a fast cycling time. Chou et al.
  • TPE materials and particularly formulations of block co-polymers such as SEBS with low oil content
  • WO2017/066869, US9,238,346, US10,369,566) enabling valves (WO2011/134038, WO2019/167031) and seals for microfluidic blisters (EP 3,269,451).
  • Applicant has also been developing pneumatic-centrifugal systems that permit pneumatic pressures to be applied directly to microfluidic chambers and channels (and contents thereof) or to apply indirectly on such chambers and channels, for example as mediated by elastomeric membranes.
  • Such systems can be provided with rotary fluid couplings or slip rings, or by various means taught in Applicant’s WO 2015/132743 the contents of which are incorporated herein by reference.
  • a technique for micro-aliquoting, i.e. filling microwells of a centrifugal microfluidic chip to separate a sample fluid into independent reaction volumes (IRVs). The technique involves: providing the sample in a chamber of the microfluidic chip, the chamber defined between a relief patterned substrate and an elastomeric membrane; and urging the membrane towards the substrate during centrifuga- tion.
  • the substrate has a relief pattern defining an array of at least 100 microwells (more preferably 1000, or even 8000), with each microwell separated from neighbouring microwells by walls, the urging of the membrane, while under centrifugation, separates air or undissolved gas from the liquid, and ensures filling of the microwells. If the membrane is urged forward until it meets tops of the walls, i.e. if rims separating the microwells contact the membrane, IRVs (contents of each microwell) are isolated in 3 dimensions.
  • the chip itself, with the membrane, substrate, and the array of microwells, is specifically designed for this novel micro-aliquoting technique and has novel features.
  • a system comprising the chip, a centrifugal microfluidic chip controller, and/or a centrifuge is also disclosed.
  • a centrifugal microfluidic chip comprising: a substrate having a relief patterned meeting surface defining an array of at least 100 wells, each well having a volume of 0.1 fL to 1 pL, and separated from at least two neigbouring wells by walls, the array provided by recesses in the meeting surface, or by a structured insert embedded in the meeting surface; and an elastomeric membrane with a first surface facing the meeting surface and a second surface, opposite the first surface, the membrane operable to define a chamber between the first and meeting surfaces, the first surface extending at least partly around the array.
  • the second surface is adapted to permit a controlled actuator to selectively press the membrane into contact with the meeting surface to fluid ically separate adjacent wells, and close the chamber.
  • a second centrifugal microfluidic chip comprising: a similarly characterized relief patterned substrate and membrane, in combination with a pneumatic enclosure surrounding the second surface, the enclosure sealed against the elastomeric layer, and coupled to a pneumatic supply line.
  • the pneumatic enclosure may be provided by a patterned layer of a polymeric material with a thermoplastic or thermoset resin base that is same, similar or dissimilar to a composition of the substrate, the patterned layer having a stiffness higher than that of the membrane.
  • Either of these chips preferably have at least 1000, more preferably at least 8000 wells.
  • each well has a respective volume, and no well has a volume greater than 0.5 pL, nor less than 0.5 fL.
  • the array may have a natural hydrophilicity or may be coated so that a contact angle of an aqueous droplet with a critical micelle concentration of surfactant is less than 60°.
  • each well is of one of at most 4 types, each type determined by: a volume, a shape, and an adjacency relationship with neighbouring well types, each type having a same volume to within at least 5%, or each type having different volumes.
  • one or more of the following characterizes each well: a cupped recess with a smooth transition between sidewalls and bottom; approximation to a minimal surface; an aspect ratio between 2:1 and 1 :5; a convexvolume, with rounded polygonal planform; a planform shape consisting of at least 3 rounded edges, or at least one arcuate edge.
  • the wells form a regular, semi-regular, or partially regular array.
  • Some embodiments further comprise an annular channel surrounding the array of wells, the annular channel defining a ledge that limits where the membrane meets the substrate from the array, and the annular channel is coupled, axis-proximally, to a microfluidic circuit for feeding the array, whereby actuation of the membrane brings the membrane into a covering position over the array without blocking flow in in the annular channel.
  • Some embodiments further comprise a cauling aligned with the micropillar array, adhered to either the first or second surface, for increasing local stiffness of the membrane.
  • Some embodiments further comprise a microfluidic reservoir for retaining a volume of liquid that is between 1.2 and 12 times a collective volume of the wells, and a channel communicating between the reservoir and the array, the reservoir defined by a relief pattern of the substrate or layer. Some embodiments further comprise a network of microfluidic chambers and channels defined by a relief pattern of the substrate or membrane for preparing a sample, including mixing the sample with one or more reagents, and supplying a test fluid to the array.
  • the chip is adapted so that actuating the membrane brings the membrane to a second state where the membrane contacts the walls surrounding the microwells, to separate the sample into independent reaction volumes that are isolated in 3 dimensions.
  • a centrifugal microfluidic system comprising a chip as characterized hereinabove, a centrifuge blade having a chip part for mounting the chip, a centrifuge mounting part for mounting to a centrifuge for rotation of at least the chip part and the chip by the centrifuge, and at least one of: the actuator identified in claim 1 , operable during centrifugation of the chip and chip part; and the pneumatic supply line identified in claim 2.
  • the blade has a fluidic interface for coupling to at least one port of the chip to: a pneumatic slip ring; a closed pressurized container via an electromechanical flow control device and an electronic controller for controlling the device; or an opening to ambience via an electromechanical flow control device and an electronic controller for controlling the device, whereby the slip ring or electronic controller can vary a pressure at a port of the chip.
  • a method for micro-aliquoting a liquid sample in a microfluidic chip comprising: providing a centrifugal microfluidic chip as characterized hereinabove, with the liquid sample loaded into the chip; rotating the chip to locate the sample within the chamber bounded the substrate and membrane; and while rotating, actuating the membrane to fluid ically separate neighbouring wells and expel excess fluid against the centrifugal field away from the array.
  • the method further comprises, prior to locating the sample in the chamber, mixing the sample with a surfactant, and/or treating the microwells to make them hydrophilic, so that a contact angle of the sample with the walls of the microwells is less than 60°.
  • actuating the membrane is pneumatic, and in others an electromagnetic, or magnetic device is mounted to the blade adjacent the chip to this end. In some embodiments the membrane actuation is reversibly controlled.
  • FIG. 1 is a partial top plan view schematically illustrating part of a centrifugal microfluidic chip in accordance with a first embodiment of the present invention, the chip having a membrane removed to show relief patterning of the substrate;
  • FIG. 1A is a partial cross-sectional side elevation view along AA of the chip of FIG. 1 , with the membrane;
  • FIGs. 1 B-G are partial cross-sectional side elevation views of respective variants of the first embodiment, with FIG. 2B showing an enlargement of an array in cross-section:
  • FIGs. 1 B,C illustrate variants without a rigid pneumatic covering layer
  • FIG. 1 C illustrates an actuating magnet as an alternative to pneumatic control
  • FIGs. 1 B,E,F illustrate variants in which the elastomeric membrane layer is patterned to provide microfluidic channels (1 B), pneumatic control chambers (1 E), or both (1 F), as opposed to the substrate and covering layer, respectively;
  • FIGs. 1 D,F,G illustrate variants in which the substrate comprises an insert and a layer for sealing against the elastomer
  • FIGs. 1 B,D,G illustrate variants without caulings, and FIG. 1 D places the cauling at the membrane adjacent the microwell array, as opposed to an actuator facing side, and FIG. 1 F provides caulings on both sides;
  • FIG. 1 G illustrates a normally closed variant
  • FIGs. 2A-D are enlarged views of the partial cross-sectional side elevation of the variant of FIG. 1 D in sequential stages: FIGs. 2B-D schematically illustrating principal timepoints of a first method of micro-aliquoting in accordance with the present invention, the timepoints corresponding to: prior to receiving sample; after the sample flows into the chamber; after some deformation of the membrane; and upon complete closure of the microwells;
  • FIG. 2C' schematically illustrates a variant of 2C, which, in comparison with FIG. 2C, shows the effects of using an actuator-side cauling as shown in FIG. 1A, on deformation of the membrane at this timepoint;
  • FIGs. 3A-C schematically illustrate 3 time points in an iterative embodiment of a method for micro-aliquoting, particularly useful if the sample viscosity, or surface affinity varies or centrifugation rate is insufficient for a particular instance;
  • FIG. 3C schematically illustrates a variant of the iterative method with a different initial wetting fluid that provides for controlled mixing within the microwells;
  • FIGs. 4A-E are schematic side elevation views of variant microwell profiles in accordance with the present invention, in which:
  • FIGs. 4A,C,D illustrate well profiles having a substantially cylindrical opening end and a tapered or slightly rounded tapered distal segment, followed by a rounded cap;
  • FIG. 4B schematically illustrates a profile with a substantially tapered, or frustoconical profile having a rounded bottom
  • FIG. 4D schematically illustrates a profile with a substantially cylindrical sidewall and a rounded bottom
  • FIGs. 5A-E are schematic top plan views of a variety of microwell array patterns that may be chosen for arranging microwells in a compact arrangement that allows for a high density of low volume microwells,
  • FIG. 5A showing a hexagonal tiling
  • FIG. 5B showing a triangular tiling
  • FIG. 5C showing a tiling defined by a triangular packing with each triangle broken into three isomorphic isosceles trapezoids to illustrate a non-edge aligned pattern
  • FIG. 5D showing a tiling of two equal area, but very different perimeter, polygonal shapes (regular hexagon and triangles: 6 triangle per hexagon: also a subdivision of a hexagonal tiling);
  • FIG. 5E showing a tiling with 4 different tiles of different areas, as can be useful in dealing with uncertain loading of samples
  • FIGs. 5F shows a cross-sectional elevation view of a regular array having a variation in volume of IRVs, shown systematically decreasing in one direction;
  • FIGs. 6A,B schematically illustrate two plan views of chips with overflow designs for handling excess fluid to permit the membrane to be opened, without risk of excess liquid contacting the IRVs as long as centrifugation is applied;
  • FIG. 5B showing an overflow design with a minimum volume retained in the chamber, allowing for IRVs to be maintained even if centrifugation stops;
  • FIG. 7A is a schematic top plan view of a chip mounted to a chip controller
  • FIG. 7B is a schematictop plan view of a chip and a chip controller for two mounting two chips, mounted to a centrifuge;
  • FIG. 7C is a schematic side elevation view of the system of FIG. 6B with two chips mounted to a chip controller having a camera and lighting system for imaging the IRV array;
  • FIG. 8A,B are exploded and top, plan views of a prototype example 7 layer chip
  • FIG. 8C is a photograph of the chip in use
  • FIG. 9 is a panel showing 4 photographs of typical enlarged images of two different
  • FIG. 10 is a panel of 4 figures illustrating fluorescence response within microwells as a function of density of DNA/RNA copies in the sample.
  • centrifugal micro-aliquoting i.e. filling respective microwells to divide a liquid into IRVs on a centrifugal microfluidic chip.
  • a membrane is provided opposite the microwell array, and is actuable to effectively enclose the microwells, displacing the liquid into adjacent microwells, or away from the array.
  • meniscus and like effects can be minimized, a more uniform volume may be aliquoted in comparison with structures that rely on an air-interface.
  • the membrane can be actuated during centrifugation, via a pneumatic blade or simpler slip ring assembly, the centrifugation to aliquot can be provided during membrane closure. As the liquid becomes enclosed by the membrane and microwells, they are effectively incapable of cross-contamination or fluid exchange.
  • FIG. 1 is a schematic top plan illustration of a patterned substrate 10 for defining a set of features of a centrifugal microfluidic chip useful for micro-aliquoting in accordance with the present invention
  • FIG. 1A is a side elevation view along section AA.
  • FIG. 1A shows a membrane 12 and an optional cover 24 that were removed in FIG. 1 to facilitate viewing of the fluidic relief pattern.
  • the membrane 12 is essential to the present invention, in that it seals around a microfluidic network (only partially illustrated) to enclose the chip, and provides a resilient wall for selectively enclosing the IRVs, however the cover 24 offers one (pneumatic) actuation mechanism that can be replaced with another mechanism in other embodiments.
  • the invention is substantially agnostic to materials and forming techniques, as long as the required resolution of the structures in the substrate are met, the sealing is provided between the membrane and substrate, and the substrate 10 is rigid compared with membrane 12.
  • the substrate at least at an array thereof, preferably has a natural hydrophilicity or is coated so that a contact angle of surfactant is less than 60°, more preferably between 5° and 50°. Nonetheless, Applicant finds rigid thermoplastic materials, glasses, ceramics, and metals are ideal for the substrate, and thermoplastic elastomers, in particular block co-polymers and preferably low oil or oil free TPEs with shore A range hardnesses are good candidates for the membrane. Mediprene OFTM and ZeonorTM have useful forming and patterning properties, and excellent adhesion.
  • FIG. 1 The features of FIG. 1 include an array limiting edge 13, circumscribed by a ridge 14 demarcating a microfluidic chamber 15.
  • the chamber 15 is vented at port 16 via a microfluidic channel 17, and communicates with a microfluidic network (not shown) via a microfluidic channel 18. Both channels extend from axis proximal directions, as the features are all on a bottom (axis distal) end of the substrate 10.
  • FIG. 1A shows a cross-section running substantially axis radially with the port 16 axis proximal of the chamber 15.
  • the port 16 is provided by a through-bore in substrate 10, although it could equally be provided through membrane 12 and cover 24 in alternative embodiments, or at an edge port between membrane 12 and substrate 10.
  • FIG. 1 shows the array of microwells, although individual microwells 20 are too small to see.
  • at least 8000 microwells are provided, each of which having a volume of 0.1 pL to 1 nL, and separated by walls from at least two neigbouring wells.
  • the volume may be tightly controlled, to ensure as much as possible, that a same volume is provided for each IRV: for example no microwell may have a volume more than 10% above or below the average volume.
  • a cross-sectional area of each microwell (in the plane imaged) is also preferably similar. Further image analysis is abetted by uniform size and shape of walls between the microwells that provide a regular spacing for the array, although these are not necessary.
  • FIGs. 3 schematically illustrate microwell depth profiles and FIGs. 4 illustrate a few tilings of a plane that permit efficient dense packings of microwells.
  • FIG. 1A shows microwells 20 of exaggerated size to permit visualization. Only a single microwell is identified by lead-line to avoid occlusion of the drawing.
  • the microwells 20 are illustrated as a relief pattern on a raised platform which provides the array limiting edge 13 in this embodiment.
  • the raised platform does not extend to ridge 14, which provides a sealing edge for the chamber 15; rather, annular channel 22 extends, in this embodiment, circumferentially around the raised platform, and array, to provide a moat around the array for circulating liquid during use. Use is further described hereinbelow in relation to FIGs. 2.
  • FIG. 1A also provides a cauling 23, adhered to the membrane 12 in alignment with the platform.
  • the cauling 23 can be any stiffening material with good adhesion to the membrane.
  • the cauling 23 may also be designed to reduce stiction of the membrane 12 with the inside surface of a pneumatic chamber 25 provided by the cover 24.
  • the cauling 23 may be specifically selected to provide a diffuse, matte, background colour that aides visual imaging of colorimetrically or fluoroscopically dyed, or pigmented constituents of IRVs.
  • the cauling 23 and platform are separated by a region 12a of the membrane 12, that has one side facing the pneumatic chamber 25, and the other side facing the microfluidic chamber 15.
  • Chamber 25 has a channel communicating with a pressure-controlled port of the chip (not in view), which may extend along a bonded interface between membrane 12 (away from part 12a) and cover 24, to a conveniently located port, that is itself coupled to a pressurized fluid supply line.
  • the port may extend through cover 24, may extend through an aligned hole in the membrane 12 and substrate 10, or may run along the bonded interface until it reaches an edge of the chip.
  • variants are understood to comprise features of the embodiment from which they deviate, and all identified features of the embodiment are understood to be a part of the variant.
  • like reference numerals identify like features and the same reference features of variants are not explained except to identify similarities and differences.
  • Each variation of each variant is to be presumed independent, and each variants is chosen to cover one or more different variations, although other variants with different combinations of the variants are equally intended. FIGs.
  • 1 B-G illustrate variants of chip 10 that all share some common features, including: that the substrate has a relief patterned surface, or has a structured insert embedded therein that has a relief patterned surface, that defines an array of at least 8000 0.1 pL to 1 nL wells; that the array is provided as a surface within a vented chamber of a part of a microfluidic network, in at least one state of the chip; and that a membrane is provided overlying the array and at least partially surrounding the array, the membrane selectively actuable to be pressed into closed contact with the array, to fluidically separate neighbouring wells.
  • the fluidic separation is particularly useful for evacuating a liquid sample within the chamber except for the IRVs contained within the microwells.
  • FIG. 1 B schematically illustrates a first variant of the chip.
  • the first variant does not have a cover 24, which allows any suitably positioned mechanical device to press against an aligned region 12a of the membrane 12.
  • This mechanical device is preferably pneumatically, or electrically controlled or modulated, and is adapted to be mounted to the centrifuge.
  • the actuator may be integrated with a cartridge containing the chip, may be integrated with a centrifugal blade to which the chip is mounted in use, may be a peelably (or otherwise) removable or permanent layer of the chip; or may be a stand-alone accessory for mounting to a centrifuge.
  • the centrifuge may have an electronic or magnetic slip ring assembly for powering an on- centrifuge controller, flow control devices, temperature control devices, sensors, etc., which can power and/or control the actuator. If the chip does not have a cover 24, or a cauling 23, the membrane may be marked to facilitate alignment with the actuator: the marking may further assist in viewing of the array by providing a suitable contrast.
  • the first variant has no cauling, although such may well be expected to be a part of the actuator, along with a system for controlling centricity of the force applied by the system.
  • the first variant has patterning of both membrane 12 and substrate 10. It is generally an advantage of the embodiment of FIG. 1A that no alignment of membrane 12 is required with respect to either the substrate or cover, and reasonably high tolerance alignment of the substrate and cover are permitted (especially if pneumatic chamber 25 is oversized relative to chamber 15), as this greatly facilitates assembly of the chip. As the elastomeric membrane 12 is compliant, perfect alignment of the membrane 12 at one corner, or even at all four corners, does not ensure feature alignment within the chip, which can be vexing compared with relatively stable alignment of rigid bodies like the cover and substrate.
  • thermoplastic or metallic substrate 10 may be provided with the high resolution relief structure, and the microfluidics may be patterned on the membrane 12 as shown in FIG. 1 B.
  • the array is provided on the substrate 10 without any platform or recess.
  • the ridge 14 is provided by patterning of the membrane 12, and while deformation of the region 12a may be preferred over deformation of the area surrounding the region 12a with some actuation schemes, there is some risk of partial collapse around the edges.
  • One preferred aspect of operation of the chip in accordance with the present invention is the provision of an annular, or partial annular channel 22 to preclude a closed volume of liquid resisting advance of the membrane 12. It will be noted that unless the actuation seals two edges, there is no enclosure. Furthermore, if the whole array is covered by the membrane prior to some sealed, liquid filled region being formed, there may be no problem with the advance of the membrane. In the first variant, to ensure the fluid has a conduit to port 16, for example, a width of the annular channel 22 is increased.
  • FIG. 1 B shows an enlarged view of the microwells 20 separated by walls 21 (only two identified to facilitate viewing).
  • the microwells 20 are shown having a well profile that has an aspect ratio of about 1 .2, however it will be appreciated that different aspect ratios can be used in various embodiments.
  • high IRV density is desired. If smaller volumes are acceptable, shallower microwells 20 can certainly be provided until the limits on accurate reproduction are met.
  • an aspect ratio below 0.2 may be provided with good regularity with some forming routes, but are generally not preferred because a substantially higher microwell density can be supplied with greater relief-depth, and good filling properties. If larger volumes are sought the depth can increase somewhat, for example to an aspect ratio of about 2. After this aspect ratio it is increasingly difficult to ensure filling of the microwell in a centrifugal environment, even with rounded and tapered profiles.
  • FIG. 1 C schematically illustrates a second variant.
  • the chip does not have a cover 24, and the array is on a level surface with the annular channel 22; and like the embodiment, the membrane 12 has a uniform thickness.
  • the channel 17 has different (greater in this case) depth than the chamber 15.
  • the membrane 12 may inherently have low affinity for substrate 10, which is helpful for avoiding excessive stiction during closure, especially if closure is to be maintained for extended periods. However, this tends to make stiction difficult to predict, and sometimes substantially higher than for other chips, depending on handling and exposure of the specific chip. This may particularly be required if favourable material selection identified hereinabove is not elected.
  • a bonding layer 26 is provided that seals readily against both the substrate 10 and membrane 12.
  • the second variant shows a mover 27, which may be a magnet held atop the chip.
  • the mover 27 is subject to centrifugal accelerations in operation, and therefore must be localized radially and azimuthally to align with the region 12a, but is preferably free to move axially, as well as to pitch and yaw about azimuthal and radial axes, at least to a limited extend, for example as mounted by an effective universal or effective spherical joint.
  • An electromagnet preferably on a centrifugal blade, although alternatively possible on the chip or a cartridge supporting the chip, controls a force exerted by the magnet on the region 12a.
  • the mover 27 may be infinitely stiff in comparison with the membrane, and will isolate bulk deformation of the membrane to a region overlying the annular channel 22. This commends a softer, more compliant, membrane, and may also suggest a requirement forthe mover 27 to reciprocate, to separate the membrane when required by a specific protocol.
  • FIG. 1 D schematically illustrates a third variant, which has the array defined on a chip insert 28 as opposed to the substrate 10.
  • This can be useful for the following reasons: if the patterning, dicing, and assembly of high finesse relief structures is more cost effective than patterning the whole substrate; if stiction between the membrane and substrate, even though the surface area of the array offers a small area, calls forthe array being formed in a second material; if the substrate is beneficially formed to receive a variety of inserts for a variety of processes; or if the substrate is surface functionalized or treated by separate providers. Typically to control stiction, one would not need both a cauling 23 and an insert 28.
  • the third variant also has a cauling 23, located between the region 12a and chamber 15.
  • the mechanical effect of the cauling 23 located on this side of the membrane 12 is largely similar, in that it changes local stiffness, and redistributes deformation of the membrane. However, the effects are more notable in terms of stiction.
  • the insert 28 and cauling 23 may be provided as one, with each protecting the other during sale and transport. By inserting the two together into the chamber 15, sealing membrane 12, and cover 24, pressurizing cavity 25 may press the insert into position, while pressing the cauling 23 against the region 12a.
  • FIG. 1 E schematically illustrates a fourth variant, which has a simplified cover 24 bearing no surface patterning.
  • the pneumatic cavity 25, and any channels communicating therewith, are patterned by relief of the membrane 12.
  • This also has an advantage of thinning the membrane 12 in the region 12a, which makes a more compliant separator that has lowered resistance to actuation of the membrane. This reduction in resistance can be useful, especially if the pneumatic cavity 25 has limited seal force that limits a rated pressure that can be supplied to cavity 25.
  • a channel 29 is shown for feeding the cavity 25.
  • FIG. 1 F schematically illustrates a fifth variant, which has a simplified assembly process, in certain respects.
  • the patterning of the entire chip, for both pneumatic control and microfluidics is provided by a single patterned membrane 12, which is patterned on both sides. Both cover 24 and a bottom cover 31 have no function but to seal against respective surfaces of the membrane12. No alignment is required, as long as the microfluidic network and pneumatic network are enclosed. All venting, all ports for loading or coupling to pressure supply lines, are provided along edges of the chip.
  • the array is provided by an insert/substrate 10 that fits within the chamber 15.
  • the cavity as shown has no spacer or features to provide a fitting of the insert/substrate 10 within the cavity.
  • the process may introduce liquid in the chamber 15 and this liquid may envelop the insert/substrate 10. Pressing of the region 12a against the insert/ substrate 10 will press it against the cover 31 , but this may not result in adhesion, sufficient to overcome the adhesion between the membrane (or cauling 23) and insert/substrate 10. If so, a process for fluid ically separating the microwells 20 is complete, but it may be irreversible.
  • choice of the cauling 23 can be made to ensure a low surface affinity for the insert/substrate 10, such that the low affinity of the substrate 10 for cover 31 dominates during a retraction of the region 12a.
  • spacers, and or features can be added to the insert/substrate 10 and/or cauling 23, the features being positioned away from the array, to provide a desired spacing.
  • Variant six is schematically illustrated as FIG. 1 G.
  • the sixth variant has an insert 28 bearing the array, similarly to the third variant, and has a mechanical interlock forthe insert.
  • the principal variation of this variant is that instead of a normally open membrane pose, the relaxed pose is closed. Any variant can be in a normally closed or normally open pose, especially if a cauling is provided to avoid stiction.
  • the insert itself is chosen to have low surface affinity for the membrane, and thus no cauling is required.
  • FIG. 2 is a set of cross-sectional images of a chip, generally according to the third variant, enlarged and centered on a zone including the array. Many elements are not identified in FIG. 2 to facilitate viewing of the chip, although the features are present.
  • the set 2A-D show principal steps in a process for fluidically separating microwells, and specifically retaining IRVs.
  • FIG. 2A specifically shows a seventh variant in which the microwells have an aspect ratio near a limit of 2:1 . Otherwise, FIG. 2A shows the chip in the same state as in FIG. 1 : prior to a liquid sample 30 entering into the chamber 15.
  • a liquid, sample, or liquid sample includes any complex mixture of solids, liquids, and gasses that flows as a liquid in microfluidics.
  • biofluids e.g. blood, milk, urine, saliva, tears, sweat, egg white or yolk, sperm
  • hydrocarbons e.g. blood, milk, urine, saliva, tears, sweat, egg white or yolk, sperm
  • wastewater, effluent, runoff e.g. water, water, effluent, runoff, and food and chemical products and byproducts.
  • biofluids e.g. blood, milk, urine, saliva, tears, sweat, egg white or yolk, sperm
  • hydrocarbons e.g. blood
  • FIG. 2B schematically shows the sample 30 in the chamber 15 pulled by a centrifugal field, and a meniscus showing different contact angles (wetting) for the sample 30 with respect to the membrane 12 vs the insert 28.
  • the region 12a is shown deflected underthe weight of the liquid sample 30, which is natural if the cavity 25 is open to ambient, or enclosed at a pressure about equal to that of the gaseous pressure in chamber 15, and the membrane 12 has sufficient compliance.
  • a volume of the sample 30 may be metered and is clearly more than sufficient to fill the IRVs.
  • FIG. 2C shows deflection of the region 12a once a pressure in cavity 25 overbears the elastic resilience of the membrane in region 12a, the centrifugal force and the surface tension on the sample 30.
  • a small deformation of the region 12a imparts a large volume movement against centrifugal force, and ensures a complete filling of the microwells 20, and displacement of ambient gas.
  • notional bubble entrapment does not include microscopic dissolved bubbles that the sample 30 may naturally contain, but rather refers to larger-scale bubbles formed by incomplete surface wetting.
  • centrifugal microfluidics essentially prevents entrapment of large scale gas bubbles in the filling of these microwells 20 as long as: sufficient centrifugal force is applied; the sample and the insert 28 have a reasonably high affinity (the sample may have a small volume ratio (e.g. 0.01-20 vol.%) additive (e.g. surfactant) to ensure this); and the shape and volume of the microwells 30 are suitably selected. Accordingly, the filling of the chamber 15 in FIG. 2B also prevents bubble entrapment, despite the fact that the centrifugation was not acted against by the deflecting membrane.
  • FIGs. 2B,C,D were chosen to suggest the progressive deformation by pressurization of the cavity 25, or logically by other actuation mechanism.
  • the membrane in the region 12a starts by deflecting into chamber 15 with a centre of the region 12a moving first, as the deflection conforms with a substantially minimal surface, or substantially uniform distribution of stress.
  • the sample 30 is forced to move around, and invariably moves against the centrifugal field within the chamber 15.
  • the progressive increase in pressure in the cavity 25 steadily increases the deflection until the centre makes contact with the insert 28.
  • the elevated platform on which the microwells 20 are disposed provides a natural limit to the contact region, and limits the amount of deformation required by the membrane, while providing greater assurance that the annular channel 22 remains open. However this is not necessary, as explained in relation to FIG. 1 B, an increased annular extent of the channel 22 is sufficient to avoid occlusion. In general, for a smaller footprint, placing the array on a raised platform is preferred. In general, to minimizing unused sample 30, and for better evacuation of sample 30 after or during this method of fluidic separation (explained further hereinbelow), an elevated platform is not preferred.
  • FIGs. 2A-D illustrate the natural deflection of a compliant membrane with some elasticity.
  • the membrane is expected to have a viscoelastic profile, and to exhibit a viscosity effect that deforms the membrane inelastically, as well as a resilient deformation.
  • the higherthe viscosity effect the more supple the membrane, and the less pressure is required to deform. Too supple a membrane, especially if affinity with the material on which the array is formed (insert or substrate) is too high, may lead to a requirement for suction to be applied by cavity 25 to effect release and retraction of the membrane.
  • FIG. 2C' illustrates a variant of the process if a cauling 23 is used.
  • the chip illustrated is that of FIG. 1A, but with a pneumatic channel 29 illustrated, the pneumatic channel 29 provided by a relief patterning of the cover 24. Only this step is illustrated as the differences in steps of FIGs. 2A,B,D are negligible.
  • the membrane 12a is deflected in this variant, in a somewhat different manner.
  • the cauling 23 tends to stiffen part of the region 12a, and as a result, to concentrate strain in the region 12a to a ring around the cauling 23.
  • the structure illustrated is a bit involved because of this deflection.
  • the cauling 23 is shown conforming somewhat as a result of a good adhesion to the membrane, under the pressurization of the cavity 25 relative to chamber 15.
  • 4 surfaces of the cauling 23 are in view: the vertically cross-hatched section showing where the cauling is sectioned; part of a top surface of the cauling, especially the top surface remote from the section line, and two curved side edges of the cauling that are partially in view.
  • a top (cavity facing) surface of the region 12a is identified, although the sectioned surface thereof occupies more surface of the page.
  • One advantage of use of the cauling 23 is to hasten the spread of the contact zone, and to make the time until completion more reliable. By concentrating strain to the ring, the strain is more extreme (at th is stage in the process) but the chamber-meeting surface of the region 12a is far more uniformly spaced from the array. Instead of an initial point of contact being somewhere near a middle of the array, and the contact line spreading, this variant allows for almost all of the array meeting the chamber-meeting surface at once. Even if one edge of the chamber-meeting surface meets first, the nearly planarized chamber-meeting surface will be resisted more substantially by the liquid, but brings more of the membrane into contact with the array at once than the progressive process of FIGs. 2A-D. As advance of a contact line may be inconvenient to monitor (although it may well be visible with video imaging), and as a cauling has substantial advantages provided by a low cost, easily assembled, insert, it use may be preferred.
  • the cauling provided on the chamber-meeting surface instead of, or in addition to, the cauling on the cavity-facing side of the region 12a affects deformation in the same manner.
  • the cauling 23, if cavity-facing offers a design option to tailor the surface affinity of the membrane to the array (substrate in this case, or insert), and can be selected for stiffness as well as a trade-off between ease of contact, and strength of contact with the substrate/insert, and ease of retraction of the membrane under negative or ambient pressure (if normally open and elastic response is enough to overbear stiction).
  • FIGs. 3A-C schematically illustrate enlarged cross-sections showing most of 4 microwells 20 in three states, in accordance with an iterative method for micro-aliquoting.
  • sample liquid properties chiefly hydro-philicity/ -phobicity
  • complete filling of each microwell 20 may not be obtained on a first contact of the sample with the membrane.
  • Most applications involve testing samples with at least something unknown in the composition, and while all candidate samples may have a narrow range of liquid properties, it may not be preferred to ensure bubble-free filling at first contact.
  • 3A shows two of the 3 filled microwells having bubbles 35 as may be produced either by the sample flowing through the chamber 15 from an axis proximal to axis distal end under centrifugation, or by forcing the liquid against centrifugal force, by actuation of the membrane (not in view). It will be appreciated that depending on how the liquid is introduced into the chamber 15 there might be a higher propensity towards air inclusion: specifically if the sample moves against centrifugation, inclusion may be discouraged. As the surface of the micro-wells 20 are hydrophilic, the liquid is encouraged to move around the previously occupying air. While not desiring to be limited by the following theory, a race condition appears to be set between branches of the liquid frontier as the frontier moves across the array.
  • FIG. 3B shows the effect of retraction of liquid from the microwells 20. If there is no bubble in the microwell, surface tension allows for much of the bulk sample to retract from the microwells under centrifugal force. This leaves the surface wetted, and where internal edges or surfaces have sufficiently small radii of curvature, films or webs 36 are locked into place against the pull of the sample 30.
  • the webs 36 are schematically shown of exaggerated dimensions that are the same for both previously bubble-filled and completely filled microwells. If there is a bubble 35 in a microwell during retraction, it offers a fragile thin connection within the body of liquid that can be spontaneously ruptured as the liquid is withdrawn and the bubble joins an air plug. The residual sample 30 in these webs 36 reshape the surface, dramatically changing the propensity for air bubble inclusion upon a second encounter with the sample.
  • FIG. 3C shows membrane 12 in a closed position against the array, providing a 3D fluidic separation of IRVs 39. No bubbles are present, as is consistent with a second, or subsequent contact with the sample 30.
  • pressing the membrane into the chamber 15 drives the sample 30 against centrifugal force to cover the array, and further pressing of the membrane allows for isolation of the IRVs 39 FIG. 2D.
  • the sample 30 is fed into the chamber 15 without contacting the array, or the part of the array as shown. If so FIG. 3C shows the first time the membrane meets the substrate 10. Alternatively it may be a second or later contact.
  • a process can be provided that supplies the sample, images the array (bubbles are readily visible if the liquid is coloured), or uses light diffraction to test index of refraction through microwells of the chip, to test for bubbles. If bubbles are detected, the process involves retracting the membrane under centrifugation, until the sample fill line extends axis distally of the array, and then pressing on the membrane again until the sample covers the array, and further until the membrane flu id ically isolates the IRVs. [0098] FIG.
  • 3C' schematically illustrates a variant where the sample or liquid first introduced into the chamber 15 is different from that which is subsequently delivered to the microwells.
  • Use cases can be provided for immiscible, selectively soluble, or miscible compositions.
  • the webs 36' are of a first composition
  • the sample 39 subsequently filling the microwells is of a different composition, depending on the miscibility of the first composition and the sample
  • the IRV composition may be dissolved, suspended, or homogenized at a given rate and diffusion may bring the liquid into a single phase, or an interface may be maintained between the two liquids.
  • the interface may be selectively broken by advective stimulus, such as a laser beam that selectively heats one of the two compositions.
  • the sample 39 is a mixture of a residual volume of the first composition and a second composition; the sample has markedly lower mass density than the first composition and a barrier interface wont to be formed with the residual volume, such that the sample overlies the residual volume; or the residual volume is microfluidically removed from the chamber 15 prior to introduction of the sample (e.g. as with chip of FIG. 6C).
  • FIGs. 4A-E schematically illustrate 5 depth profiles for the microwells 20.
  • Each microwell 20 of a given array may have a same profile, but preferably has a same volume (obviously up to limits of fabrication), and also preferably has a same planar surface area for easier readout.
  • Each of FIGs. 4 have sidewalls and a floor that may be rotationally symmetric at all angles of rotation about an axis thereof, or may have a number of angles of rotational symmetry. All feature an opening end 20a, and floor with a rounded bottom 20b, by which a centre axis defines a point of highest depth.
  • the opening end’s mean cross-sectional area (plane perpendicular to axis) and highest cross-sectional area are both greater than those of any other segment as the profile’s cross-sectional area generally monotonically decreases from the opening end 20a to the bottom. In general, it is easier to manufacture rounded smooth recesses that monotonically decrease in cross-sectional area with greater depth.
  • FIGs. 4A,B show profiles defined by two segments, the rounded bottom and a uniform sidewall
  • FIGs. 4D,E show three segment profiles
  • FIG. 4C shows a 4 segment profile.
  • the opening end 20a of the profile may be chosen to account for a planform (i.e. perpendicular to axis of the profile as shown) shape of the microwell 20, such as those illustrated in FIGs. 5. While a circular opening end geometry may be provided, with an optimal hexagonal packing as shown in FIG. 9 (right side), the present invention is not limited to such an in-plane geometry of the opening end.
  • FIG. 4A schematically illustrates a profile having two segments: an opening 20a, and bottom 20b.
  • the opening end 20a is defined by two facing walls that are essentially parallel in the section shown. This is consistent with a cylindrical opening end 20a, as well as many other geometries such as a triangular, square, rectangular, hexagonal or other polygonal form, or any rounding, or smoothing thereof, or such a polygonal form with arcuate webs replacing the line segments, although some vertical edges might be in view, depending on the specific planform shape of the microwell 20. In general, such edges would be rounded and provide no visible edge to avoid corners that may not be completely filled.
  • opening end 20a with vertical walls provides a highest volume forthe microwell 20, but provides a higher resistance to liquid entry than a chamfered, filleted, or beveled edge.
  • the surface affinity for the sample 30 is obviously important for ensuring that the microwell 20 is wetted, both by selection of the material of the substrate I insert, and by use of surfactants or contact-angle modifiers, but by providing a smooth, reasonably edge-free, microwells that can be densely packed, filling can be facilitated.
  • FIG. 4B shows a first profile variant of the embodiment of FIG. 4A in which the vertical side walls are canted from vertical.
  • An angle of the bevel is shown to be about 11 °, although the angle could be otherwise (e.g. 0-60°) with a selected trade-off between volume and ease of entry.
  • the volume of first profile variant without sacrificing density can be made equal only by increasing microwell 20 depth.
  • FIG. 4C schematically illustrates a second variant profile structured to reduce barrier to sample entry.
  • the second variant profile comprises side walls (two shown) of shallower cant (bevel angle ⁇ 8° (epitomized by linearsection 20c) with a flared opening end 20a having an angle ⁇ 23°.
  • the relatively steep angle of 23° does substantially reduce a volume otherwise attainable with the microwell, but it’s very small vertical extent reduces the effect.
  • a transition between the bottom 20b and segment 20c is naturally smooth as the bottom 20b has a curvature selected for this.
  • the only transition left to smooth for such a profile is the meeting with the wall at the top surface. This too can be filleted or otherwise rounded to avoid interface pinning that arises on sharp edges, as it can for all of the embodiments.
  • Such a shape may offer a good trade-off between volume and reduced barrier to entry.
  • FIG. 4D A third variant profile is schematically illustrated in FIG. 4D.
  • This profile includes vertically walled opening end 20a, and a ⁇ 15° canted section that smoothly meets bottom 20b. The transition between opening and mid sections is smoothed by a fine rounding.
  • FIG. 4E A fourth variant profile, schematically illustrated in FIG. 4E, has a vertically walled opening end 20a, and a rounded midsection. The rounding itself provides smooth transitions with the bottom 20b, and vertical opening end 20a.
  • FIGs. 5A-D illustrate 4 regular patterns for packing microwells 20 onto the array. Each pattern provides a respective planform shape for the microwell 20.
  • FIG. 5A shows a conventional hexagonal packing suitable for use with any of the profile embodiments or variants illustrated herein. In general there are several strategies for producing shapes with spherical, or ellipsoidal bottoms 20b, and arbitrary planform shapes.
  • each microwell 20 is indistinguishable from any other microwell of the array, except of course for a number of edge-adjacent or directly neighbouring microwells along edges of the array, as a volume, shape, and orientation of each microwell is identical.
  • FIG. 5B schematically illustrates a triangular packing pattern. While triangular packings are not particularly preferable, they are suited to some uses, and the figure is particularly useful for showing one strategy for reconciling the planform shape with an essentially hemispherical bottom.
  • the first concern with some low order polygonal planform such as triangular planforms, or any other irregular polygon having a vertex with an angle less than about 120°, is that they may trap an unpredictable volume of air, resulting in incomplete filling. These can generally be addressed by rounding the internal corners with webbing (shown in FIGs. 5C, D).
  • the microwells 20 of FIG. 5B can have a profile such as that of one of the profile variants, along one section of the side wall. For example, if microwell 20 were sectioned along a bisector of the equilateral triangle (along the axis passing through one of the vertices), the bisected edge would have a profile of the form of FIG. 4A, whereas the vertex could have the form of any of FIGs. 4B,C,D,E.
  • FIG. 5C schematically illustrates a pattern with trapezoidal planform, derived by a regular division of a triangular tessellation.
  • FIG. 5B already explained that a same microwell 20 may have different profile types at different locations. If the planform shape is elongated, there is space for curving the side walls in one direction, more than the other.
  • the trapezoidal planform illustrates a down-side of any polygonal planform with angles that meet acutely.
  • the webbing of the corners of the trapezoid are excellent features for guiding liquid into the microwell, as they can be curved in relief for good smoothness, but they do take up more of the surface area of the array than may be desired.
  • this design could be further optimized by curving the radial wye arms that now extend from a centre of the triangle, so that each bent arms reaches its edge of the triangle closer to 90°. This can greatly reduce the webbing of one of the two ends of each trapezoid, leaving only one preferred entrance for each microwell (the corner at the edge of the triangle).
  • An elongated well structure such as this, or as modified, maybe advantageous for trapping cells of a defined size in each microwell. This example also shows that the regular pattern may be non-vertex aligned.
  • FIGs. 5B,C illustrate microwells that are respectively planform triangular, and trapezoidal, which respectively have only one of two, or one of six orientations. As orientation has generally no effect on filling, one can consider all of the microwells of these arrays to be of a same type, except for adjacencies of microwells on the edges of the array.
  • FIG. 5D shows a two tile pattern with two distinct types of microwells 20.
  • FIG. 5D schematically illustrates a two tile pattern with 1 hexagon for every six triangles.
  • the pattern is a subdivision of a hexagonal pattern with the hexagon at a centre, whereby each hexagonal microwell 20' has a same orientation, and the triangular microwells 20" have one of six orientations.
  • the triangles were selected for having a same surface area as the hexagon prior to rounding (the hexagon tile size was chosen for an area 1 /7 th that of the hexagonal unit pattern). Patterns of two or more tiles can be efficient, for example, if it is desired to spatially constrain cells of one shape to microwells that are spaced apart.
  • a first use for the IRVs is provided by the previous embodiments: to isolate IRVs for example, to subject them to a thermal cycling and subsequent readout. This requires that all of the chemical additives required for PCR, or other processing, and dying or labelling provided in the sample: a very mature chemical process. This is not the only use case, however, and it may be desired to divide the sample into IRVs, and then subsequently process them, expose them to another reagent, or outgas the IRVs, for example.
  • FIGs. 6 schematically illustrate 3 modifications of the chamber 15 patterned into substrate 10 that permit removal of excess sample 30.
  • FIG. 6A is a schematic top plan view of a variant substrate 10, the variant featuring an overflow 32 for trapping excess sample 30.
  • FIGs. 6A,B both illustrate variants that allow for this trapping without any further valves, or pressure control ports, and that use the membrane displacement itself to effect the drainage.
  • the overflow 32 is provided by a bifurcation of channel 17, and is also vented by port 16, via a second channel that prevents a liquid plug of the sample from blocking the overflow 32.
  • FIG. 6A also shows the array shifted axis proximally relative to the chamber 15.
  • FIG. 2B Three lines h-h are illustrated to show fluid fill levels at three respective states in a process according to the present invention: a first state, h, corresponds with that of FIG. 2B; a second state, l 2 , corresponds with that of FIG. 2D; and the third state, l 3 , corresponds with a release of the membrane after the second state.
  • the fill levels are assigned by an assumed axis centred on the substrate 10 at a reasonable distance above the axis distal part of the substrate 10 illustrated, although it will be appreciated that a range of axis positions relative to this part of the substrate could be used. Assuming the sample enters chamber 15 under centrifugal force, through channel 18, the sample 30 will fill the chamberto an initial fill line h.
  • the cavity 25 is coupled to a suitable pressurized fluid supply, it is capable of both over pressure and under pressure relative to ambience. If so, a negative pressure in the cavity 25 (relative to ambience) can be used to expand the chamber 15 further, and may appreciably lower this fill line, for example to below a bottom edge of the array. Alternatively, the location of the array can be shifted axis proximally to achieve the same ends. This would ensure that microwells 20 are only filled at a time when the membrane forces the sample 30 to move against centrifugal force.
  • a fill level of the sample rises to a high-water mark l 2 .
  • the sample exceeds a branch threshold in the channel 17, and the sample begins to flow into the overflow 32.
  • the continued actuation of the membrane will continue at least until the contact area surrounds the array, which may occur before or after the high-water mark is met.
  • the annular channel 22 may be thinned, but is never occluded, and consequently the extraction of excess sample is not impaired.
  • the membrane is released, to return to a relaxed pose, with much of the sample in the IRVs and overflow 32.
  • a trace volume i.e.
  • a volume of the sample in the thinned annular channel pools at a bottom of the chamber 15 during the gradual release of the membrane, under the centrifugal field, thus before the contact line retracts to expose the first peripheral IRVs, the trace volume has pooled axis distally of the array. Once the membrane releases and top surfaces of the IRVs are open to ambience, the pooling of the trace volume has a fill level l 3 .
  • Variant substrate 10 shown in FIG. 6B has the array on a raised platform defined by limiting edge 13, which extends, by a bridging 33, to ridge 14, to provide a single occlusion of annular channel 22.
  • the bridging 33 is preferably webbed and arcuate to facilitate filling and avoid entrapment of air at nooks where it meets the annular channel 22.
  • the overflow 32 is coupled by a respective channel 34 to the chamber 15, which is still co-vented with the chamber at port 16.
  • This substrate 10 in use with layers to form a chip has a built-in overflow control, which prevents centrifugal filling from exceeding a fill line l 4 , although this would not be leveraged for precious sample applications, rather the sample is metered into the chip, and flows into the chamber via channel 18 with a low dead-volume path. From the arc of the fill line l 4 it should be clear that the axis for this chip is right of centre.
  • the sample in the chamber 15 comes to a fill volume (h not illustrated) that extends axis proximal of the bridging 33.
  • the design of the location of the bridging to balance a rate of advance of the membrane doesn’t apply too high a flow rate across a gap between the membrane and array that resists closure until a sufficient closure of the bridge is established, which thereafter forces the sample to move clockwise, azimuthally around the annular channel 22, and across the gap outside of the contact zone, to the outlet channel 34.
  • a position of the outlet channel 34 may also be lowered or raised, or provided with a fluid-dynamic restrictive element to retard flow to ensure complete filling of the mi crowe I Is.
  • FIG. 6C schematically illustrates an air-plug valved waste chamber 34 that precludes sample from entering throughout the process, even under pressure from the membrane, until an air vent of the waste chamber 34 is opened, which is only performed after the fluidic isolation of the IRVs is complete.
  • An air-plug valve is disclosed as FIG. 11 of Applicant’s US Patent 10,702,868, the contents of which are incorporated herein by reference (in both figures, port 16 may be pressure controlled to allow liquid to move axis distally, although use of vents to the axis distal chamber’s port can equally be used for this purpose).
  • a variety of valving structures can be used, including siphon valves, Quake style valves, and Venturi valves to block flow into waste chamber 34 until the fluidic isolation process is complete.
  • FIG. 7A schematically illustrates a chip 40 mounted to a pneumatically controlled blade 45 for rotation by a centrifuge.
  • the blade 45 controls pneumatic supply to chip 40 and has three pressure supply lines 42, coupled to respective pneumatic and fluidic ports of the chip 40.
  • Flow control elements 43 supplied in the pressure supply lines 42 may be: binary open/close valves; graduated valves with 3 or more states between wide open, and closed; or three way valves with open, closed, orvented states, forexample.
  • An electronic controller 44 controls each flow control element 43, and may be powered by an electronic slip ring, as it is for mounting to a centrifuge.
  • the flow control elements 43 control coupling of the ports of the chip 40 to a pressurized supply, such as a pressurized canister, a pump, or a channel to a pneumatic slip ring (also known as a rotary fluid coupling). Specifically by selectively coupling the pneumatic port to a pressurized supply, the membrane can be controlled to fluidically separate a sample into a multitude of IRVs.
  • a pressurized supply such as a pressurized canister, a pump, or a channel to a pneumatic slip ring (also known as a rotary fluid coupling).
  • FIG. 7B schematically illustrates a variant pneumatically controlled blade 45 having two controllers 44, one for each of two chips 40, although one chip is removed for better viewing of the provisioning of the blade 45.
  • the blade 45 has two chip receiving areas thereon.
  • the blade 45 also has, in a region underlying the array of microwells 20, a thermal control unit 46, and a sensor 47, which may be a thermocouple for example.
  • the sensor 47 provides feedback to the TCU 46 and is controlled by thermal controller 48, which may be a processor autonomous from the controller 44 or may share control over the process with controller 44 in any convenient manner, or may be a power regulation chip responsive to the electronics controller 44, in which case the sensor feedback goes to the controller 44.
  • thermal controller 48 which may be a processor autonomous from the controller 44 or may share control over the process with controller 44 in any convenient manner, or may be a power regulation chip responsive to the electronics controller 44, in which case the sensor feedback goes to the controller 44.
  • FIG. 7C schematically illustrates the blade 45 and two chips 40 according to FIG. 7B mounted on a centrifuge 50 for rotation about an axis 55.
  • the centrifuge 50 spins the blade 45, and the chips mounted thereto.
  • a cover 51 of the centrifuge is partially illustrated. It has a set of lights 53, such as strobing lights and a camera 52 for imaging the arrays on the chips 40. It should be noted that strobing lights with controlled timing are preferred for imaging from the stationary cover 51 , but this can be avoided if the camera 52 is mounted on the blade, or a cartridge of the chip, for example.
  • the electronic slip ring between the blade 45 and centrifuge 50 may allow for feedback between the camera and lights to provide feedback to the process, and control flow control elements 43 to change a pressurization of a cavity via the pneumatic port coupling it to a pressurized supply line 42.
  • the controller 44 can also direct a thermal treatment, and image the IRVs thereafter.
  • FIG. 8A,B illustrate a layout of a 5 layer microfluidic chip used to demonstrate the present invention, respectively in exploded and assembled form.
  • This chip is composed of a top cover 10a bearing no patterning except for an array of througholes for ports, an upper relief patterned layer 10b, an elastomeric membrane 12, a lower relief patterned middle layer 24b and bottom cover 24a.
  • the lower layer 24b and bottom cover 24a define a pneumatic control layer such as that provided by FIGs. 1A,D,F, and together the top cover 10a and upper layer l Ob provide a substrate patterned to produce a microfluidic layer bearing a relatively simple network(that together define).
  • Each layer has top and bottom sides, each of which (except for a top surface oftop layer 24a, and a bottom surface of bottom cover 10a) providing meeting surfaces forsealed assembly that permits pressurization of respective chambers, without undue leakage or rupture.
  • the chip has a plurality of ports 19 for providing fluidic access to the network of the chip (i.e. the chambers and the microchannels that interconnect them) once assembled.
  • all ports are provided on the top surface of the top layer 24a.
  • This is a common strategy that allows for the chip to require only a single surface to be accessible for venting and fluid control. This can be provided by placing the top surface down on a meeting surface of a cartridge with integrated fluid supply conduits, or onto a blade or chipholder of a centrifuge, for fluid-tight sealed coupling of the chip, after loading of the chip (injection of sample, reagents, and all other liquids) has been performed. Typically the chip is clipped, snapped, or pressed into position on the blade or chip mounting.
  • each layer has a same 8 port row 61 , the ports thereof are spaced uniformly along a minor axis edge of each layer (except the bottom 10a).
  • This minor axis edge is the axis proximal edge of the chip and each layer.
  • the alignment of these 8 ports is simplified by this position, and alignment of the layers is provided by alignment of these ports, even if there are further ports of the chip. Only 3 of the 8 ports are actively used in this design (19a, b,c).
  • each of these ports 19a-c is coupled to at most one of the relief patterned layers, and serves the chip as a sample loading port, vent and/or pressure control port, although in other instances these ports could serve as vias, interconnecting two or more relief patterned layers.
  • the cover 24a provides through-holes for reagent injection ports 62 to allow injection of reagent into the chip (these ports are closed after sample loading).
  • the upper layer 24b has a relief structure defining part of a pressurization cavity 25, some of a reagent supply 63 (directly coupled to the injection ports 62) and most of a channel 64 coupling port 19c with the cavity 25.
  • the membrane 12 is patterned with a through-hole to define a thin part of the reagent supply 63.
  • the membrane 12 further retains cauling 23 in alignment with the cavity 25 on the cavity-facing side thereof.
  • membrane 12 is unpatterned and unstructured.
  • the lower layer’s relief structure defines: an excess chamber 68; a chamber 15 defined as a through-hole; the reagent supply 63, also defined as a through-hole; and various channels defined in low relief (as opposed to through-holes) including: a second channel 65 interconnecting the first pressure inlet port 19a to an axis proximal end of the reagent supply 63; a third channel 66 interconnecting the reagent supply 63 (axis distally) and the chamber 15 (axis proximally); a fourth channel 67 interconnecting the chamber 15 (axis proximally) and the excess chamber 68 (axis distally), and a fifth channel 69 interconnecting the excess chamber 68 (axis proximally) and the second pressure inlet port 19b (which detours around the inlet port 19a).
  • Bottom cover 24a has only a fine patterned array of microwells 20.
  • the assembled chip 40 as seen in FIG. 8B, has a deep reagent supply 63 (contributed to by the through-bores of upper and lower layers 10b/24b as well as the smaller thickness of membrane 12), and the cavity 25 and chamber 15 aligned, but separated by membrane 12.
  • the array of microwells 20 is provided at a bottom surface of the chamber 15.
  • the appearance of intersection 71 of the channels 64,65 is illusory, as 64 is provided by upper layer 10b, whereas channel 65 is provided by lower layer 24b.
  • Each layer of the assembled chip 40 was fabricated by computer numerical control (CNC) machining and laser cutting, although other microfabrication methods can be used e.g. soft lithography, hot embossing, and injection molding.
  • Thermoplastic layers 10a, 10b, 24a, 24b were designed with 3D CAD software and patterned by CNC milling.
  • Elastomer layer 12 was prepared by cutting chambers with laser cutter. Bonding between thermoplastic layers was achieved with medical grade, double sided, adhesive and elastomer layer 12 was bonded to thermoplastic layers by thermal bonding by curing at 60°C overnight. During lamination, gentle positive pressure was applied from the top to ensure uniform bonding of adhesive across the whole chip.
  • the chip as manufactured and assembled was loaded with sample and reagent.
  • the sample and a reagent (PCR master mix containing Taq DNA polymerase, dNTPs, MgCI 2 and reaction buffers) were injected into the device through one of the reagent injection ports 62.
  • the reagent was stored in the reagent supply 63, and then reagent injection ports 62 are closed.
  • the chip was then mounted to a pneumatically controlled centrifugal system.
  • Each of ports 19a-c was coupled to a respective pressurized fluid supply line 42 as in FIG. 7B in accordance with teachings of WO 2015/132743.
  • the pneumatically controlled centrifugal system included a centrifuge (1-1000 rpm), a camera with LED lights for the imaging of chip during the spinning, heater meeting with bottom surface of chip and control the temperature inside, pneumatic ports to control the pressure inside of connected microfluidic chip.
  • the pneumatically controlled centrifugal system had a blade 45 for mounting one or two chips 40 at once, equipped a pressurized supply maintained by an on-board pump and a controller 44 for applying pneumatic pressure to the pressurized supply lines 42.
  • FIG. 8C is a photo of the assembled, mounted, chip.
  • the reagent was transferred to the chamber 15 via the third channel 66.
  • This chamber 15 has a same radial and azimuthal position as the cavity 25 but is physically separated by the elastomeric membrane 12.
  • the transferred reagent is held in the chamber 15.
  • Positive pressure is released at port 19a and the rotation speed can be adjusted to apply various centrifugal accelerations to achieve the filling wells of the membrane 12 by removing bubbles.
  • Positive pressure was then applied through the third pressure inlet port 19c to pressurize the first channel 64 and the cavity 25. As a result the membrane 12 deflects downwardly to cover the microwells 20 with the membrane 12. The micro-aliquoting was achieved, with discretization of the reagent.
  • the cauling 23 was found to assist the sealing by pressing the elastomeric membrane more uniformly. To avoid the excessive pressures on the reagent, the excess chamber 68 was provided between the inlet port 19c and the chamber 15 and this provides a space for the reagent to move into during the deflection of the membrane 12.
  • FIG. 9 is a panel showing microscopic imaging of the microwells 20.
  • Different chips were produced, that had, respectively 12,000 and 8,000 microwells 20. Both chips were based on hexagonal tilings, one having slightly rounded hexagonal planform shape, and the other had cylindrical planform (microwell size in hex tiling is 50 pm and diameter of cylindrical tiling was 75 pm).
  • a bottom most image shows the chip of hex tiling that was manual filled.
  • the surface of the membrane 12 was treated with a hydrophilic reagent to lower the contact angle and a surfactant (0.1wt% of Twin20) was added in the reagent to reduce surface tension, the majority of wells still have trapped bubbles.
  • a surfactant 0.1wt% of Twin20
  • the two middle drawings are uniform across the imaged surface, and offer little to see, as no bubbles are produced.
  • the sample was micro-aliquoted using the structure defined hereinabove, subject to the method described above. Each IRV is well metered and the sample was discretized. On the same materials were used as in the bottom panel, and indeed the same chip as used on the right-hand side (hydrophilic treated membrane and surfactant added sample reagent).
  • the proposed method involved centrifugation under 160 g-force and sealing after the filling by the pressurization of the elastomeric membrane 12 was implemented and the uniform filling without air-trapping could be achieved as shown.
  • the top panel image in comparison with the similarly cylindrical planform array from the middle, demonstrates the filling result at different gravitational acceleration.
  • the chips were mounted at different radial positions and the centrifuge was operated to apply a same rotation speed (1000 rpm).
  • the top represents the filling results at 95 g-force and middle shows a 168 g-force, respectively. Cleary 95 g’s were insufficient to achieve uniform filling across the entire array while 168 g’s were successful in filling the array’s microwells. Consistent with our calculations, the higher gravitational acceleration provides the higher Bo, resulting in better filling.
  • FIG 10 is a panel of images showing an example of digital PCR implementation on microwells.
  • Microwell array of 12,000 IRV was utilized for this digital PCR with the filing method proposed in this invention and various concentrations were tested ( ⁇ 1 copy/pL to ⁇ 1 ,000 copies/uL).
  • Bulk PCR master reaction mixture was found to wet microwells without bubbling issue and was submitted to thermal cycling between 65°C and 95°C. Amplified fluorescence signals at each well was observed if target nucleic acid exist in the well. Consistent with theory, a number of positive wells (fluorescent wells) were increased as target concentration was increased and target detection was possible at single copy level.
  • the panel illustrates the merits of arrays of microwells having at least a few different volumes (such as FIG. 5E). If a smaller loading of DNA or RNA is provided in a sample than expected, a very sparse number of well will light up, which could be difficult to distinguish from a failure of the reagent. Largerwells are suited to smaller loading, just as smaller wells are suited to larger loadings, as larger wells will saturate and generally over represent sample loads.

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Abstract

L'invention concerne une technique de micro-aliquotage centrifuge sur une puce microfluidique centrifuge comprenant une membrane disposée à l'opposé d'un réseau de micropuits, la membrane pouvant être actionnée pour enfermer efficacement des micropuits, déplaçant le liquide dans des micropuits adjacents, ou à l'opposé du réseau. Comme les effets du ménisque et les effets similaires peuvent être réduits à un minimum, un volume plus uniforme peut être aliquoté par rapport à des structures qui reposent sur une interface aérienne. Comme la membrane peut être actionnée pendant la centrifugation, par l'intermédiaire d'une lame pneumatique ou d'une bague collectrice, des aliquotes peuvent être isolées avant l'arrêt de la centrifugation. Comme le liquide est enfermé par la membrane et les micropuits, ils sont effectivement incapables d'être à l'origine d'une contamination croisée ou d'un échange de fluides, et des bulles entraînées peuvent être évitées.
PCT/IB2024/051767 2023-02-24 2024-02-23 Micro-aliquotage centrifuge Ceased WO2024176188A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050226779A1 (en) * 2003-09-19 2005-10-13 Oldham Mark F Vacuum assist for a microplate
US9546932B2 (en) * 2009-11-23 2017-01-17 Cyvek, Inc. Microfluidic assay operating system and methods of use
EP3269451A1 (fr) * 2016-04-11 2018-01-17 National Research Council of Canada Film à motifs pour former un blister rempli de fluide, blister microfluidique et kit et procédé de formation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050226779A1 (en) * 2003-09-19 2005-10-13 Oldham Mark F Vacuum assist for a microplate
US9546932B2 (en) * 2009-11-23 2017-01-17 Cyvek, Inc. Microfluidic assay operating system and methods of use
EP3269451A1 (fr) * 2016-04-11 2018-01-17 National Research Council of Canada Film à motifs pour former un blister rempli de fluide, blister microfluidique et kit et procédé de formation

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