Microfluidic chip, kit, and system for displacing independent reaction volumes of emulsion

Abstract

To provide a microfluidic chip for presenting reaction volumes (IRVs) in discretization, processing, and a monolayer without collapsing the IRVs.SOLUTION: A deep treatment chamber (tc) is provided for heat treating emulsion-divided independent reaction volumes (IRVs) within a centrifugal microfluidic chip, and 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 to 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 to a window transparent to a wavelength for inspecting the pc.SELECTED DRAWING: None

Description

[Technical Field]

[0001]

[0001] The present invention generally relates to the processing and handling of microfluidic emulsions of regularly sized independent reaction volumes (IRVs), and more particularly to techniques for discretizing, processing, and presenting IRVs in a monolayer on a low-cost microfluidic chip without causing the IRVs to collapse. [Background technology]

[0002]

[0002] The Covid-19 pandemic has highlighted the need for low-cost, readily available, and rapid sample-to-answer systems for genetic testing and quantification, which is addressed, among other things, by the present invention.

[0003]

[0003] Emulsions are a useful, sophisticated, and efficient solution for analyzing complex samples. 3 ~10 5 (and possibly 10 6 By dividing the emulsion into isolated, independent reaction volumes (IRVs) (as described above), a simplified reaction with fewer confounding reactions and a simplified readout process for evaluating the components of the subdivided sample become possible. Isolated by a thin web of oil or other immiscible fluid, each emulsion-divided IRV can be processed simultaneously in the same microfluidic chamber.

[0004]

[0004] Currently, there are several applications that utilize IRVs. For example, nucleic acid amplification assays such as digital droplet PCR (ddPCR) and isothermal digital droplet amplification (e.g., ddLAMP) inevitably involve emulsified PCR, or isothermal amplification master mix (containing primers and probes, as well as amplification buffer and appropriate polymerase), and a sample (i.e., template nucleic acid) which is then distributed into thousands, e.g., nanoliter scale IRVs, followed by heat treatment (thermal cycling in the case of ddPCR, or single-temperature application in the case of isothermal amplification) and readout (e.g., fluorescence or colorimetric analysis).

[0005]

[0005] Unfortunately, the means for forming and manipulating regularly sized IRVs are technically quite complex and require expensive, large-scale equipment that limits IRV-based technologies. Commercial systems (such as the QX100 and QX200 from Biorad®) or custom-made research-grade devices can perform the complete workflow, but typically require manual steps: (1) generating droplets, (2) transferring the emulsified droplets to PCR tubes for thermal cycling, and (3) further transferring the droplets from the PCR tubes to the imaging chamber or droplet readout channel (Malic et al., 2019). These manual steps are laborious and can be prone to pipetting errors that can impair emulsion integrity and assay reproducibility. Furthermore, it should be noted that these (large) sample volumes further limit the use of the technology.

[0006]

[0006] As with all service equipment, there is a trade-off between the ability to run a very limited number of protocols with full automation and the ability to run more protocols with limited automation. In order to provide a high level of automation, the protocols need to be steady enough and have sufficiently high throughput to be worth the investment.

[0007]

[0007] For example, the BioRAD QX200 ddPCR System (trademark) clearly requires a 28 × 36 × 13 (cm) droplet generator, a 66 × 52 × 29 (cm) reader, a C1000 Touch Thermal Cycler (trademark) with a specific well reaction module, and a PX1 PCR Plate Sealer (trademark). Loading is manual or automatic, and the well plates need to be shuffled among these four devices to complete the process.

[0008]

[0008] Sophisticated instruments incorporating robotic systems, such as Biorad's QX One Droplet Digital PCR System (trademark), can automate the entire workflow, but the system requires a considerable amount of laboratory space (122 x 66 x 38 cm) and costs hundreds of thousands of dollars. Therefore, it is out of reach for some laboratories. The system exhibits sample-reaction capability for microliter samples.

[0009]

[0009] Another commercially available platform, the Naica System (trademark) from Stilla Technologies (trademark), automates emulsification and thermal cycling but requires separate imaging equipment (Madic et al., 2016). The Naica System manages fluid movement using pressurized chambers within a hydrodynamic network. This requires sophisticated instrumentation and pressure control systems, further complicating the design of the equipment and devices and increasing installation and maintenance costs. There are limited ways in which a pressure control system can be integrated into centrifugal microfluidics and achieve similar effects with modern technology. This system is based on a single, defined droplet size, which limits customization. Furthermore, the system uses a 20 ml sample container, similar to BioRAD's, and is essentially macrofluidics.

[0010]

[0010] Several centrifugal microfluidic systems have been developed to integrate droplet generation with heat treatment 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 allow for the compacting of assays, reduction of reagent consumption and therefore the overall cost of the assay, and the integration of different microfluidic functions for processing samples before emulsification, during processing, or after imaging. All of these extend capabilities on closed process lines, such as those presented by pump-based fluid systems. Disposable centrifugal microfluidic tips can be designed for a variety of processes, integrating smaller volumes of reagents and presenting a wider range of protocols. A reusable chip controller technology (referred to herein as pneumatic or "P-" blade), such as that claimed in International Publication No. 2015 / 132743, whose entire content is incorporated herein by reference, can provide an addressable pressurized fluid supply to one or more ports of a chip, further extending the processing capacity of the chip while keeping all contaminated material confined to the chip. Unlike dedicated machines that can perform only one protocol, centrifugal microfluidic devices are versatile devices that can be used to provide a very large number of different protocols depending on the chip structure, load contents, and several peripheral devices (e.g., those for imaging and thermal control).

[0011]

[0011] There are several microfluidic strategies for generating IRVs, including those demonstrated in several centrifugal microfluidic chips (Clime et al., 2020; Schuler et al., 2015). While centrifuges are adapted to naturally form IRVs, maintaining natural formation during many processes depends on the fragility of the oil web separating the IRVs and the pressure on the droplets. For example, as noted by Schuler 2016, the gas solubility of a liquid decreases with increasing temperature, and therefore the amount of gas released during sample heating to 95°C can be at least 18 v / v%. Gas bubbles in a centrifugal environment can disrupt the oil web and mix the IRVs.

[0012]

[0012] Automating the complete protocol, including droplet generation, heat treatment, and monolayer formation, remains challenging in microfluidic environments. This is primarily due to the difficulty in controlling the stability of IRVs during heat treatment and the need for IRV monolayer formation for imaging. While this may not be a problem for isothermal amplification at lower temperatures (e.g., 37°C for RPA and possibly 65°C for LAMP), tightly packed IRVs in a monolayer (required for droplet imaging) typically cannot withstand heat treatment at high temperatures (e.g., 95°C) required for PCR thermal cycling assays without IRV mixing, inducing assay errors and reducing sampling uniformity and efficiency.

[0013]

[0013] Furthermore, most devices described in the literature are manufactured using PDMS (siloxane). While PDMS is commonly used in academic research and has good transparency and biocompatibility, this material is unsuitable for large-scale manufacturing and is rarely used in industry (e.g., pharmaceutical and clinical research), with biocompatible rigid thermoplastics such as polystyrene (PS) and cyclic olefin copolymers (COC) being preferred. PDMS can adsorb proteins and small molecules, biasing the final assay results, and its gas permeability can lead to sample evaporation over time, which is particularly problematic during thermal cycling.

[0014]

[0014] The centrifugal cartridge described by Schuler et al. allows for complete workflow integration on a single platform, but the device has a single chamber for droplet generation, thermal cycling, and IRV readout, which requires the fabrication of a complex pyramidal structure with an inclined angle formed in the chamber floor to allow for the formation of the IRV monolayer required for imaging and bubble removal. This increases the complexity of device fabrication and further provides only a limited surface area for monolayer formation. Only 500 IRVs were read out in the imaging chamber, an order of magnitude lower than the minimum required for most assays.

[0015]

[0015] Therefore, there is a need for centrifugal microfluidic chips, devices, and systems that are particularly compact, easy for operators to use, robust and accurate, and chips manufactured from materials compatible with mass production techniques such as thermoplastics and thermoplastic elastomers for generating, processing, and imaging IRVs. [Overview of the project]

[0016]

[0016] The applicant has devised a complete protocol sample-reaction relating to a low-occupancy, low-power centrifugal microfluidic system which may be fabricated at low cost and may even be fabricated in a portable form. The system may have an occupancy area of ​​less than 30 × 30 × 30 (cm) and may operate within a range of microfluidic sample volumes that can be reliably supplied and is suitable for more complete automation. The system essentially consists of a general-purpose centrifugal microfluidic device with a suitably supplied and loaded tip. The general-purpose centrifugal microfluidic device may be used for several alternative purposes, in particular if it has a pressure supply port controlled so that the centrifugal microfluidic device becomes a P-blade platform, or if it has at least a pneumatic slip ring or rotary coupling for pressurized fluid transfer for stator base control to pressure supplied at one or more ports of the tip.

[0017]

[0017] The suitably provided chip has a network of chambers and interconnecting channels, including a processing chamber (tc), a presentation chamber (pc), a sample path for delivering a sample to the tc, and a draw-in path connecting a draw-in chamber (rc) to the pc. The network is provided by relief patterning of the (at least one) substrate on at least one side of the substrate and sealing the relief-patterned surface with another substrate or a single cover. The chip has a window transparent to the inspection wavelength to enable imaging of the pc over the length and width of the pc, preferably without passing through several layers of the substrate and the cover, either through the cover or the substrate. The window may be substantially the entire cover or substrate, indistinguishable and unmarked, effectively enabling imaging of any part of the network within the field of view.

[0018]

[0018] The suitably provided chip has a nozzle in the sample path for generating IRV from a sample and an immiscible fluid, which may be one or more nozzles at the inlet of the tc, a relatively deep tc enabling processing of IRV while in a 3D lattice or packing that is substantially more robust than 2D packing, a shallow pc for single molecule layer imaging, and a low-resistance aperture between the pc and the tc for transporting the IRV intact. Specifically, the nozzle is adapted to deliver to the tc a sample discretized as emulsion-split IRV surrounded by an immiscible fluid and having a hydrodynamic radius r of 2 to 120 μm [[ID=,7]] n having.

[0019]

[0019] The tc has an average length (l tc ) defined by a straight line that intersects the tc at its maximum extension from the intended axis of rotation of the chip, an average width (w tc ) in the plane of the chip perpendicular to l tc , and an average depth (d tc ) in the relief direction. The pc and the aperture similarly have l pc , w pc , dpc ,l o , w o d o As shown by the art, d tc <l tc d tc <w tc d pc <l pc d pc <w pc , and d o <w o The volume of tc (v tc ) can typically be 10-800 μL depending on the application, but a smaller regular radius (r IRV Further reductions are expected as nozzle precision improves to form IRVs having ). To ensure that the emulsion can withstand processing, d tc is 5 × r n It is ~2 mm. To present a monolayer, d pc is 1.2 × r n ~7×r n And more preferably, 1.6 × r n ~6×r n and d tc >2d pc And more preferably, d tc >3d pc or d tc >5d pc Therefore, d pc is 1.5 × r IRV ~3×r IRV And more preferably, 1.6 × r IRV ~2.8×r IRV , or 1.8 × r IRV ~2.5×r IRV It is possible.

[0020]

[0020] The opening is 8 × r nIt has a larger minimum hydrodynamic radius and a smooth surrounding wall that can be planar or gradually curved. Generally, the closer the surrounding wall is to a smooth cylindrical wall, the closer it is to the minimized perimeter relative to the cross-sectional area, resulting in less resistance to flow through the opening and the smallest shear stress experienced by the IRV. It has been found that a flat and smooth wall of the slit is sufficient.

[0021]

[0021] The pc may have a large occupied area of ​​at least 1 × 3 (cm) to image, for example, at least 1000 IRVs (at least 5000 are required in some assays, and at least 10-12K are required in other assays), and may have a volume of 0.6 to 2 times the volume of the tc so that the entire emulsion can be imaged at once. Alternatively, the pc may have a smaller occupied area, enabling continuous imaging of IRVs, saving space on the chip, and eliminating the need to transport delicate IRVs over longer distances. The pc is aligned with a window for imaging the IRV aggregate.

[0022]

[0022] All channels and chambers other than the PC may have the same depth, which is convenient for chip design and manufacturing and allows for efficient use of the chip's occupied area. pc Because the occupancy area can be sufficiently small relative to the chip's occupancy area, the film and cover may not be rigid enough to ensure a perfectly constant working depth across the expected range of pressure differences between the external environment of the chip and the inside of the chip. It is well known to reinforce the chip with an array of pillars inside the chip and / or external structures. The need for this reinforcement is, d pc This is reduced by increasing the pressure difference between the PC and the surroundings, and by reducing the area occupied by the PC. None of these may be suitable for reduction if it is desired that all IRVs be imaged within the PC at once. In such cases, pillars or other support structures can be used. pc It is possible to support a pc that guarantees that the parameters are maintained within the given range.

[0023]

[0023] Alternatively, the pc can provide imaging of a small percentage of IRVs at any given moment, but rather the IRVs are arranged in a matrix across the pc. If so, augmentation may be avoided and more extreme aspect ratios can be provided, such as a length of less than 1 / 5 of the width (in radius from the axial direction). Measuring the color variation of multiple consecutive photographs presents some problems with the accuracy of the count if the timing between photographs is too long relative to the transverse speed, but the movement of the IRVs is slow enough to allow inexpensive, low-shutter-speed imaging with substantial overlap of images, making it possible to capture and register multiple images of each IRV. Furthermore, much lower frame rate captures may be used for statistical purposes or for inspection recording. Image analysis of consecutive photographs can also provide a more accurate assessment across incomplete or intentionally varied spatiotemporal illumination patterns, for example, the color state of some IRVs may show better contrast in some illumination bands, and the color state of other IRVs may show better contrast in other bands.

[0024]

[0024] w pc By increasing this, more IRVs are captured per frame, and the minimum l pc This is determined by the imaging method. If 80% of IRVs are imaged only once and 10% overlap is perfectly sufficient for identifying multiple imaged IRVs, then 10% could be the entire bottom row of 10 rows of pc, in which case l pc is 20 × r IRV This is possible. By providing a gradual or stepwise decrease in depth across the opening, and optionally an increase in width depending on the distance from tc, shear strain on the emulsion can be reduced, taking into account the required difference in depth between tc and pc, and the degree of IRV reconstruction required to conform to the form of a monolayer for presentation.

[0025]

[0025] tc dimension, especially d tcIn this configuration, if completely surrounded by a suitable medium such as heavy oil (i.e., denser than the sample), the chip may be able to undergo thermal cycling up to a temperature of at least 99°C without observing the collapse of the IRV. The system is preferably designed to bring the chip into close contact with a heater or other energy transfer device (e.g., ultrasonic, acoustic, electromagnetic, or thermal), preferably providing close contact between the device and a cover or substrate that partially forms tc.

[0026]

[0026] Centrifugal microfluidic systems enable sample-reaction testing, such as genetic testing of emulsion-divided independent reaction volumes (IRVs). Most prior art references refer to IRVs as "droplets," a term that can seem vague, lacking in teleological character and ambiguity, given that the entire sample can be the volume of a droplet. Specifically, since it has been observed that IRVs disintegrate when IRV assemblies are moved within a microfluidic chamber, the present invention solves the problem of having to avoid handling IRVs, which typically tend to result in IRV fusion, which is essential for many assays, or having to present monolayer IRVpcs with a very limited surface area.

[0027]

[0027] The applicant has made many attempts to move IRV assemblies that have failed. The present invention solves this problem by moving IRV assemblies from a deep tc to a monolayer depth pc without disintegrating the IRVs, even if they are large assemblies, under centrifugal force. As a result, centrifugal microfluidic systems, particularly pneumatically assisted centrifugal microfluidic systems such as the centrifugal microfluidic system described in the P-Blade patent, can be utilized for the production, processing, and presentation of IRVs.

[0028]

[0028] The applicant has found that the emulsion can be moved with virtually no loss due to a suitable interface between tc and pc, and a sufficiently low pressure applied to the corresponding port of the tip to drive the movement. An unprecedented number of IRVs have been imaged in a centrifugal microfluidic system in which the system becomes automatic once the sample is loaded onto the tip and the tip is coupled to the tip controller. A suitable opening between pc and tc allows movement when a suitably controlled pressure is applied.

[0029]

[0029] One of the advantages of using a P-blade platform over non-pneumatically assisted (i.e., traditional) centrifugal microfluidics is that the dimensions of the IRV can be precisely controlled by varying both the centrifugal speed and the applied pressure, as described by the applicant in the paper entitled "Bouyancy-driven step emulsification on pneumatic centrifugal microfluidic platform" (Lab on Chip 2020, 20, 3091, June 22, 2020). Specifically, the paper shows a chip configuration in which the IRV diameter can be varied by at least 50 μm with centrifugal speed variations of 300–700 rpm and applied pressures of 0–40 kPa above ambient. The entire contents of the paper and all supplementary disclosures thereof are incorporated herein by reference.

[0030]

[0030] Therefore, a centrifugal microfluidic chip to be attached to a centrifuge to rotate about an axis, comprising a network of chambers and interconnecting channels, the network having a volume of 10 to 800 μL (v tc ), as well as average length (l tc ), average width (w tc ), and mean depth (d tc ) has, d tc <l tc , and d tc <w tcThe system comprises a processing chamber (tc) and a first path for delivering a sample to the tc, wherein the first path has a hydrodynamic radius r of 2 to 120 μm. n A first path comprising a nozzle having a nozzle, adapted to deliver a discretized sample as emulsion-partitioned independent reaction volumes (IRVs) to a tc when the nozzle is filled with a suitable medium; a presentation chamber (pc) coupled to the tc by an opening, which allows for the selective movement of IRVs from the tc to the pc through the opening; a wavelength-transparent window for inspecting the pc, provided through the tip over at least the length and width of the pc; and a retraction chamber (rc) coupled to the pc by a second path, d tc However, 5×r n It is ~2mm, and the PC is 1.2×r n ~7×r n thickness d pc It has, d tc >2d pc The flow path passing through the opening is 8 × r n A centrifugal microfluidic chip having a smaller minimum hydrodynamic radius is provided.

[0031]

[0031] PC is 3cm 2 ~This represents 80% of the chip's surface area, 0.6~1.2V tc Volume (v pc ) may have. The network of chambers and channels may be formed in relief on at least a first surface of a first film having a nominal thickness of 20 μm to 5 mm, and the chip may have a cover film covering the first surface to seal the chambers and channels away from the chip ports. Despite the pressure inside the pc compared to the ambient pressure, d pc To ensure that it is nominally maintained, an array of supporting microstructures may be included.

[0032]

[0032] The nozzle may be located at the entrance of the first path to tc. The first path may branch to provide one or more additional entities of the nozzle.

[0033]

[0033] The opening between tc and pc is preferably wider than it is longer. The opening has a depth of d tc from d pc The slope that changes up to, or the intermediate depth d tc from d pc It may include one or more steps. The inclined section may have a slope of 30° to 75°.

[0034]

[0034] The chip may comprise a stack of two or more films, at least one of which has a relief structure forming a network, each film having a nominal thickness of 20 μm to 3 mm, each film being composed of a cured or solidified polymer compound other than siloxane, the chip having a thickness of 0.1 to 12 mm, the planar extension of the chip being 3 to 25 cm, or the chip having at least two ports.

[0035]

[0035] The first or second path may include a metering chamber having an overflow chamber for volume-controlled dispensing.

[0036]

[0036] The tip may be a tip that forms a microfluidic system by being loaded with one or more of the following: a sample preparation reaction mixture, such as a PCR mixture, in dry or liquid form in a first chamber in a first pathway; a sample, in dry or liquid form in a second chamber in a first pathway; a buffer, solvent, or liquid for dissolving or suspending the sample or reaction mixture in the first pathway; or a dispersion medium adapted to support an IRC loaded in tc, pc, or dc. The system may further comprise a low-density medium in the chamber of the tip, the low-density medium chamber being coupled to tc by a third pathway that merges with tc at the axial proximal end of tc, and the low-density medium chamber being loaded with a liquid having a density lower than the density of the sample, buffer, solvent, or liquid and the dispersion medium.

[0037]

[0037] The chip may be housed in a cartridge, the cartridge having a rigid structure to facilitate operation and alignment while providing access to the chip's ports and avoiding obstruction of the chip's vents.

[0038]

[0038] The chip may be a chip equipped with a chip controller and mounted on or mounted on a centrifuge, provided in a kit for rotating both the chip and the controller, wherein the chip controller is equipped with an off-chip flow control device for selectively moving fluid in the pc to the dc. The flow control device may be equipped with a pressurized fluid supply line for coupling to a port on the chip. The chip controller may be a chip controller equipped with a chip holding surface dimensioned to support the chip on one side of the chip controller, wherein the chip holding surface is equipped with an energy device for selectively exposing the processing chamber of the chip to an energy field. The energy field may be a thermal energy source or sink powered by an ultrasonic transducer or electromagnetic field generator adjacent to the processing chamber and in contact with the chip. The kit may further be equipped with an illumination and imaging system for imaging the pc during centrifugation or when the chip is stationary. The kit may be assembled to form a centrifugal microfluidic system.

[0039]

[0039] A method is also provided for simultaneously processing emulsion-divided reaction volumes (IRVs) to present the processed IRVs in a monolayer, the method comprising: providing the IRVs into a processing chamber (tc) of a centrifugal microfluidic chip; energizing the IRVs in the tc by activating an energy device attached to the chip on a centrifuge while the chip is being centrifuged, wherein the tc has a minimum size that accommodates 3 to 100 IRVs; and operating a flow control device to arrange the IRVs in a substantial monolayer, moving the IRVs from the tc to a presentation chamber (pc) through an opening, wherein the opening has a width greater than the average diameter of the IRVs and the pc has a depth, and the flow control device applies a pressure difference of less than 15 kPa.

[0040]

[0040] Further features of the present invention will be described or become apparent in the course of the following detailed description.

[0041]

[0041] To make the present invention easier to understand, embodiments of the present invention will be described in detail here by reference to the accompanying drawings. [Brief explanation of the drawing]

[0042] [Figure 1] This is a schematic top view of a patterned substrate for a centrifugal microfluidic chip according to a first embodiment of the present invention. [Figure 1A] Figure 1 is a partial cross-sectional view of the tip, focusing on the wall, which has a cover and generally provides an exit from TC to PC, and the nozzle. [Figure 1B] Figure 1 is a strip illustrating six steps in a process for forming IRVs, processing the formed IRVs, and then moving the processed IRVs into tc, using the chip shown. [Figure 2] This is a schematic top view of a patterned substrate for a chip having a smaller pc, according to a second embodiment of the present invention. [Figure 2A]This is a partial cross-sectional view of the tip in Figure 2, showing the transition in the depth of the opening between tc and pc. [Figure 2B] This strip shows an enlarged view of Figure 2, illustrating the three steps in the process of forming, processing, and presenting IRVs. [Figure 3] This strip shows, in 12 steps, how IRVs are formed and separated from the nozzle in order to form IRVs. [Figure 4] This is a schematic top view of a patterned substrate for a chip according to a third embodiment of the present invention, which includes an additional chamber for providing a complete ddPCR protocol. [Figure 5] This is a schematic diagram of a system comprising a chip controller mounted on top of a fourth chip embodiment. [Figure 5A] This is a magnified view of the chip. [Figure 5B] This is a magnified view of the arrangement of nozzles merging into the TC and the arrangement of support microstructures within the PC. [Figure 6] This is a photograph of the chip used to demonstrate the present invention. [Figure 7] Figure 7 is a photograph of the presentation chamber taken during centrifugation, and Figure 7A is a magnified view of a small area of ​​the image. [Figure 8] This is a bar graph showing the IRV diameter distribution of an aggregate, created using an example prepared to demonstrate the present invention. [Modes for carrying out the invention]

[0043]

[0054] This specification describes centrifugal microfluidic technologies (including tips, cartridges, kits, systems, and methods) that enable sample-reaction testing, such as genetic testing of emulsion-partitioned independent reaction volumes (IRVs). Specifically, tips, tips loaded with fluid for forming microfluidic systems, cartridges supporting tips and facilitating mounting to centrifuges or tip controllers, kits comprising tips and tip controllers, and kits assembled to form centrifugal microfluidic devices address the problem of how to move the IRV sequence from a processing chamber (tc) having the necessary depth to ensure that the IRVs are in a 3D arrangement that is far more resilient to heat treatment, into a presentation chamber (pc) with a monolayer depth for imaging, where this movement induces less or substantially less destruction of the IRV partitioning.

[0044]

[0055] Centrifugal microfluidic systems, particularly pneumatically assisted centrifugal microfluidic systems, such as the conventional rotary coupler / pneumatic slip ring described in the aforementioned International Publication No. 2015 / 132743, which allows pressure control at one or more ports of the tip during centrifugation, can supply a controlled pressure and centrifugal field that cooperate to allow the movement of the dispersion medium and IRV from tc to pc, with excess dispersion medium flowing into the retraction chamber (rc).

[0045]

[0056] Figure 1 is a schematic diagram of a patterned film 10 of a chip, illustrating a simplified embodiment of the present invention. The chip is formed of the film 10 and a suitable cover, not shown, to allow visualization of a microfluidic network formed in a relief pattern on the surface of the film 10. The relief pattern provides a network that enables the method of the present invention and even a little more. More complex protocols require more chambers and channels, which may require multiple interconnected films. To accommodate more complex protocols, it is known to provide a chip as a stack of layers having vias for interconnecting the layers. Alternatively, similarly, the film 10 can be understood as a single layer of a multilayer chip having, for example, a sample port 12a which is considered a via from another layer of the chip from which a sample is prepared.

[0046]

[0057] Typically, the chamber is vented, meaning there is fluid in each of the vents 12 coupled to the chamber. In particular, if the chamber is designed so that each liquid is loaded into the chamber at the start of the protocol, the vents may also function as loading ports. As the terminology is commonly used, “port” is any opening to the environment on the tip, while vent, via, loading port, or pressure control / pressure supply port specifies the intended function of the port. A vent can raise (move axially proximal) or lower (move axially distal) the fluid level within the chamber without trapping the fluid (compressing or expanding a fixed volume of air plug). A via communicates with a through-hole to another tip layer. A loading port is a port that allows the introduction of liquid into the chamber (there are several processes for off-tip loading during centrifugation, as described in the aforementioned International Publication 2015 / 132743, but often this is performed while the tip is stationary). The pressure control port is a port coupled to a pressure supply source, which may be coupled to the tip via a slip ring (fluid-coupled rotary joint), or, if not supplied via a slip ring, to a pressure supply chamber or pressurized canister of the tip controller, which may be pumped (attached). In some cases, the pressurized fluid supply source can be ambient pressure, and before the valve is opened, the chamber may be in a positive or negative pressure state, for example, considering an air plug in the network, and when the valve is opened, this pressure can be released. Similarly, closing an opened valve can establish a positive or negative pressure as a result of centrifugal separation. In principle, liquids may also be used, but typically, pressure is supplied via gas.

[0047]

[0058] According to the present invention, the chip comprises a processing chamber (tc) 15, a presentation chamber (pc) 20, and a retraction chamber (rc) 25, which do not necessarily have to be arranged as shown in the figure. The chambers are interconnected by channels, and by paths comprising one or more channels and optionally further chambers. Specifically, a delivery path is provided for delivering a sample to the tc 15, and a retraction path is provided between the pc 20 and the rc 25, through which fluid can be supplied into and withdrawn from the pc 20.

[0048]

[0059] The discharge path shown in Figure 1 includes a sample port 12a that supplies a sample to a sample chamber 14 having a discharge path to an overflow chamber 13 vented by port 12c. The sample port 12a is wider than the other ports to facilitate sample loading. For example, it is logical for the tip to have a smaller loading port for mechanically loading reagents, for example, for alignment and automatic injection (which may be performed at vents 12b or 12d to the medium), and a larger loading port for manually supplying samples or other fluids injected by the user, in a controlled environment.

[0049]

[0060] By providing a discharge channel in the sample chamber 14, the sample volume can be introduced in excess of the desired volume without accurately pre-quantifying the sample volume. When centrifugation is applied, the excess volume will overflow the sample chamber 14, and the remainder in the sample chamber 14 will be efficiently weighed with a certain degree of accuracy.

[0050]

[0061] The sample chamber 14 is coupled to the processing chamber 15 for sample delivery via a J-shaped channel 16. In some embodiments, although not shown in this embodiment except for a pressure control port 12a, it is preferable to provide a flow control device for stopping or starting delivery and / or controlling the delivery rate of the sample. The J-channel 16 merges with tc 15 at nozzle 18, but in principle, nozzle 18 may be provided at other locations in the delivery path. For example, the applicant's U.S. Patent No. 10,836,918 and the cited prior art documents teach a microfluidic structure for inline production of encapsulated droplets. It is possible to generate IRVs further up the delivery path using inline production, and in alternative embodiments, different IRVs may be subjected to different processing before entering tc 15.

[0051]

[0062] The nozzle 18 has a hydrodynamic radius r, which is the only most definitive feature of the tip's intended IRV size. n It has the average radius (r) of the IRV for typical centrifugal speed and pressure. IRV ) is 1 to 5 times r n And more typically, about 1.1–3.5r n , or 1.2~2.8r n Therefore, since IRV is not part of the chip itself (although it may be part of the system), n This is used to characterize the patent claims for the chip.

[0052]

[0063] Therefore, the nozzle 18 is adapted to feed the sample into tc 15 by splitting the sample into IRV when tc is partially filled with a denser, immiscible fluid such as oil. tc is aerated (12b) and provides volume for accommodating the emulsion of IRV. Optimally, the medium fills tc by the volume metered by the sample chamber 14 minus the volume metered by the sample chamber 14, and there may be a filling line on the substrate or cover to indicate this.

[0053]

[0064] The depth of tc(d tc ) is preferably at least five times the average radius (r IRV ) of the IRV for which the chip is designed, but in principle it is 3.5 times smaller than r IRV , and still it can function to distribute the IRV in the 3D packing lattice. pc has a depth (d IRV ) of 1.3 - 3.2r pc , more preferably 1.6 - 2.5r IRV , or 1.8 - 2.3r IRV . As understood with respect to r IRV rather than r n , d pc is 1.3 - 15r n as the outer - outer range, and 2.16 - 8.05r n as the most preferred range, but all intermediate ranges within all ranges herein are intended. r IRV can be in the range of 30 - 150 μm, more typically 40 - 110 μm, and currently typically 50 - 100 μm in order to promote a monolayer arrangement, so d pc is from 48 - 450 μm to 95 - 230 μm, including all intermediate ranges intended herein. With the progress of technology, smaller IRVs are expected, and smaller r n and d pc are required.

[0054]

[0065] The opening 22 between tc and pc is a slit corresponding to the change in depth between d tc with the inclined portion 23 and d pc , which is best understood by referring to FIGS. 1 and 1A together.

[0055]

[0066] FIG. 1A shows a partial cross - sectional view taken from the line AA of a chip including the film 10 and the cover layer 11. This figure focuses on the axial - distal wall of tc and shows both the nozzle 18 at the upper edge of tc and the change in depth of the opening 22 extending between tc15 and pc20. Specifically, from a depth of about 0.3d tc to dpc This shows a smooth inclined section 23 that extends to [the specified point]. pc Approximately 1 / 4d tc As indicated, for most applications, 1 / 5 to 1 / 10 d tc The ratio of r may be more preferable. IRV The smaller d pc It becomes smaller, but d tc It does not necessarily need to be small. The inclined part is 0.3d as shown. tc It will be understood that it may start lower or higher than and, furthermore, if the inclined section is not easily formed, one or preferably more steps may be provided. The density of IRV at different depths may depend on the distance from the opening 22 into tc. If the cover faces upward, and the medium is sufficiently dense, the density of IRV at the opening 22 will be due to gravity (0.3d tc The inclined portion 23 may be lower than the lower edge. The applicant found that the inclined portion is not necessary for good operation.

[0056]

[0067] From Figure 1, it can be seen that the run of the inclined section 23 is approximately 15% of the opening width at tc 15, and the opening widens from tc to pc at an angle of approximately 38°. Thus, the opening expands continuously in one direction and narrows in the other. Partial compensation for the high degree of narrowing in the depth direction by the expansion in the width direction reduces the shear stress on the IRV as it moves across the opening 22. The substantial reorganization of the IRV necessary to adopt the presented monolayer from the 3D packing can be achieved by the design of the opening, with appropriate control over the centrifugal speed and the application of a nominal pressure difference at the tip port. It is an unexpected result that the IRV can pass through this gate intact while being biased forward by the pneumatic supply applied by port 12.

[0057]

[0068] The intake path includes a channel 19 connecting the axial distal ends of rc25 and pc20, and a pressurized port 12d that allows controlled fluid movement between pc and rc. Port 12d is shown as an edge port, which may be advantageous in tip assembly and mounting for sealing coupling to the pressure supply port. For example, if all ports are edge ports, manual coupling of various ports to various supply lines can be provided at once by a suitable sealing structure with some tension, with minimal equipment and appropriate sealing.

[0058]

[0069] Film 10 can be relief patterned by any of the methods known in the art, including injection molding, thermoforming, 3D microprinting, and micromachining. Each of these forming routes has economic advantages and disadvantages that depend mainly on the production volume, but some are particularly suitable for producing channels and chambers with relatively constant depth floors, while some require special effort to vary the depth within a single chamber. Thus, the pyramidal features required on the floor of the chamber, as described by Schuler, impose some limitations on forming, which can result in wasted chips at the required production scale. All methods offer a variety of chambers with varying depths.

[0059]

[0070] Figure 1B is a strip of six planar frames of film 10, showing the sequential steps in the IRV generation, processing, and presentation process. The upper left frame shows the sample in the sample chamber 14 (with a small amount overflowing into the overflow chamber 13) and the heavy medium loaded into the draw chamber 25. The sample is preferably a biological fluid, or an aqueous carrier of air, water, food, or soil samples (or, for example, a filtrate, retention solution, lysate, fraction, or other purification of the processed sample or fluid) and a buffer, an all-in-one PCR (or similar) reaction mixture, and a colorimetric analysis target or functionalized particle that will enable ddPCR. This frame corresponds to the chip imaged after the first spin, after loading and mounting into the centrifuge.

[0060]

[0071] The second frame (upper right) shows the heavy medium being supplied into tc and filling pc when the heavy medium is under continuous centrifugation, provided that port 12d is not blocked. Port 12d can be coupled to an circumferentially open valve, as shown in Figure 12 of the P-Blade patent, to control the timing of the heavy medium supply, if timing is desired. Otherwise, the continuous supply process may begin with centrifugation when the sample takes its position in the first frame. Centrifugation will naturally draw the sample through the J-shaped channel 16 at a given rate and the high-density medium at another rate, but the competition between these two fluids for the nozzle 18 is not significant because the hydrodynamic resistance in the J-shaped channel 16 and nozzle 18 will prevent the sample from entering tc 15, whether empty or full, until a pressure difference is applied to 12b at ports 12a,c, to overcome the hydrodynamic resistance in the supply path. As a result, an air plug is present between the heavy medium and the sample, and before the sample is delivered through the nozzle 18, it is released through the oil as bubbles, as shown in the third frame (center left).

[0061]

[0072] The third frame shows the formation of the first IRV30 and its buoyant rise through the heavy medium. The heavy medium has high surface tension and a suitable contact angle for encapsulating the sample. The IRV rises and forms foam by flotation in tc15. IRV formation continues in the fourth frame (center right) until tc is substantially filled or the sample chamber 14 is sufficiently empty. It is undesirable to introduce any bubbles into tc15 after the IRV has formed, and therefore it is preferable to allow visual (or machine vision) inspection of the sample chamber 14 for manual or automatic feedback-based control of the duration of the pressure difference applied between ports 12a and 12b, in which case a second window may be provided for camera-based imaging of the sample chamber 14. Alternatively, and more efficiently, once the sample chamber 14 is metered, the programmed control to the pressure supply can be calibrated to supply volume for a provided time, allowing the risk of excessive release into tc15 to be sufficiently low. d tc This ensures that the IRVs form a three-dimensional packing within the tc, which provides a thicker set of heavy medium webs that more reliably separates adjacent IRVs than two-dimensional packing.

[0062]

[0073] The supply of heavy medium in tc15 should also be ensured. The heavy medium shown in the second frame is more than sufficient for initial IRV formation, but as the IRV rises due to buoyancy, the additional mass of the sample pushes down the heavy medium, raising the heavy medium level in the draw chamber 25. If the heavy medium level falls below the level of the nozzle 18, the IRV may continue to rise, but the heavy medium can only thin the web of all IRVs until some of the IRVs in the most depleted zones fuse together. Therefore, in use, the pre-loaded amount of heavy medium first fills pc, and tc is filled sufficiently so that the filling level does not fall below the nozzle 18. Another option is to monitor and control the heavy medium by, for example, visualizing tc15 and selectively applying positive pressure at port 12d to apply a gentle push that lifts the heavy medium before it flows to the axial distal side of the nozzle 18 before the IRV collapses, and stopping the pressure at port 12a when tc is full. If there are large fluctuations in the volume ratio of IRV to encapsulating fluid, monitoring may be preferable. This monitoring may be provided online with image analysis and feedback, or it may be provided manually by a human operator.

[0063]

[0074] Therefore, between frame 4 and frame 5, the sample chamber 14 is emptied, all IRVs are formed, and the assembly of IRVs is subjected to heat treatment. For this purpose, the blade to which the chip (or cartridge supporting the chip) is attached is preferably provided with a temperature control unit such as a Peltier heater / cooler, or a heat-capturing conductive surface in close proximity to the tc 15. The heat-capturing conductive surface is preferably in close proximity to the tc and may have a built-in temperature sensor, but is preferably attached to the blade or the chip controller of the blade and powered by the blade or the chip controller, as opposed to being part of the film 10 or the chip, or permanently fixed, thereby making the chip a lower-cost disposable type. However, the film 10 or cover 11 may be selected for a slight improvement in thermal conductivity. The purpose of the heat-capturing conductive surface is to responsively heat the tc, for example, via an on-blade laser, LED, or light-emitting bulb heat source, or a strobe-fixed laser, LED, or light-emitting bulb heat source, intermittently aligned with the tc.

[0064]

[0075] Once the heat treatment is complete, the heavy medium is retracted, and the IRV30, accompanied by buoyancy, is provided. The fifth frame indicates that this process has begun, and by the sixth frame, the retraction is complete, the retraction chamber 25 is full, and a monolayer of IRV is presented to the pc20. The tip controller or centrifuge may, of course, have a camera for imaging the pc, which may be mounted on the blades in some cases, which may require special optics and illumination, or which may be provided by strobe light and a camera inside the centrifuge, or further provided to the tip after centrifugation. If imaging is performed away from the centrifuge or after centrifugation, the contents of the tip may shift between the stationary state of the contents of the tip during centrifugation and when the tip is stationary, in particular if a chamber without vents is allowed to deploy a pressurized air plug while in operation. In extreme cases, this shift may break the IRV boundary. Controlled deceleration of the tip (with or without a pressurized air plug) may affect the movement of the IRV to the pc without damage.

[0065]

[0076] Most aspects of the microfluidic network of the chip, including the shape, orientation, and dimensions of the chamber, the layout of the channels and ports, and the material or composition of the layers (i.e., the film 10 and the cover 11), are not essential. Hereinafter, variations of the first embodiment are provided to illustrate the features of several variations that may be modified in particular ways. These variations are understood to be substantially independent of each other in that any combination of features can be assembled in each embodiment of the present invention.

[0066]

[0077] Figure 2 shows a layout of the substrate 10 with changes in key respects, where pc is a flow-through chamber for video graphics imaging or continuous imaging of the IRV, and a light medium chamber is provided in tc to replenish the IRV and reduce gas release during heat treatment. Figure 2 further shows the heat treatment area 28 and the window area 26 from which pc 20 can be imaged in ghost view. There are also minor changes to the chip layout, for example, with ports 12a and 12c joined together.

[0067]

[0078] The light media chamber 30 is preferred for IRVs that are prone to volatilization during heat treatment, such as aqueous samples. Capping light media such as oil have been shown to be useful in preventing gas release that would otherwise affect the volume of IRVs, the dissolution of sample components, and impair the reaction.

[0068]

[0079] The flow-through pc20 has a somewhat symmetrical form in that both the opening 22 and the outlet have symmetrical flares. As can be seen from Figures 2 and 2A, the inclined section 23 has a slope of about 20° in the first modified form, which is much gentler. tc and d pc Since the modified form is comparable to the embodiment, this is achieved by providing a run of inclined section 23 that is approximately four times longer. The flow through the opening 22 is expected to impart lower shear stress to the IRV 30 in the modified form, and the transition from the 3D packing of tc to the monolayer of pc is gradually provided by the inclined section 23 of this lower slope. The inclined section 23 terminates at the beginning of the window region 26 and at the beginning of pc 20. Although not required, a flare at the pc outlet leading to the retraction channel 19 may reduce shear forces in abrupt changes in the flow direction. Any collapse of the IRV before entering the retraction chamber 25 is substantially preferable to avoid, in which case the aggregated aqueous IRV contents may rise buoyantly through the heavy medium without interacting with any IRV in the pc. For this purpose, the channel 19 may be further modified to provide a smoother transition to the heavy medium carrying the IRV.

[0069]

[0080] Figure 2A shows a marked partial cross-section of a tip consisting of a film 10 and a cover 11, as shown in Figure 2. Figure 2B is a strip illustrating three steps of the process according to the present invention, where each frame of the three steps shows a magnified view of the tip in Figure 2A, with the first frame (top) showing the tc, opening, and pc filled with heavy medium, the second frame (middle) showing the IRV beginning to enter the pc, and the third frame (bottom) showing the IRV in a matriculation. To operate the tip, the tip is mounted on a platform and set to spin. The sample and heavy medium are moved and enter the J channel 16, respectively, and fill the pc to the filling level in the tc (top frame). When complete, the heavy medium covers the nozzle in the tc, and the sample is transferred to the sample weighing chamber by applying a positive pressure high enough to overcome the siphon valve connecting the supply chamber 14 and the tc 15 at port 15a (15c is closed or co-pressurized). Once the sample is transferred, the emulsification process begins when nozzle 18 introduces the sample into tc as individual IRVs, which is further illustrated and described below with reference to Figure 3. Thus, the IRVs are produced in tc. Once the IRVs are produced, the light medium from chamber 30 is transferred to tc by applying positive pressure at port 12e. The light medium rises to the top of the chamber (because it is less dense than the sample). Heat treatment is then initiated by applying a set temperature (or temperature cycle sequence) using a controlled heater. As the cartridge spins during heat treatment, the emulsion is trapped between the heavy oil and light oil, thereby preventing evaporation and maintaining IRV stability. After the heat treatment is complete, without stopping centrifugation, the IRVs are transferred into the shallow pc 20 by applying negative pressure at the port of the draw-in chamber 25. The central frame shows this initiation, and the lower frame shows random moments of the IRV procession across the window. Thus, the first frame in Figure 2B corresponds to one of frames 2-4 in Figure 1.The second frame shows how, once a medium of sufficient density is drawn into the retraction chamber 25, a portion of the IRV is drawn into the opening 22 and passes through the opening 22. The IRV is then made to march in a matrix across the pc. The movement is slow, and this process is expected to take some time, as this facilitates imaging and the prevention of IRV collapse. Unlike the embodiment, the variant allows for the accumulation of IRV within the retraction chamber 25, or other storage or collection of IRV contents.

[0070]

[0081] Figure 3 shows a strip illustrating IRV formation in nozzle 18. Note that each of the 12 frames is shown laterally, with the lower edge of the frame corresponding to the axial distal wall of tc 15 and the right edge corresponding to the surface of cover 11. In each successive frame, a larger volume of sample is injected into the heavy medium, increasing buoyancy and pulling it upward (i.e., away from the centrifugal field) as it is drawn in. By frame 9, it can be said that an IRV has formed but is still tethered to the nozzle by a string of sample. Buoyancy moves the IRV further and further away from the nozzle, drawing more sample into tc, but in the 12th frame the tether breaks, and the string of sample springs back into a spherical shape favorable to free surface energy, sowing the next IRV.

[0071]

[0082] Figures 1 and 2 illustrate two embodiments of the present invention that have no features other than those directly cooperating in the IRV formation and presentation process, but it will be understood by those skilled in the art that many chips preferably perform multiple steps of sample preparation and processing to generate a sample for testing. The chips of Figures 1 and 2 are suitable for any application requiring IRV generation, thermal manipulation, and subsequent monolayer formation for imaging, and include ddPCR, ddLAMP, ddRPA, or single-cell based assays in which a single cell is encapsulated with a specific reagent, cultured for a specified period, and then imaged. To control up to one cell per IRV, the simplest method is to dilute the sample to a suitable cell concentration that provides up to one cell per IRV as predicted by the Poisson distribution, for a given sample and IRV volume. For example, about 15% of the IRV may contain one cell, while the other IRVs will contain only aqueous buffer and lysed or supported particles and seeds. As those skilled in the art will understand, flow focusing and other techniques can be provided to control or position cells within a channel at specific intervals before they reach a nozzle that forms droplets.

[0072]

[0083] Figure 4 schematically shows a film 10 that is patterned and provides a complete sample tip for assays such as nucleic acid extraction from various clinically relevant biofluids including blood, saliva, and urine. In addition to providing all the chambers and channels necessary for IRV generation, heat treatment, and presentation, an additional chamber is provided for nucleic acid isolation from biofluids. When assembled for intended use, the sample chamber 14 is pre-loaded with dry reagents, and the following chambers are loaded as follows: heavy medium in the draw-in chamber 25; light medium in the chamber 30; binding buffer in the chamber 36; washing solution in the chamber 37; and elution buffer in the chamber 38. The binding buffer, washing solution, and elution buffer chambers 36-38 are coupled to a mixing chamber 31 containing a bead bed, the beads functionalized for nucleic acid extraction. The mixing chamber 31 is provided for sample dissolution. Furthermore, there is an off-tip sample vial 40 coupled to the mixing chamber 31 by piping. The mixing chamber 31 is also in fluid communication with the sample chamber 14 and the off-tip waste reservoir 39, as taught in the applicant's concurrently pending international publication No. 2020 / 100039.

[0073]

[0084] The sample is transferred to the mixing chamber 31, and the binding buffer is also transferred to the chamber 31 by applying positive pressure in the chamber 36 through a separate port in the chamber 36. The binding buffer dissolves the sample and extracts nucleic acids from the sample. Functionalized beads (preferably porous, high-surface-area beads such as silica) capture the nucleic acids, preferably with the assistance of temperature control provided by a Peltier device. The liquid contents of the chamber 31 (unbound lysate) are then drained by applying positive pressure at the port of the mixing chamber 31 for a period of time long enough to prime the channel to the off-tip waste 39, but not long enough to prime the channel to the sample chamber 14. The beads are retained while the unbound lysate is drained, as the diameter of the beads is larger than the diameter of the opening of the mixing chamber to the off-tip waste 39. The chamber 31 is then washed at least once by moving a washing solution into the chamber 31 and draining the chamber 31 again. The template nucleic acid is eluted from the beads by transferring the elution buffer into the chamber 31. This may include heating the mixing chamber 31 to promote nucleic acid release. The eluted template is then transferred to the sample chamber 14 by applying negative pressure to ports 12a, b, and d with sufficient pressure and duration to overcome, for example, the siphon valve connection chambers 31 and 14. A channel with a siphon valve, a J-channel with a nozzle 18 in the sample chamber 14, and tc15 form the delivery path in this embodiment. At this point, the elution buffer supporting the nucleic acid fills the sample chamber 14 to hydrate and dissolve the dry premixture. If useful, pressure can be applied to ports 12a, b, and d to induce bubble mixing of the sample and ensure sufficient dissolution and sample homogeneity. Once this is complete, the emulsification, heat treatment, and presentation processes can be initiated as in the previous example.

[0074]

[0085] Figure 5 shows a specific example of a centrifugal microfluidic system for the heat treatment and display of emulsion splitting IRVs. The system comprises a centrifugal blade 50 adapted to be mounted on a centrifuge around an axis (not shown). The blade 50 comprises two tip receiving portions 53 at both ends and a flared or rounded plate body extending substantially midway between the tip receiving portions 53. The flared plate body portion provides a place to apply a mass and balances the centrifuge.

[0075]

[0086] The two tips 55 are designed to be attached to the blade 50 at the tip receiving section 53, but in one example, the cover is removed for illustrative purposes (i.e., the patterned film 10). It should be noted that the tips are often reinforced to form a cartridge, which tends to facilitate operation, prevent the movement of pre-loaded fluid due to unintended operation of the tip, and allow for precise alignment of the tip with corresponding features of the blade 50, such as heating systems, observation systems, and pressure supply ports. However, structures to provide such reinforcement tend to obstruct the tip and are therefore omitted.

[0076]

[0087] At the center of the blade 50 is a controller 52 for supplying fluid, power, or signals, which may have an electrical circuit, power supply, pressurized fluid source, pump, flow control element, or pneumatic, hydraulic, and / or electrical slip ring, eight of which are shown, controlling the pressure supplied to each fluid supply line 56. A supply coupler 54 connects each port 12 of the tip to the respective fluid supply line 56. Two examples of tips may, for example, perform overlapping tests or have different sample sources that perform the same tests, but typically have overlapping pressure supply lines 56 to operate both tips simultaneously.

[0077]

[0088] Figure 5A shows the film 10 in a third modified form, with the coupler 54 disassembled to provide a complete view of the film 10. In the third modified form, there are several minor changes. For example, chamber 13 is a separate chamber for dry reagents instead of an overflow chamber, into which the untreated sample is supplied. The sample supplied into chamber 13 before centrifugation remains with the dry reagents for a sufficient time for mixing by diffusion and is metered into the sample chamber 14 by control of the port of chamber 13. The J channel 16 has hydrodynamic resistance and opens into an array of nozzles 18, as shown better in Figure 5B. tc 15 has an overflow chamber 15' that provides a buffer space for the light medium supplied from chamber 30 through hydrodynamic resistance, improving control of the supply rate. The fact that introducing the sample into tc expands the occupied volume of tc, while the heavy medium is returned to the draw-in chamber 25, presents a supply problem with respect to this volume. While the light medium helps to address volume differences, its ability to overfill the tc to the point where the light medium is substantially removed from the tc without accompanying any IRV is useful. In an alternative embodiment, the light medium may be returned to chamber 30 instead of moving air in the overflow chamber 15'. The heavy medium chamber 25 is provided with a vented metering channel to supply a more precise amount of heavy medium. The metering channel provides a useful location for verifying the level of heavy medium filling in the tc during centrifugation before IRV formation. pc20 is shown with an array of pillars for support and reinforcement. d pc Because the pressure fluctuations on the tip are very small, pillars are necessary to ensure that these fluctuations do not substantially change the depth during use. These are better illustrated in Figure 5B.

[0078]

[0089] Figure 5A also shows how a single aggregate structure can press against each of the provided ports of the tip 55 and seally connect to the respective supply line 56 via the coupler 54.

[0079]

[0090] Figure 5B shows a partially enlarged view of a particularly sensitive portion of the patterning of film 10 in the third deformation form. There are seven nozzles 18 shown coupled to an extended manifold segment of the J channel 16 after the hydrodynamic constraint. Thus, the sample pressed through the constraint is pushed into the manifold, distributing approximately equal flow rates to each of the seven nozzles. Not far away, an opening 22 is shown, and a pillar 59 is visible, positioned within pc 20 and extending appropriately to the opening 22. The pillar 59 has dimensions, pitch, and offset to minimize flow interaction with the IRV and avoid shear stress in the flow. [Examples]

[0080]

[0091] The present invention has been demonstrated by several experiments. Figure 6 is a photograph of the chip used to examine the present invention. The patterning most closely resembles the third variant, and the chip has a large pc with a field of view for presenting all IRVs at once for imaging. The chip was manufactured by hot embossing a film in thermoplastic elastomer (Mediprene OF®) and bonding it to a flat COC sheet. Subsequently, using the device, IRVs were generated, the emulsion was thermally cycled, and the emulsion was transferred to the pc using the protocol described below. In this chip, d pc is 60 μm, d tc It was 500 μm.

[0081]

[0092] First, the chip was filled with all the liquids necessary to generate IRV and perform droplet PCR. Specifically, 200 μl of heavy oil was loaded into the intake chamber, followed by 20 μl of sample (PCR mixture with template) being filled into chamber 13, and 20 μl of mineral oil being filled into the light medium chamber 30. Next, with the inlet sealed at the pressure supply port of the blade, the device was placed on the centrifuge blades and the platform was rotated at 400 rpm.

[0082]

[0093] A positive pressure of 1.5 psi is applied at port 7 (in the illustrative section, all ports are numbered and identified from left to right in Figure 5B or Figure 6) to transfer heavy oil to the pc, opening, and tc until tc15 (not the overflow chamber 15') is completely filled. The rotation speed is then increased to 600 rpm. At this point, the contents of tc have decreased to approximately half-filled tc. Subsequently, the sample is transferred from chamber 13 to sample chamber 14 by applying +1 psi in a 200 ms pulse at port 3.

[0083]

[0094] Using the sample in chamber 14, an IRV can be generated in the next step by applying a constant pressure greater than +1.5 psi at ports 2 and 3 (and further controlling the rotational speed), which is sufficient to overcome the resistance of the J channel and initiate the IRV generation process through the nozzle. The precise pressure and rotational speed, along with the provided hydrodynamic radius of the nozzle, determine the diameter and volume of the IRV, which can be set on demand and customized for specific applications. In this case, the nozzle had a width of 10 μm and a depth of 8 μm, so the hydrodynamic radius was 2(10 × 8) / (2(8 + 10)), which is approximately 4.5 μm.

[0084]

[0095] Upon applying pressure, IRV was generated to completely fill the tc all the way up to the overflow chamber 15', but the IRV did not enter the overflow chamber. This was important to avoid IRV collapse during heat treatment. After IRV generation, diesel fuel was supplied into the overflow chamber by applying a pressure of +1 psi at port 5. This step was used to prevent evaporation during thermal cycling and to maintain IRV stability at the high temperatures required for the specific PCR process being applied (i.e., 95°C).

[0085]

[0096] Next, a thermal cycling sequence was applied using a Peltier heater located on the lower blade of the tc. After the thermal cycling was complete, a negative pressure of -1 psi was applied to ports 6 and 7 to draw in heavy oil and initiate the transfer of the emulsion to the pc. During this process, when the platform stopped spinning, the rotation speed was slowly reduced to 300 rpm (in 10 rpm increments) to prevent backflow of oil into the pc. Once the transfer was complete, the pressure was turned off, the rotation stopped, and subsequent IRV imaging was enabled.

[0086]

[0097] Figure 7 is a photograph of the captured pc, with the arrangement of support pillars shown as dark spots. Some banding of IRVs is observed, and several areas of IRV decay are visible, particularly near the top of the pc, but the majority of the pc is filled with roughly hexagonal packing of uniformly sized IRVs. Adjacent IRVs have different colorations, demonstrating effective separation of IRVs during heat treatment. Figure 7A shows a portion (top) of the enlarged region showing a densely packed hexagonal monolayer, with each IRV independently transmitting a PCR fluorescence response.

[0087]

[0098] Figure 8 is a histogram showing the number of IRV diameters, giving the applicant the opportunity to calculate the mean and CV of the distribution, which are 49 μm and 2.38%, respectively. This demonstrates that the volume of each IRV is sufficiently constant for most analyses. As described in the LOC paper incorporated by reference above, a chip configuration for generating IRV diameters with a moderately narrow distribution centered on various diameters from a few microns to at least 100 μm can be generated with variations in centrifugation from 300 to 700 rpm and positive pressure from 0 to 40 kPa.

[0088]

[0099] The proposed chip and system applications have been demonstrated for automated IRV generation, thermal cycling, and emulsion transfer for imaging.

[0089]

[0100] Essential features of the chip and system for heat treatment and IRV emulsion transfer are described herein with minimal alternative elements that obstruct the view. It will be understood by those skilled in the art that several sensors and devices can be added to the blade or chip controller to provide additional process control and feedback for further improvement. Other advantages inherent in the structure will be obvious to those skilled in the art. Embodiments are described herein by example and are not intended to limit the scope of the claimed invention. Variations of the embodiments described herein will be obvious to those skilled in the art and are intended to be included by the inventors in the following claims.

[0090]

[0101] References Malic, Lidija et al., “Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR.” Analyst 144.22 (2019): 6541~6553. Schuler, Friedrich, et al., "Digital droplet PCR on disk." Lab on a Chip 16.1(2016):208~216. Schuler, Friedrich, et al., "Digital droplet LAMP as a microfluidic app on standard laboratory devices." Analytical Methods 8.13(2016):2750~2755. Li, Bin et al., “Miniaturized Continuous-Flow Digital PCR for Clinical-Level Serum Sample Based on the 3D Microfluidics and CMOS Imaging Device.” Sensors 20.9(2020):2492. Hu, Fei et al., “Smartphone-based highly droplet digital LAMP device with rapid nucleic acid isolation for sensitive point-of-care detection.” Analytical Chemistry 92.2 (2019): 2258~2265. Madic, J., A. Zocevic, V. Senlis, E. Fradet, B. Andre, S. Muller, R. Dangla, and MEDroniou, “Three-color crystal digital PCR.” Biomolecular detection and quantification 10(2016):34–46. Clime, Liviu et al. “Bouyancy-driven step emulsification on pneumatic centrifugal microfluidic platforms.” Lab on a Chip 20(2020):3091~3095. Schuler, Friedrich, et al., "Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA." Lab on a Chip 15 (2015):2759~5766. [Explanation of symbols]

[0091] 2...Port, 3...Port, 5...Port, 6...Port, 7...Port, 10...Film, Substrate, 11...Cover layer, Cover, 12...Port, Vent, 12a...Sample port, Pressure control port, 12b...Vent, 12c...Port, 12d...Vent, Pressurization port, 12e...Port, 13...Overflow chamber, 14...Sample chamber, Supply chamber, Siphon valve connection chamber, 15...Processing chamber (tc), 15'...Overflow chamber, 15a...Port, 16...J-shaped channel, J-channel, 18...Nozzle, 19...Retraction channel, 20...Presentation chamber (pc), flow-through pc, 22…opening, 23…inclined section, 25…retraction chamber (rc), heavy medium chamber, 26…window area, 28…heat treatment area, 30…light medium chamber, 31…mixing chamber, siphon valve connection chamber, 36…elution buffer chamber, 37…chamber, 38…elution buffer chamber, 39…off-tip waste reservoir, off-tip waste, 40…off-tip sample vial, 50…centrifuge blade, 52…controller, 53…tip receiving section, 54…supply coupler, 55…tip, 56…fluid supply line, pressure supply line, 59…pillar, r n ...The hydrodynamic radius of the nozzle, r IRV ...average radius of IRV, l tc ...average length of tc, w tc ...average width of tc, d tc ...average depth of tc, v tc ...volume of tc, l pc ...average length of PC, w pc ...average width of PC, d pc ...average depth of pc, v pc ...PC volume, l o ...average length of the opening, w o ...average width of opening, d o ...Average depth of the opening.

Claims

1. 1. A centrifugal microfluidic chip for mounting in a centrifuge for rotation about an axis, the chip comprising a network of chambers and interconnecting channels, the network comprising: Volume (v ｔｃ ), and the average length (l ｔｃ ), average width (w ｔｃ ), and the average depth (d ｔｃ ) and d ｔｃ <l ｔｃ , and d ｔｃ <w ｔｃ a processing chamber (tc) where A first pathway adapted to deliver a sample to tc, the first pathway having a hydrodynamic radius r between 2 and 120 μm. ｎ a first path including a nozzle having a diameter of about 1 mm and adapted to deliver the sample discretized as an emulsion divided independent reaction volume (IRV) to the tc when the nozzle is filled with a suitable medium; a presentation chamber (pc) coupled to the tc by an opening through which the IRV can be selectively moved from the tc to the pc; a window through the chip spanning at least the length and width of the pc, the window being transparent for inspecting the pc; a retraction chamber (rc) coupled to the pc by a second path; Including, d ｔｃ But, 5 x r ｎ 2 mm, and the pc is 1.2 × r ｎ ~7×r ｎ Thickness d ｐｃ d ｔｃ >2d ｐｃ and the flow path through the opening is 8×r ｎ A centrifugal microfluidic chip having a minimum hydrodynamic radius greater than .

2. The PC is 0.6 to 1.2 V ｔｃ The volume (v ｐｃ 2. The centrifugal microfluidic chip of claim 1, having a surface area of ​​at least 3 cm2 that is 80% or less of the surface area of ​​the chip.

3. 2. The centrifugal microfluidic chip of claim 1, wherein the network of chambers and channels is produced in relief on at least a first surface of a first film having a nominal thickness of 20 μm to 5 mm, and the chip comprises a cover film covering the first surface to seal the chambers and channels away from the ports of the chip.

4. d ｐｃ 2. The centrifugal microfluidic chip of claim 1, wherein the pressure is nominally maintained by an array of supporting microstructures despite pressure relative to the surroundings.

5. The nozzle is disposed at the inlet of the first path to the tc. the inlet of the first path to the tc branches to further provide one or more additional instances of the nozzle; The opening between the tc and pc is wider than it is long; the floor of the opening comprises a slope having a depth that varies from d tc to d pc ; the floor of the opening comprises a slope having a slope of 30° to 75°; the first or second pathway includes a metering chamber having an overflow chamber for volumetrically controlled delivery; 5. The centrifugal microfluidic chip according to claim 4, which corresponds to at least one of the above.

6. 2. The centrifugal microfluidic chip of claim 1, comprising a stack of two or more films, at least one of the films having a relief structure forming the network, each film having a nominal thickness of 20 μm to 3 mm, each film being composed of a cured or solidified polymeric compound other than siloxane, the thickness of the chip being 0.1 to 12 mm, the planar extension of the chip being 3 to 25 cm, or the chip having at least two ports.

7. The centrifugal microfluidic chip according to any one of claims 1 to 6, wherein one or more of the following are loaded: a sample preparation reaction mixture, such as a PCR mixture, in a first chamber in the first path, in dry or liquid form; a sample, in a second chamber in the first path, in dry or liquid form; a buffer, solvent, or liquid for dissolving or suspending the sample or the reaction mixture in the first path; or a dispersion medium adapted to support an IRC loaded in the tc, the pc, or the rc.

8. 8. The centrifugal microfluidic chip according to claim 7, further comprising a low density medium chamber connected to the tc by a third path that joins the tc at an axially proximal end of the tc, wherein the low density medium chamber is loaded with a liquid having a density lower than that of the sample, the buffer solution, the solvent, or the liquid and the dispersion medium.

9. 2. The centrifugal type microfluidic chip of claim 1, provided in a kit mounted on the centrifuge or for mounting on the centrifuge, comprising a chip controller, for rotating both the chip and the controller, the chip controller comprising an off-chip flow control device for selectively moving fluid in the pc into the rc, and a pressurized fluid supply line for coupling to a port of the chip.

10. the chip controller comprising a chip support surface dimensioned to support the chip on one side of the chip controller; the chip-supporting surface comprises an energy device for selectively exposing the processing chambers of the chip to an energy field, such as a thermal energy source or sink, an ultrasonic transducer, or an electromagnetic field generator; or The kit of claim 9 , wherein the chip-holding surface holds the chip such that an illumination and imaging system images the pc through the window.

11. The kit of claim 10 assembled to form a centrifugal microfluidic system.

12. 1. A method for co-processing emulsion separated reaction volumes (IRVs) to present the processed IRVs in a monolayer, the method comprising: Providing the IRV in a processing chamber (tc) of a centrifugal microfluidic chip; energizing the IRVs in the tc by activating an energy device attached to the tip on a centrifuge while the tip is being centrifuged, the tc having a minimum dimension to accommodate between 3 and 100 IRVs; operating a flow control device to move the IRVs from the tc into a presentation chamber (pc) through an opening to position the IRVs in a substantial monolayer, the opening having a width greater than an average diameter of the IRVs and a depth of the pc, the flow control device applying a pressure differential of less than 15 kPa; The method includes:

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