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US-12618104-B2 - Microfluidic chip, kit, and system for displacing independent reaction volumes of an emulsion

US12618104B2US 12618104 B2US12618104 B2US 12618104B2US-12618104-B2

Abstract

A centrifugal microfluidic technique for heat treating emulsion-divided independent reaction volumes (IRVs) within a centrifugal microfluidic chip, and displacing the emulsion into a monolayer presentation chamber (pc) for imaging. A deep treatment chamber (tc) is provided for the heat treatment, a nozzle having a hydrodynamic radius for forming the IRVs is provided for injecting a sample for the IRVs into the tc filled with a dense immiscible medium. The tc is adjacent a heat controlled element for collectively heat treating the IRVs within the tc, where the IRVs form a 3d packing arrangement. The tc is coupled to a presentation chamber (pc) by an opening through which the IRVs can be selectively displaced without collapsing. The pc is adjacent a window transparent to a wavelength for inspecting the pc.

Inventors

  • Liviu Clime
  • Lidija Malic
  • Teodor Veres

Assignees

  • NATIONAL RESEARCH COUNCIL OF CANADA

Dates

Publication Date
20260505
Application Date
20220414

Claims (20)

  1. 1 . A centrifugal microfluidic chip for mounting to a centrifuge for rotation about an axis, the chip comprising a network of chambers and interconnecting channels including: a treatment chamber (tc) having: a volume (v tc ) of 10-800 μL; and a mean length (l tc ), mean width (w tc ) and mean depth (d tc ), with d tc <l tc , and d tc <w tc ; a first path adapted to deliver a sample to the tc, the first path comprising a nozzle having a hydrodynamic radius r n of 2-120 μm, adapted to deliver the sample to the tc discretized, as emulsion-divided independent reaction volumes (IRVs) if the nozzle is filled with a suitable medium; a presentation chamber (pc) coupled to the tc by an opening through which the IRVs can be selectively displaced from the tc to the pc; a window transparent for inspecting the pc, provided through the chip at least across a length and width of the pc; and a retraction chamber (rc) coupled by a second path to the pc, wherein: d tc is between 5×r n and 2 mm; the pc has a depth (d pc ) between 1.2×r n to 7×r n ; d tc >2 d pc ; and a flow path through the opening has a minimum hydrodynamic radius greater than 8×r n .
  2. 2 . The chip of claim 1 wherein the pc has: a volume (v pc ) of 0.6 to 1.2 v tc ; and a footprint between 3 cm 2 and 80% of a footprint of the chip.
  3. 3 . The chip of claim 1 wherein the network of chambers and channels are produced in relief on at least a first side of a first film having a nominal thickness between 20 μm and 5 mm, and the chip comprises a covering film that covers the first side to enclose the chambers and channels, away from ports of the chip.
  4. 4 . The chip of claim 1 wherein d pc is nominally maintained despite a pressure relative to ambient by an array of supporting microstructures.
  5. 5 . The chip of claim 4 wherein the nozzle is located at an entry of the first path to the tc.
  6. 6 . The chip of claim 1 wherein the entry of the first path to the tc branches to further provide one or more additional instances of the nozzle.
  7. 7 . The chip of claim 1 wherein the opening between tc and pc is wider than long.
  8. 8 . The chip of claim 1 wherein a floor of the opening comprises a ramp with a depth varying from d tc to d pc .
  9. 9 . The chip of claim 8 wherein the ramp has a slope of 30° to 75°.
  10. 10 . The chip of claim 1 comprising a stack of two or more films, at least one of the films having a relief structure defining the network, wherein: each film has a nominal thickness between 20 μm and 3 mm; each film is composed of a cured or set polymeric compound other than a siloxane; the chip has a thickness of 0.1 to 12 mm; the chip has planar extents of 3 to 25 cm; or the chip has at least 2 ports.
  11. 11 . The chip of claim 1 wherein the first or second path comprises a metering chamber with an overflow chamber, for volume-controlled delivery.
  12. 12 . The chip of claim 10 loaded with one or more of: a sample-ready reaction mix, in dried or liquid form in a first chamber within the first path; a sample in dried or liquid form, in a second chamber within the first path; a buffer, solvent, or liquid for dissolving, or suspending the sample or reaction mix within the first path; a dispersion medium adapted to support IRCs loaded within the tc, pc, or rc.
  13. 13 . The chip of claim 12 further comprising a low density medium chamber coupled to the tc by a third path that meets the tc at an axis-proximal end of the tc, the low density medium chamber loaded with a liquid having a density lower than that of the sample, the buffer, solvent or liquid, and dispersion medium.
  14. 14 . The chip of claim 1 encased in a cartridge, the cartridge having a rigid structure for ease of manipulation and registration, while providing access to ports of the chip, and avoiding occlusion of vents of the chip.
  15. 15 . A kit comprising the chip of claim 1 and a chip controller, mounted, or for mounting to, the centrifuge, for rotation of both the chip and controller, the chip controller comprising an off-chip flow control device for selectively displacing fluid in the pc into the rc.
  16. 16 . The kit of claim 15 wherein the flow control device comprises a pressurized fluid supply line for coupling to a port of the chip.
  17. 17 . The kit of claim 15 wherein the chip controller, or a cartridge of the kit for encasing and rigidifying the chip, comprises a chip holding surface dimensioned to support the chip on one side thereof, the chip holding surface comprising an energy device for selectively exposing the treatment chamber of the chip to an energy field.
  18. 18 . The kit of claim 17 wherein the energy field is a thermal energy source or sink, an ultrasonic transducer, or an electromagnetic field generator, in contact with chip adjacent the treatment chamber.
  19. 19 . The kit of claim 15 further comprising a lighting and imaging system for imaging the pc, during centrifugation, or when the chip is at rest.
  20. 20 . The kit of claim 15 assembled to form a centrifugal microfluidic system.

Description

FIELD OF THE INVENTION The present invention relates in general to treating and handling microfluidic emulsions of regular-sized independent reaction volumes (IRVs), and in particular to a technique for discretizing, processing and presenting in a monolayer, the IRVs without collapsing the IRVs, on a low-cost microfluidic chip. BACKGROUND OF THE INVENTION The Covid-19 pandemic has underscored a need for low-cost, available, fast sample to answer systems for genetic testing and quantification, and this, inter alia, is addressed by the present invention. An emulsion is a useful, elegant and efficient solution to analyzing complex samples. Dividing an already small sample volume into 103-105 (and potentially 106 or more) isolated, independent reaction volumes (IRVs), allows for simplified reactions with fewer confounding reactions, and a simplified readout process for assessing constituents of the subdivided sample. Isolated by thin webs of an oil or other immiscible fluid, each emulsion-divided IRV can be treated, at the same time, in a same microfluidic chamber. There are currently several applications leveraging IRVs. For example, nucleic acid amplification assays such as digital droplet PCR (ddPCR), and isothermal digital droplet amplification (e.g. ddLAMP) entail emulsifying PCR or isothermal amplification master mix (which contains primers and probes as well as amplification buffer and appropriate polymerase) as well as sample (i.e. nucleic acid template) which is partitioned into thousands of, for example, nanoliter-scale, IRVs, followed by thermal processing (thermal cycling in the case of ddPCR, or single temperature application in the case of isothermal amplification) and readout (for example, fluorescence or colorimetric). Unfortunately, the means for forming and manipulating regular sized IRVs are rather technologically involved, requiring expensive, large-footprint, equipment which limits IRV-based technologies. Commercial systems (such as QX 100™ and QX 200™ from Biorad™) or custom research grade devices, can implement complete workflows but typically require manual steps to (1) generate the droplets, (2) transfer the emulsified droplets to PCR tubes in order to perform thermal cycling, and (3) transfer the droplets again from PCR tubes to imaging chambers or droplet readout channels (Malic et al., 2019). These manual steps are laborious and may be prone to pipetting errors which can compromise emulsion integrity and assay repeatability. Furthermore, it must be noted that sample volumes on these (large) scales further limit use of the technologies. As with all service equipment, there is a trade-off between capabilities to perform a very limited number of protocols, with complete automation, vs. being able to perform a wider number of protocols with limited automation. To provide high levels of automation, the protocol needs to be a sufficiently routine and sufficiently high throughput process to warrant the investment. For example QX200 ddPCR System™ by BioRAD apparently requires a droplet generator that is 28×36×13 (cm), and a reader that is 66×52×29 (cm), as well as a C1000 Touch Thermal Cycler™ with a particular well reaction module, as well as a PX1 PCR Plate Sealer™. Thus the loading is manual or automated and the well plate needs to be shuffled around between these 4 devices to complete a process. Sophisticated instruments with embedded robotic systems, such as QX One Droplet Digital PCR System™ from Biorad are capable of automating the entire workflow, however the systems takes significant lab footprint (122×66×38 (cm)) and costs several hundreds of thousands of dollars. As such, it is beyond the reach of some research laboratories. The system performs sample to answer capabilities on microliter samples. Another commercial platform, the Naica System™ from Stilla Technologies™, automates emulsification and thermal cycling, however it requires a separate imaging instrument (Madic et al., 2016). The Naica System uses pressurized chambers within a fluid-dynamic network to direct movement of fluids. This requires sophisticated instrumentation and pressure control systems, inducing further complexity in instrument and device design, and adding to costs of equipment and maintenance. There are limited ways that pressure control systems can be integrated into centrifugal microfluidics to achieve similar effect in the state of the art. This system is based on a single prescribed droplet size, which limits customization. Furthermore, the system, like BioRAD's, uses 20 ml sample vessels and is essentially macro-fluidic. A few centrifugal microfluidic systems have been developed to integrate droplet generation with thermal processing and post-PCR imaging for ddPCR and ddLAMP assays (Schuler et al., 2016a and 2016b, Li et al., 2020, Hu et al, 2019). Centrifugal microfluidic systems miniaturize assays and reduce reagent consumption and thus the overall cost of the assay, and permit integration of other