Microfluidic reaction structures

Abstract

The rotatable device includes a plurality of reaction structures arranged circumferentially over at least a portion of the device. Each reaction structure in the plurality of reaction structures includes a preparative chamber and a reaction chamber located radially outward from the preparative chamber. The preparative chamber has an outlet, and the reaction chamber has an inlet connected to the outlet of the preparative chamber. The rotatable device also includes one or more connecting siphons, which are radially horizontal to each other and radially inward from the outlets of the preparative chambers of the plurality of reaction structures. Each connecting siphon of the one or more connecting siphons connects the preparative chambers of the corresponding adjacent reaction structures in the plurality of reaction structures.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 489,422, filed on March 9, 2023, and U.S. Provisional Patent Application No. 63 / 489,677, filed on March 10, 2023. The disclosure of each application is hereby incorporated by reference in its entirety for all purposes.

[0002] This disclosure relates to devices and methods that enable reactions to be performed in parallel in centrifugal microfluidics.

Background Art

[0003] Currently, 70% of all medical decisions rely on laboratory - based diagnoses. However, today, the diagnostic process is detached from how care is provided. In primary healthcare systems, patients need to travel to an external blood collection site to have their blood drawn, and that blood is sent to the laboratory by courier and processed overnight. That is, the test results reach healthcare providers long after the patient has returned. Such frictions in care delivery and disease management result in significant waste in the healthcare system. a. Patients often delay getting tested in the laboratory or fail to follow up on laboratory tests and subsequent care recommendations. b. Gaps in the diagnostic process lead to missed tests, missed diagnoses, insufficient interventions, and ultimately poor outcomes. c. Healthcare providers waste time tracking test orders and patient examination notes. When an intervention is required, more time is wasted contacting the patient and advancing the subsequent steps in the patient's care pathway.

[0004] Such problems are even more severe when caring for patients living in rural areas or belonging to groups facing adverse social determinants of health, and there are many challenges to ensuring the success of follow - up from the initial patient examination.

[0005] To bridge this gap, several companies are manufacturing point-of-care devices. However, these devices are limited to a single type of test and fail to fully meet the workflow needs of primary care providers for a single system that produces simple, comprehensive, and rapid test results. A product that meets these needs is currently under development. This product achieves this through a highly automated workflow realized by the use of centrifugal microfluidic disks.

[0006] Centrifugal microfluidics are used in clinical chemistry, immunoassays, hematology, medicine, biomedical research, and other fields. Many of these applications involve numerous reactions. To enable numerous reactions, existing microfluidics often have configurations in which multiple structures are arranged sequentially to avoid contamination. Such configurations require radial offsets between each structure, leading to at least two problems: a limitation on the number of structures per cartridge and slightly different operating parameters for each structure.

[0007] Therefore, there remains a need for improved devices and methods for carrying out numerous reactions in centrifugal microfluidics. [Overview of the project]

[0008] This disclosure addresses these and other needs in the art by providing devices and methods that allow reactions to be carried out in parallel, often involving controlled reconstitution of dried reagents and intermediate light-based readout.

[0009] In one embodiment, the present disclosure provides a device rotatable around a rotation axis. The device includes a reaction chamber having an inlet for receiving fluid and a first well that serves as both a reagent well and a readout well. When the device is fabricated, a first reagent is placed in the first well. The radial position of the fluid meniscus in the reaction chamber depends at least in part on the rotation speed of the device. The first well has substantially flat walls perpendicular to the rotation axis to allow reproducible light transmission.

[0010] In some embodiments, the reaction chamber further includes a second well and an intermediate chamber between the first well and the second well. In some such embodiments, when the device is fabricated, the second reagent is placed in or set in the second well.

[0011] In some embodiments, the first and / or second reagents are lyophilized. In some embodiments, the first and / or second reagents are in bead form. In some embodiments, the second reagent is different from the first reagent.

[0012] In some embodiments, the reaction chamber is non-aerated to facilitate homogeneous mixing of the first reagent and the fluid. For example, in some such embodiments, the reaction chamber includes an outlet connected to an air chamber. In some embodiments, a first well is connected to an air chamber. In some embodiments, a second well is connected to an air chamber.

[0013] In some embodiments, the device further includes an upstream chamber located radially inward of the reaction chamber and connected to the inlet of the reaction chamber. In some embodiments, the inlet of the reaction chamber is located in a first well, and a second well is located radially inward of the first well. In some embodiments, the outlet is located in the second well.

[0014] In some embodiments, the reaction chamber, upstream chamber, air chamber, or a combination thereof is configured to allow the initial filling of the first well with fluid without wetting the second well.

[0015] In another embodiment, the disclosure provides a device rotatable about a rotation axis. The device includes a plurality of reaction structures arranged circumferentially over at least a portion of the device. Each reaction structure in the plurality of reaction structures includes a preparative chamber and a reaction chamber located radially outward from the preparative chamber. The preparative chamber has an outlet, and the reaction chamber has an inlet connected to the outlet of the preparative chamber. In some embodiments, the device also includes one or more connecting siphons that are radially horizontal to each other and radially inward from the outlets of the preparative chambers of the plurality of reaction structures. Each of the one or more connecting siphons connects the preparative chambers of corresponding adjacent reaction structures in the plurality of reaction structures.

[0016] In some embodiments, for each reaction structure in a plurality of reaction structures, the reaction chamber includes a first well. In some embodiments, for each reaction structure in a plurality of reaction structures, a first reagent is placed in the first well when the device is fabricated. In some embodiments, for each reaction structure in a plurality of reaction structures, the first well serves as both a reagent well and a readout well. In some embodiments, the first wells of the plurality of reaction structures are radially horizontal to each other.

[0017] In some embodiments, for each reaction structure in at least a subset of multiple reaction structures, the reaction chamber includes a second well and an intermediate chamber between the first well and the second well. In some embodiments, for each reaction structure in at least a subset of multiple reaction structures, a second reagent is placed in the second well when the device is fabricated. In some embodiments, the second wells of at least a subset of multiple reaction structures are radially horizontal to each other. In some embodiments, the inlet of the reaction chamber is located in the first well, and the second well is located radially inward of the first well.

[0018] In some embodiments, each of the multiple reaction structures includes an air chamber, and each reaction chamber of the reaction structure has an outlet connected to the air chamber.

[0019] In some embodiments, the device further includes an overflow chamber and an overflow channel connecting the preparative chamber of the last reaction structure in a plurality of reaction structures to the overflow chamber. In some such embodiments, the overflow channel includes a siphon portion, the siphon portion including an inlet and a apex. In some embodiments, the inlet of the siphon portion of the overflow channel is radially horizontal to the inlets and outlets of each connecting siphon in one or more siphons. In some embodiments, the apex of the siphon portion of the overflow channel is horizontal to or radially outward from the apex of each connecting siphon in one or more siphons. In some embodiments, the overflow channel further includes a U-channel having an outlet connected to the overflow chamber. In some embodiments, a portion of the U-channel is outside the overflow channel outlet.

[0020] In a further embodiment, the Disclosure provides a method comprising (A) obtaining a device rotatable about a rotation axis. The device comprises a plurality of reaction structures and one or more connecting siphons. The reaction structures in the plurality of reaction structures are arranged circumferentially over at least a portion of the device. Each reaction structure in the plurality of reaction structures comprises a preparative chamber and a reaction chamber located radially outward from the preparative chamber. The preparative chamber has an outlet, and the reaction chamber has an inlet connected to the outlet of the preparative chamber. One or more connecting siphons are radially horizontal to one another and radially inward from the outlets of the preparative chambers of the plurality of reaction structures. Each connecting siphon of the one or more connecting siphons connects the preparative chambers of corresponding adjacent reaction structures in the plurality of reaction structures. The Method also comprises (B) rotating the device at a certain speed to fill a first portion of the reaction chamber of each reaction structure in the plurality of reaction structures with fluid. The Method further comprises (C) increasing the speed to discharge fluid from each corresponding connecting siphon in the one or more connecting siphons. Furthermore, the method includes (D) reducing the rate to create a gas volume confined within each corresponding connecting siphon in one or more connecting siphons, thereby interrupting the fluid communication between the multiple reaction structures.

[0021] In some embodiments, raising (C) moves at least a portion of the fluid from the preparative chamber of each of the multiple reaction structures so that the fluid contained in the preparative chamber of each of the multiple reaction structures is positioned outside the inlet and outlet of each corresponding connecting siphon in one or more connecting siphons, thereby discharging the fluid from each corresponding connecting siphon in one or more connecting siphons.

[0022] In some embodiments, lowering (D) moves at least a portion of the fluid contained in the reaction chamber of each of the multiple reaction structures to the respective reaction chamber of each of the multiple reaction structures, so that the fluid contained in the preparative chamber of each of the multiple reaction structures is positioned inside the inlet and outlet of each corresponding connecting siphon in one or more connecting siphons, thereby wetting the inlet and outlet of each corresponding connecting siphon in one or more connecting siphons with the gas volume trapped inside each corresponding connecting siphon.

[0023] In some embodiments, the method further includes (E) raising (C) and lowering (D) one or more times to facilitate mixing of the fluid and the first reagent, in which case the first portion of the reaction chamber of each reaction structure in the plurality of reaction structures includes a first well containing a first reagent, the method further includes (F) detecting light transmitted through the first well or the wall of the first well of the reaction chamber for each reaction structure in the plurality of reaction structures, the method further includes (F) detecting light transmitted through the first well or the wall of the first well of the reaction chamber. In some embodiments, the detection (F) is carried out without repetition (E) (e.g., without mixing).

[0024] In some embodiments, the method further includes (G) increasing the rate to fill the second portion of the reaction chamber of each reaction structure in at least a subset of the plurality of reaction structures, and (H) decreasing the rate (D). In some embodiments, where the second portion of the reaction chamber of each reaction structure in at least a subset of the plurality of reaction structures comprises a second well having a second reagent, the method further includes (I) increasing the rate (G) and decreasing the rate (H) one or more times to promote mixing of the fluid and the second reagent. In some embodiments, the method further includes (J) detecting the light transmitted through the first well or the wall of the first well of the reaction chamber for each reaction structure in at least a subset of the plurality of reaction structures.

[0025] The devices, systems, and methods of the present disclosure will become apparent from the accompanying drawings incorporated herein and the following detailed description, or have other features and advantages that will be more particularly described, both of which together serve to explain the specific principles of exemplary embodiments of the present disclosure.

[0026] The accompanying drawings incorporated herein and constituting a part hereof show one or more exemplary embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and implementations of the exemplary embodiments of the invention. The accompanying drawings are not necessarily to scale. For example, certain design features of the invention disclosed herein, including specific dimensions, directions, positions, and shapes, are determined in part by the particular intended use and operating environment. Further, the components illustrated in the figures can be combined in any useful number and combination.

[0027] The drawings are as follows.

Brief Description of the Drawings

[0028] <0​​​​​​​​​​​​​​​​​​​​​​​​This is a schematic diagram illustrating a process performed using the device shown in Figure 2A, according to some exemplary embodiments of this disclosure. [Figure 4B] This is a schematic diagram illustrating a process performed using the device shown in Figure 2A, according to some exemplary embodiments of this disclosure. [Figure 4C] This is a schematic diagram illustrating a process performed using the device shown in Figure 2A, according to some exemplary embodiments of this disclosure. [Figure 4D] This is a schematic diagram illustrating a process performed using the device shown in Figure 2A, according to some exemplary embodiments of this disclosure. [Figure 4E] This is a schematic diagram illustrating a process performed using the device shown in Figure 2A, according to some exemplary embodiments of this disclosure. [Figure 4F] This is a schematic diagram illustrating a process performed using the device shown in Figure 2A, according to some exemplary embodiments of this disclosure. [Figure 5A] This is a schematic diagram showing a device (e.g., a disk) according to some exemplary embodiments of the present disclosure. [Figure 5B] This is a schematic diagram illustrating a loading process according to some exemplary embodiments of the present disclosure. [Figure 5C] This is a schematic diagram illustrating separation and metric processes according to some exemplary embodiments of the present disclosure. [Figure 5D] This is a schematic diagram illustrating a weighing process according to some exemplary embodiments of the present disclosure. [Figure 5E-1] This is a schematic diagram illustrating a dilution process according to some exemplary embodiments of the present disclosure. [Figure 5E-2] This is a magnified view of a portion of Figure 5E-1. [Figure 5F-1] This is a schematic diagram illustrating a transfer and dissolution process according to some exemplary embodiments of the present disclosure. [Figure 5F-2] This is a magnified view of a portion of Figure 5F-1. [Figure 5G]This is a schematic diagram illustrating a mixing process according to some exemplary embodiments of the present disclosure. [Figure 5H] This is a schematic diagram illustrating a dissolution process according to some exemplary embodiments of the present disclosure. [Figure 5I] This is a schematic diagram illustrating a mixing and detection process according to some exemplary embodiments of the present disclosure. [Figure 6] A block diagram showing workflows according to some exemplary embodiments of this disclosure. [Figure 7] This is a schematic diagram showing a device (e.g., a disk) according to some exemplary embodiments of the present disclosure. [Modes for carrying out the invention]

[0029] This disclosure provides systems and methods for performing multiple reactions in parallel. In various embodiments, the systems and methods of this disclosure enable controlled reconstitution of dried reagents and intermediate light-based readout, such as in the first step of two reagent reactions. This can be achieved by controlling reaction timing and volume while minimizing dead volume (e.g., liquid volume not read by the detection method). This is very important because even a slight increase in dead volume can lead to a large amount of space (e.g., space in a cartridge) being allocated to non-critical functions, limiting the number of reactions that can be performed in a single device (e.g., a cartridge).

[0030] Referring here to the drawings, similar reference figures indicate similar elements throughout, and Figures 1A–1C show exemplary devices 100 according to several embodiments of the present disclosure. Device 100 is rotatable around a rotation axis 102. Device 100 includes a reaction structure 104 configured to control the position of the liquid using pneumatic and centrifugal force, enabling the reconstitution of the reagent within the same chamber in which incubation and detection take place. This eliminates dead volume and variability associated with the transfer of liquid from the reconstitution chamber to a separate detection chamber.

[0031] The reaction structure 104 includes a reaction chamber 110 having an inlet 112 for receiving liquid from, for example, an upstream chamber or pipette port. The reaction chamber 110 is configured such that the radial position 114 of the fluid meniscus within the reaction chamber depends at least partially on the rotational speed of the device. The reaction chamber 110 includes one or more wells. As a non-limiting example, Figure 1A shows a reaction chamber 110 having a first well 121 and a second well 122, connected between the first and second wells by an intermediate chamber 123. In some embodiments, the inlet is located in the first well, and the second well is located radially inward from the first well. As another non-limiting example, Figure 1C shows a reaction chamber 110 having a single well, such as the first well 121. Note that the reaction chamber may have more than two, more than three, more than four, or more than five wells.

[0032] At least one of the one or more wells is configured to serve as both a reagent well and a readout well. For example, as shown in Figure 1B, in some embodiments, a first well 121 is configured to serve as both a reagent well and a readout well. In some such embodiments, as shown in Figure 1B, a first reagent 151 is placed in the first well when the device is fabricated. The first well 121 is made with substantially flat walls 124 perpendicular to the axis of rotation to allow for reproducible light transmission. Alternatively, in some embodiments, a second well 122 is configured to serve as both a reagent well and a readout well, or both the first and second wells are configured to serve as both a reagent well and a readout well. In some embodiments, a second reagent 152 is placed in the second well 122 when the device is fabricated. The second reagent may be the same as or different from the first reagent.

[0033] In some embodiments, the first and / or second reagents are lyophilized. In some embodiments, the first and / or second reagents are in bead form. In some embodiments, a device including a reaction chamber or at least a portion of a device is made of an injection-molded thermoplastic piece having one or more cavities into which the first and / or second reagents (e.g., lyophilized beads) are placed, followed by a bonding step with a pressure-sensitive adhesive lining laminate to define the upper surface of the chamber, as shown in Figure 1B.

[0034] In some embodiments, as shown in Figure 1A, the reaction chamber 110 is non-aerated to facilitate homogeneous mixing of the first and / or second reagent with the fluid. For example, in some embodiments, the reaction structure 104 includes a pressure chamber (e.g., an air chamber) 130, and the reaction chamber includes an outlet 114 connected to the pressure chamber. The outlet may be located in the second well as shown in Figure 1A, in the intermediate chamber as shown in Figure 1C, or in any other suitable location. This allows the liquid meniscus position within the reaction chamber to be controlled using the rotational speed, ensuring homogeneous mixing of the liquid within the reaction chamber with the first or second reagent. For example, changing the rotational speed causes the liquid to move inward or outward, promoting homogeneous mixing of the reagents. This also has the advantage of allowing precise control of the liquid position simultaneously across all reaction chambers in multiple reaction structures, as will be described in more detail. The only drivers for the liquid position are the rotational speed, the shape of the chamber, and the reaction volume.

[0035] In some embodiments, the reaction structure 104 includes an upstream chamber 140 (e.g., a preparative chamber) located radially inward of the reaction chamber 110, and has outlets (e.g., outlet 145, outlet 146, or both) connected to the inlet of the reaction chamber to supply fluid to the reaction chamber. In some embodiments, the reaction chamber, upstream chamber, pressure chamber (e.g., an air chamber), or a combination thereof, is configured to allow initial filling of the first well 121 with fluid without wetting the second well 122, if a second well 122 is present. For example, in some embodiments, the upstream chamber 140 is configured to have a first deeper portion 141, a second deeper portion 142, and a shallow portion 143 between the first and second deeper portions. In some embodiments, the intermediate chamber 123 of the reaction chamber is configured to have a relatively small volume but a relatively long radial length. This can ensure minimal variation in reaction volume during metering, even with large volume fluctuations in the upstream chamber. This ensures that fluctuations in the radial position of the liquid meniscus 116 within the reaction chamber 110 do not coincide with large fluctuations in the volume within the reaction chamber 110, thereby guaranteeing minimal fluctuations in the reaction volume during metering, even in the event of large fluctuations in the volume of the upstream chamber.

[0036] The reaction structure 104 can be configured to include additional, optional, or alternative components. For example, in a non-limiting example, Figure 1D shows the reaction structure 104 including a vent port 160, where a first well 121 is connected to the vent port and a second well 122 is connected to the first well 121, the air chamber 130, and the upstream chamber 140. In this embodiment, the first well 122 is filled during initial filling, leaving a minimum volume in the upstream chamber 140. By reducing the rotation speed, the liquid meniscus moves inward, wetting the top of the siphon and filling the second reagent well 122.

[0037] Referring to Figures 2A and 2B, exemplary devices 200 according to several embodiments of the present disclosure are shown. The device 200 is rotatable about a rotation axis 202. The device 200 includes a plurality of reaction structures, such as reaction structure 104 disclosed herein. The reaction structures in the plurality of reaction structures are arranged circumferentially over at least a portion of the device. The device 200 can include any suitable number of reaction structures. For example, the device may include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 reaction structures. As a non-limiting example, Figures 2A and 2B show a device having four reaction structures, for example, reaction structures 104-1, 104-2, 104-3, and 104-4.

[0038] A reaction structure in a plurality of reaction structures can be configured to be the same as or different from another reaction structure in the plurality of reaction structures. In some embodiments, each reaction structure in a plurality of reaction structures is configured to be substantially the same as another reaction structure in the plurality of reaction structures. In some embodiments, at least one reaction structure in a plurality of reaction structures is configured differently from one or more other reaction structures in the plurality of reaction structures. For example, Figures 2A and 2B show that reaction structures 104-2, 104-3 and 104-4 are configured to be substantially the same or identical to each other, while reaction structure 104-1 is configured differently from reaction structures 104-2, 104-3 and 104-4.

[0039] Each reaction structure in the plurality of reaction structures includes a preparative chamber, such as the upstream chamber 140 disclosed herein, and a reaction chamber, such as the reaction chamber 110 disclosed herein. The preparative chamber has an outlet (e.g., outlet 145, outlet 146, or both), and the reaction chamber has an inlet (e.g., inlet 112) connected to the outlet of the preparative chamber. The reaction chamber is located radially outward from the preparative chamber. In some embodiments, each reaction structure in the plurality of reaction structures includes an air chamber, such as the air chamber 130 disclosed herein. For each reaction structure, the reaction chamber has an outlet (e.g., outlet 114) connected to the air chamber.

[0040] In some embodiments, for each reaction structure in a plurality of reaction structures, the reaction chamber includes a first well, such as a first well 121 disclosed herein. In some embodiments, for each reaction structure in a plurality of reaction structures, a first reagent, such as a first reagent 151, is placed in the first well when the device is fabricated. In some embodiments, for each reaction structure in a plurality of reaction structures, the first well serves as both a reagent well and a readout well. In some embodiments, the first wells of the plurality of reaction structures are radially horizontal to each other (e.g., at the same radial position). In some embodiments, for each and all of the plurality of reaction structures, the reaction chamber consists of a single well, such as a first well 121 disclosed herein. In some embodiments, for each reaction structure in at least a subset of the plurality of reaction structures, the reaction chamber includes a second well, such as a second well 122 disclosed herein, and an intermediate chamber, such as an intermediate chamber 123 disclosed herein, between the first well and the second well. In some embodiments, for each reaction structure in at least a subset of the multiple reaction structures, a second reagent, such as a second reagent 152, is placed in a second well when the device is fabricated. In some embodiments, for each and all of the multiple reaction structures, the reaction chamber includes both a first and a second well. For example, Figures 2A and 2B show that the reaction chamber of reaction structure 104-1 includes a single well (e.g., a first well 121), and the reaction chambers of reaction structures 104-2, 104-3, and 104-4 each include a first and a second well.

[0041] In some embodiments, the second wells of at least a subset of the multiple reaction structures are radially horizontal to each other. In some embodiments, for each of the multiple reaction structures, the inlet of the reaction chamber is located in the first well, and the second well (if present) is located radially inward of the first well, as shown in Figures 1A and 2A.

[0042] In some embodiments, the reaction structures in a plurality of reaction structures are positioned within a range of radii. For example, in some embodiments, the reaction structures in a plurality of reaction structures may be positioned between a first radius and a second radius with respect to the axis of rotation. The first radius may be less than about 30 mm, less than about 35 mm, less than about 40 mm, less than about 45 mm, or less than 50 mm. The second radius may be greater than about 35 mm, greater than about 40 mm, greater than about 45 mm, greater than 50 mm, greater than 55 mm, or greater than 60 mm. In some specific embodiments, the plurality of reaction structures may be positioned within a range of radii between about 25 mm and about 45 mm, between about 30 mm and about 50 mm, between about 35 mm and about 55 mm, or between about 40 mm and about 60 mm. In some specific embodiments, the plurality of reaction structures may be positioned within a range of radii between about 43 mm and about 50 mm.

[0043] The volume of the first well (e.g., the reaction volume for the first reaction 1R in the reaction structure) may be the same as or different from the volume of the second well (e.g., the reaction volume for the second reaction 2R in the reaction structure). "1R" indicates a reaction that occurs in the first well or with a first reagent placed in the first well. Similarly, "2R" indicates a reaction that occurs in the second well or with a second reagent placed in the second well. In some embodiments, the reaction volume for reaction 1R is smaller than the reaction volume for reaction 2R. For example, in some specific embodiments, the reaction volume for reaction 1R may be less than about 10 μL, less than about 15 μL, less than about 20 μL, less than about 25 μL, or less than about 30 μL, and the reaction volume for reaction 1R may be greater than about 20 μL, greater than about 25 μL, greater than about 30 μL, greater than about 35 μL, or greater than about 40 μL. In a specific embodiment, the reaction volume for the 1R reaction may be approximately 15 μL, and the reaction volume for the 2R reaction may be 29 μL. In another specific embodiment, the reaction volume for the 1R reaction may be approximately half the reaction volume for the 2R reaction.

[0044] In some embodiments, the channel (for example, the channel connecting the upstream chamber to the reaction chamber) may have a width of about 100 μm and 150 μm, about 150 μm and 200 μm, about 200 μm and 250 μm, or about 250 μm and 300 μm, and a depth of about 50 μm and 100 μm, 100 μm and 150 μm, about 150 μm and 200 μm, or about 150 μm and 250 μm. In a specific embodiment, the channel may have a width of about 120 μm and 175 μm, and a depth of about 100 μm and 175 μm.

[0045] The chamber may have a constant or varied depth. For example, the reaction chamber may have an intermediate chamber depth different from the depth of the first and / or second wells. Similarly, the upstream chamber may have a shallower section depth different from the depth of the first and / or second deeper sections. In some embodiments, the chamber may have a depth between about 0.2 mm and about 5 mm. In some embodiments, the chamber may have a depth between about 0.4 mm and about 3 mm.

[0046] Device 200 also includes one or more connecting siphons 220 that are radially horizontal to each other (e.g., located in the same radial position) and radially inward from the outlets of the preparative chambers of the multiple reaction structures. Each of the one or more connecting siphons connects the preparative chambers of corresponding adjacent reaction structures in the multiple reaction structures. For example, in some embodiments, the device includes two reaction structures and one connecting siphon connecting the two reaction structures. In some embodiments, the device includes three reaction structures and two connecting siphons connecting two adjacent reaction structures, respectively. In the embodiment shown in Figure 2A, where four reaction structures are present, the device includes three connecting siphons, e.g., connecting siphons 220-1, 220-2, and 220-3. Connecting siphon 220-1 connects reaction structure 104-1 and reaction structure 104-2. Connecting siphon 220-2 connects reaction structure 104-2 and reaction structure 104-3. The connecting siphon 220-3 connects reaction structure 104-3 and reaction structure 104-4. The connecting siphons 220-1, 220-2, and 220-3 are radially horizontal to each other (for example, the radial positions of the siphons are the same with respect to the axis of rotation 202) and are radially inward from the outlets of the preparative chambers of the multiple reaction structures.

[0047] In some embodiments, the reaction structures (e.g., reaction chambers, air chambers, and / or preparative chambers) and / or connecting siphons are adjusted to satisfy reaction requirements and minimize dead volume. For example, in some embodiments, the reaction structures and / or connecting siphons are adjusted to minimize dead volume while keeping the reaction volume within a desired range (e.g., 1% to 10% or 5% to 15%) according to reaction kinetics. In some embodiments, the reaction structures and / or connecting siphons are adjusted to ensure that the liquid position in the reaction chamber of each reaction structure completely fills the readout / reconstitution chamber (e.g., the first well) during the initial filling step without wetting the second reagent chamber (e.g., the second well, if present).

[0048] For example, referring to Figures 2A and 2B, in some embodiments, for each reaction structure in at least a subset of multiple reaction structures, the upstream / preparative chamber includes a shallow portion adjacent to the corresponding connecting siphon, e.g., a shallow portion 143 as shown in Figure 1B. In some embodiments, the shallow portion may have a depth of less than 1000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, or less than 500 μm. In some embodiments, the shallow portion may have a depth between about 100 μm and about 500 μm. In some embodiments, the shallow portion may be located within a radius of about 2 mm, about 1.5 mm, about 1 mm, or about 0.5 mm of the connecting siphon radius. In some embodiments, to accommodate all the required reaction volumes, the preparative chamber includes deeper portions, such as a first deeper portion 141 and a second deeper portion 142, located radially both outward and inward from the shallow portion. In some embodiments, the first and / or second deeper portions may have a depth of at least 1 mm, at least 1.5 mm, at least 2 mm, at least 2.5 mm, at least 3 mm, at least 3.5 mm, or at least 4 mm. In some embodiments, the first and / or second deeper portions may have a depth of about 2 mm to about 3 mm.

[0049] In some embodiments, for each reaction structure in at least a subset of multiple reaction structures, the maximum angular length (e.g., arc length) of the upstream / preparative chamber is determined by the total reaction volume and the need to minimize the space having a minimum boundary defined by the minimum angular length occupied by the connecting siphon and the downstream features of the reaction structure. In some embodiments, for each reaction structure in at least a subset of multiple reaction structures, the angular length of the upstream / preparative chamber may be between about 1 mm and about 5 mm, between about 2 mm and about 6 mm, between about 3 mm and about 7 mm, or between about 4 mm and about 8 mm.

[0050] In some embodiments, the device 200 includes an overflow chamber 230 and an overflow channel 240. The overflow channel 240 connects the preparative chamber of a reaction structure, for example, the last reaction structure of a group of reaction structures, to the overflow chamber. For example, in the illustrated embodiment, the overflow channel 240 connects the preparative chamber of reaction structure 104-4 to the overflow chamber. The overflow channel allows for the measurement of the reaction volume within the reaction structure by overflowing the excess volume. In some embodiments, these reaction structures are loaded with excess volume, and only the target volume is retained, with the remainder being allowed to overflow.

[0051] In some embodiments, the overflow channel includes a siphon section 241. The siphon section 241 has an inlet 242 and a apex 243. The inlet 242 is radially horizontal with respect to the inlets 221 and outlets 222 of each connecting siphon in one or more siphons. The apex 243 is at the same height as the apex 223 of each connecting siphon in one or more siphons, or slightly radially outward therefrom. In some embodiments, the overflow channel further includes a U-channel 244 having an outlet connected to the overflow chamber. In some embodiments, the U-channel 244 includes an outer segment and an inner segment, as shown in Figures 2A and 2B. In some embodiments, the U-channel traps the liquid after overflow. The device 200 allows for simultaneous and precise control of the liquid position across all reaction chambers. In particular, it allows for parallel control of the liquid position during each reaction step across all reaction chambers of multiple reaction structures using pneumatic and centrifugal force.

[0052] For example, during initial filling, each connecting siphon is filled, forming a connection to the next reaction structure, which simply allows for liquid overflow from reaction structure (e.g., preparative chamber) N to reaction structure N+1. After this initial filling, a known volume of liquid is present in each reaction structure. Increasing the rotation speed introduces more volume of liquid into the reaction chambers, raising the radial position of the liquid on the upstream chamber. This ensures that the connecting siphons are emptied. Then, decreasing the speed lowers the radial position of the liquid on the upstream channels, wetting the connecting siphons again. Because this happens simultaneously in all reaction structures, both ends of the connecting siphons (e.g., inlet and outlet channels) are filled simultaneously, creating a void within the connecting siphon. This void prevents liquid movement between reaction structures during the mixing process. This also has the advantage of improving mixing efficiency by widening the speed range during mixing. The minimum speed can be lower than the initial filling speed, which is not possible if there is a simple overflow connection between the chambers.

[0053] The overflow channel allows for the metering of the reaction volume for each reaction structure. When all reaction structures are filled to a certain inner radius, and that radius is inside the apex 243 of the siphon portion of the overflow channel, the siphon portion of the overflow channel is primed, and all liquid inside the inlet 242 of the siphon portion of the overflow channel can be discharged into the overflow chamber. By adding a U-channel 244 to the outlet portion of the overflow channel, while defining the radial position of the apex 243 to be close to the desired target radial position of the meniscus on the upstream chamber during filling, the need for additional dead volume to enable initial discharge is minimized. This ensures that sufficient volume remains in the overflow channel when the siphon ruptures and a void is formed.

[0054] Furthermore, the device may have different reaction structures for single-reagent reactions and multi-reagent reactions that operate at the exact same operating speed and can be used in parallel on the same fluid system. For example, in some embodiments as shown in Figure 2A, the device has at least one structure (1R structure) having a single well (e.g., the first well) for a single reagent reaction, and at least one structure (2R structure) having two wells (e.g., the first and second wells) for two reagent reactions on the same device. This allows the reaction volume to be very close to the minimum volume required to reconstitute the lyophilized beads (e.g., the 1R structure uses about half the volume of the 2R structure). In addition, this provides extremely high flexibility when designing new cartridges. Referring to Figures 3A and 3B, flowcharts are shown illustrating exemplary methods 300 for performing multiple reactions in parallel according to some embodiments of the present disclosure. In the flowcharts, preferred parts of the method are shown in solid boxes, while additional, optional, or alternative parts of the method are shown in dashed boxes. It should be noted that the processes disclosed herein and illustrated in the flowcharts may be performed in their entirety or in the order presented, but are not necessarily required to be performed in that order.

[0055] Referring to block 302, in some embodiments, method 300 includes (A) obtaining a device such as device 200 disclosed herein (for example, as shown in Figure 2A). The device is rotatable about a pivot axis. In some embodiments, the device includes a plurality of reaction structures and one or more connecting siphons. The reaction structures in the plurality of reaction structures are arranged circumferentially over at least a portion of the device. Each reaction structure in the plurality of reaction structures includes a preparative chamber having an outlet and a reaction chamber having an inlet connected to the outlet of the preparative chamber. The reaction chamber is located radially outward of the preparative chamber. One or more connecting siphons are radially horizontal to one another and radially inward of the outlets of the preparative chambers of the plurality of reaction structures. Each connecting siphon of the one or more connecting siphons connects the preparative chambers of the corresponding adjacent reaction structures in the plurality of reaction structures.

[0056] Referring to block 304, in some embodiments, method 300 includes (B) rotating the device at a certain speed to fill a first portion (e.g., a first well) of the reaction chamber of each reaction structure in a plurality of reaction structures with fluid. In some embodiments, the device is rotated at a relatively low speed (e.g., about 2000 rpm to about 2500 rpm, or any other suitable speed depending on the embodiment). On the one hand, this speed is slow enough not to wet the R2 well prematurely, and fast enough to ensure that the first wells of all reaction structures are filled within an acceptable time in the workflow. On the other hand, this speed is fast enough to avoid surface effects and ensure a reproducible meniscus position during overflow.

[0057] This process is shown in Figures 4A to 4C. Initially, due to the rotation of the device, the fluid fills the first well of the first reaction structure (e.g., reaction structure 104-1), as shown in Figure 4A.

[0058] As more fluid flows into the upstream chamber of the first reaction structure, it overflows into the next reaction structure (e.g., reaction structure 104-2 via connecting siphon 220-1), and so on, as shown in Figure 4B (e.g., reaction structure 104-3 via connecting siphon 220-2, and then reaction structure 104-4 via connecting siphon 220-3). The time it takes to fill the first well of all reaction structures depends at least in part on the number of reaction structures, the upstream flow rate, the rotation speed, or a combination thereof. It may be long or short. In some embodiments, it may take several seconds, tens of seconds, a minute, or more than a minute. During this period, the position of the fluid within the reaction structures may fluctuate; for example, the radial position of the fluid meniscus in the upstream chamber may fluctuate between radial positions 402-1 and 402-2, and the radial position of the fluid meniscus in the reaction chamber may fluctuate between radial positions 404-1 and 404-2. Note that the radial positions shown in Figure 4B are illustrative and not limiting. The actual radial position depends at least in part on the application (e.g., fluid volume, applied rate) and the configuration of the reaction structure (e.g., size and shape of the first well and / or intermediate chamber).

[0059] When the last reaction structure (e.g., reaction structure 104-4) is filled with fluid, the liquid position in the preparative chamber (e.g., upstream chamber 140) is inside the top 406 of the overflow siphon in Figure 4C (e.g., the radial position of the top 243 of the siphon portion of the overflow channel 240). In this way, the fluid overflows and all excess volume is discharged from all reaction structures into the overflow chamber (e.g., overflow chamber 230). This leaves each reaction structure with a metered reaction volume defined by the connecting siphon and overflow channel.

[0060] Referring to block 306, in some embodiments, method 300 includes (C) increasing the rate to discharge fluid from each corresponding connecting siphon in one or more connecting siphons. In some embodiments, increasing (C) moves at least a portion of the fluid from the preparative chamber of each of the multiple reaction structures so that the fluid contained in the preparative chamber of each of the multiple reaction structures is located outside the inlet and outlet of each corresponding connecting siphon in one or more connecting siphons, thereby discharging the fluid from each corresponding connecting siphon in one or more connecting siphons.

[0061] For example, as a non-limiting example, Figure 4D shows an increase in rotational speed (e.g., between 2000 rpm and about 2500 rpm to 2500 rpm and about 3000 rpm, or any other suitable speed depending on the embodiment) that moves the meniscus position 404 of the fluid in each reaction chamber radially inward and the meniscus position 402 in each upstream chamber (e.g., preparative chamber) radially outward. In some embodiments, the meniscus positions 402 and / or 404 are moved so as not to wet the R2 chamber (e.g., the second well).

[0062] Referring to block 308, in some embodiments, method 300 includes (D) reducing the rate to create a gas volume confined inside each corresponding connecting siphon in one or more connecting siphons, thereby interrupting fluid communication between the multiple reaction structures. In some embodiments, reducing (D) moves at least a portion of the fluid contained in the reaction chamber of each of the multiple reaction structures to the preparative chamber of each of the multiple reaction structures, so that the fluid contained in the preparative chamber of each of the multiple reaction structures is positioned inside the inlet and outlet of each corresponding connecting siphon in one or more connecting siphons, thereby wetting the inlet and outlet of each corresponding connecting siphon in one or more connecting siphons with the gas volume confined inside each corresponding connecting siphon.

[0063] For example, as a non-limiting example, Figure 4E shows that fluid moves from the reaction chamber to the preparative chamber due to a decrease in rotational speed (e.g., from between 2500 rpm and about 3000 rpm to about 900 rpm, or any other suitable speed depending on the embodiment). In some embodiments, the entire volume of fluid in the reaction chamber is moved out of the reaction chamber. In some embodiments, only a portion of the volume of fluid in the reaction chamber is moved out of the reaction chamber. For example, in some specific embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the volume of fluid in the reaction chamber is moved out of the reaction chamber. In some other specific embodiments, at most 90%, at most 80%, or at most 70% of the volume of fluid in the reaction chamber is moved out of the reaction chamber. In some embodiments, the change in speed promotes mixing. Typically, the higher the percentage of volume moving in and out, the more efficient the mixing.

[0064] As the fluid returns to the preparative chamber, it wets the inlets and overflow channels of one or more connecting siphons, creating voids between the reaction volumes and between the last reaction volume and the liquid trapped in the overflow channel. These voids separate the reaction structures from each other and from the overflow chamber, as shown in Figures 2A and 2B.

[0065] Referring to block 310, in some embodiments, method 300 includes (E) raising (C) and lowering (D) one or more times. For example, in some embodiments, the first part of the reaction chamber of each reaction structure in a plurality of reaction structures includes a first well having a first reagent, and the method includes raising (C) and lowering (D) one or more times to facilitate mixing of the fluid and the first reagent. In some embodiments, to facilitate mixing, raising (C) and lowering (D) are repeated at least once, at least five times, at least ten times, at least fifteen times, at least twenty times, or any suitable number of times to satisfy the requirements of the application. In some embodiments, the method repeats the mixing step(s) after detection, which can be performed any number of times.

[0066] Referring to block 312, in some embodiments, method 300 includes (F) detecting light transmitted through a first well or the wall of the first well in the reaction chamber for each reaction structure in a plurality of reaction structures. For example, in some embodiments, the device is rotated at a speed after R1 homogenization, as shown in Figure 4F, or at any desired time or stage during the process. Light absorption at one or more wavelengths is read through the reading well (e.g., the first well). This may take less than one minute, about one minute, several minutes, or any period of time, depending on the reaction used and its reaction kinetics.

[0067] Referring to block 312, in some embodiments, method 300 includes (G) increasing the rate to fill a second portion (e.g., a second well) of the reaction chamber for each reaction structure in at least a subset of the multiple reaction structures. For example, in a non-limiting example, Figure 4G shows increasing the rotational speed (e.g., to about 7500–8000 rpm or any other suitable speed depending on the application) to fully fill the second well and start a second incubation.

[0068] Referring to blocks 314 and 316, in some embodiments, Method 300 performs (H) lowering (D) following raising, and repeats (I) raising (G) and performing (H) one or more times. For example, in some embodiments in at least a subset of a plurality of reaction structures, the second portion of the reaction chamber of each reaction structure includes a second well having a second reagent, the Method includes repeating raising (G) and performing (H) one or more times to facilitate mixing of the fluid and the second reagent.

[0069] In some embodiments, raising (G) and running (H) are repeated at least once, at least five times, at least ten times, at least fifteen times, at least twenty times, or any appropriate number of times to satisfy the requirements of the application, in order to promote mixing. Note that in running (H), the rate can be reduced to the same rate as the rate used to promote mixing of the fluid in the first well, or to a different rate.

[0070] Referring to Block 318, in some embodiments, Method 300 includes (J) detecting light transmitted through a first well or the wall of the first well in the reaction chamber for each reaction structure in at least a subset of the multiple reaction structures. For example, in some embodiments, the device is rotated at a speed after R2 homogenization or at any desired time or stage during the process. Light absorption at one or more wavelengths is read through a reading well (e.g., a first or second well). This may take less than one minute, about one minute, several minutes, or any period of time, depending on the reaction used and its kinetics.

[0071] The devices and methods of this disclosure may have additional, optional, or alternative components. For example, in some embodiments, the device may contain more than two reagents per reaction chamber (e.g., one or more additional wells in addition to the first and second wells). Each reaction well may contain more than one bead. There are no strict limitations; most are trade-offs between available radial distance, maximum rotation speed, and available space for volume dispensing. In some embodiments, both the first and second wells may be configured as readout wells to allow reading of both reagent wells on the 2R reaction structure. In some embodiments, emptying transfers may be controlled by an additional channel after 2R reconfiguration to allow reading while the device is not rotating. In some embodiments, the device may not include a connecting siphon, and the reaction chambers of each reaction structure are loaded individually with a controlled volume. In some embodiments, some components are arranged differently to further minimize the reaction volume.

[0072] Referring to Figure 7, several exemplary embodiments of the Device 700 (e.g., a disk) of the present disclosure are shown. The Device 700 (e.g., a disk) comprises a plurality of units, such as units 710-1, 710-2, 710-3, arranged circumferentially. In some embodiments, the Device 700 comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 units. In some embodiments, unit 710 comprises one or more features / components / devices disclosed herein (e.g., reaction structure 104). In some embodiments, each unit 710 comprises one or more features / components / devices disclosed herein (e.g., reaction structure 104). In some embodiments, each unit is identical to another unit in the plurality of units. In some embodiments, at least one unit is different from other units in the plurality of units. The Device 700 can be used in a variety of applications, including but not limited to clinical chemistry.

[0073] Example workflow(s)

[0074] Figures 5A–5I collectively illustrate exemplary workflows according to several exemplary embodiments of this disclosure. Note that while specific specimens (e.g., whole blood) are used in the workflow descriptions, this disclosure is not limited to these. Other specimens, such as those disclosed herein, may also be used. Furthermore, the workflows can be automated.

[0075] Referring to Figure 5A, a schematic diagram showing a device (e.g., a disk) according to some exemplary embodiments of the present disclosure. For clarity, only a portion of the device is shown. The device 500 is rotatable about an axis of rotation, such as a vertical rotation axis 503. In some embodiments, the device 500 may rotate at a speed of at least about 1000 rpm, at least about 1200 rpm, at least about 1400 rpm, at least about 1600 rpm, at least about 1800 rpm, at least about 2000 rpm, at least about 2200 rpm, at least about 2400 rpm, at least about 2600 rpm, at least about 2800 rpm, at least about 2900 rpm, at least about 3000 rpm, at least about 3500 rpm, at least about 4000 rpm, at least about 4500 rpm, at least about 5000 rpm, at least about 5500 rpm, at least about 6000 rpm, at least about 6500 rpm, or at least about 7000 rpm during one or more processes. In some embodiments, the device 500 may rotate at a speed of at most about 500 rpm, at most about 600 rpm, at most about 700 rpm, at most about 800 rpm, at most about 900 rpm, at most about 1000 rpm, at most about 1200 rpm, at most about 1400 rpm, at most about 1600 rpm, at most about 1800 rpm, at most about 2000 rpm, at most about 2200 rpm, at most about 2400 rpm, at most about 2600 rpm, at most about 2800 rpm, at most about 2900 rpm, at most about 3000 rpm, at most about 3500 rpm, at most about 4000 rpm, at most about 4500 rpm, or at most about 5000 rpm during one or more processes.

[0076] The device 500 includes one or more chambers, one or more channels, and / or other features / components. For example, in the illustrated embodiment, the device 500 includes a ventilation channel 501, a sample chamber 502, a buffer chamber 504, a blood separation chamber 506, a sample overflow chamber 508, an additional plasma overflow chamber 509, a sample weighing chamber 510, a mixing chamber 512, a diluted sample overflow chamber 514, a pneumatic chamber 515, a pneumatic and diluted buffer overflow chamber 516, one or more 1R / detection chambers 518 (e.g., the first well 121 of the reaction chamber 110 shown in Figure 1C), one or more 2R R1 / detection chambers 520 (e.g., the first well 121 of the reaction chamber 110 shown in Figure 1A), one or more 2R R2 chambers 522 (e.g., the first well 122 of the reaction chamber 110 shown in Figure 1A), one or more dispensing chambers 524, and a mixing / weighing chamber 526.

[0077] While device 500 is shown using specific components (e.g., specific chambers, channels), it should be noted that these are illustrative and non-limiting. In some embodiments, device 500 may not include one or more of these specific components. In some embodiments, device 500 may include additional or alternative components as disclosed herein. Furthermore, device 500 can be used in a variety of applications, including but not limited to clinical chemistry.

[0078] Referring to Figure 5B, loading processes according to some exemplary embodiments of the present disclosure are shown. In some embodiments, a buffer and a sample (e.g., whole blood) are loaded into the device 500. For example, in some embodiments, the buffer is loaded into the buffer chamber 504 and the whole blood is loaded into the sample chamber 502.

[0079] Referring to Figure 5C, separation and metering processes according to some exemplary embodiments of the present disclosure are shown. In some embodiments, whole blood is separated and buffer is metered in this process. For example, in some embodiments, the device 500 is rotated around a vertical rotation axis 503. Whole blood flows from the sample chamber 502 to the blood separation chamber 506, where it is separated into plasma (radially inward) and cell fraction (radially outward). Buffer flows from the buffer chamber 504 through the mixing chamber 512 to the mixing / metering chamber 526, where it is metered. If there is an excess of buffer, the excess buffer flows into the pneumatic and dilution buffer overflow chamber 516. In this process, back pressure is accumulated by what is trapped inside the pneumatic and dilution buffer overflow chamber 516. The back pressure in the pneumatic and dilution buffer overflow chamber 516 is a function of the rotational speed of the device 500.

[0080] Referring to Figure 5D, a weighing process is shown according to several exemplary embodiments of the present disclosure. In some embodiments, plasma is weighed in this process. For example, in some embodiments, the rotation speed of disk 500 is reduced. Reducing the rotation speed of disk 500 causes the weighed buffer to flow from the mixing / weighing chamber 526 to the mixing chamber 512 as the air pressure and pressure in the dilution buffer overflow chamber 516 decrease. Furthermore, as the speed of disk 500 decreases, the plasma in the blood separation chamber 506 flows through the connecting siphon toward the sample weighing chamber 510. In some embodiments, the plasma completely fills the sample weighing chamber 510, and any excess plasma overflows into the additional plasma overflow chamber 509. At this point in the process, both the plasma and the buffer are weighed.

[0081] Referring to Figures 5E-1 and 5E-2, dilution processes according to some exemplary embodiments of the present disclosure are shown. In some embodiments, in this process, metered plasma is diluted and / or mixed with metered buffer. For example, in some embodiments, the rotation speed of device 500 is increased. By increasing the rotation speed of device 500, the metered plasma flows from the sample weighing chamber 510 to the mixing chamber 512. The rotation speed of device 500 is then decreased, increasing the air pressure and expanding the trapped air in the dilution buffer overflow chamber 516, thereby pushing the metered buffer from the mixing / metering chamber 526 to the mixing chamber 512. To mix the metered plasma and the metered buffer, the rotation speed of device 500 is increased and decreased, increasing and contracting the air pressure and the trapped air in the dilution buffer overflow chamber 516, forcing the mixture of metered buffer and metered plasma to reciprocate between the mixing / metering chamber 526 and the mixing chamber 512. The rotation speed of device 500 can be increased or decreased as desired, as programmed, or repeatedly until the solution is sufficiently / thoroughly mixed.

[0082] Referring to Figures 5F-1 and 5F-2, transfer and dissolution processes are shown according to some exemplary embodiments of the present disclosure. In some embodiments, in this step, the diluted plasma buffer mixture is transferred to the outermost detection chambers to dissolve the lyophilized reagents in those chambers. For example, in some embodiments, the rotation speed of device 500 is first reduced to prime the connection channel(s) between the mixing chamber 512 and the dispensing chamber 524(s). The rotation speed of device 500 is then increased to transfer the diluted plasma buffer mixture to the dispensing chamber 524(s), the 2R R1 / detection chamber 520(s), and the 1R / detection chamber 518(s), with any excess (if any) flowing into the diluted sample overflow 514. Figure 5F-1 shows the process before completion, and Figure 5F-2 shows the complete transfer. Due to the back pressure in the pneumatic chamber 515, the diluted plasma buffer mixture does not reach the 2R R2 chamber 522 in this step.

[0083] Referring to Figure 5G, a mixing process is shown according to several exemplary embodiments of the present disclosure. In some embodiments, in this step, the dissolved lyophilized reagent and the diluted plasma buffer mixture are thoroughly or completely mixed. For example, in some embodiments, the rotation speed of the device 500 is increased or decreased to expand and contract the trapped air in the pneumatic chamber 515 (and / or other pneumatic chambers connected to the 2R R1 / detection chambers 520(or more) and 1R / detection chambers 518(or more)) and force the mixture to reciprocate between the 2R R1 / detection chamber 520, 1R / detection chamber 518, and preparative chamber 524. Increasing / decreasing the rotation speed of the device 500 can be repeated as desired, as programmed, or until sufficient / thorough mixing is achieved. Once mixing is complete (e.g., once sufficient mixing is achieved), the reaction mixture can be returned to the 2R R1 / detection chambers 520(or more) and 1R / detection chambers 518(or more), where optical measurements can be performed as desired.

[0084] Referring to Figure 5H, a dissolution process is shown according to several exemplary embodiments of the present disclosure. In some embodiments, the lyophilized reagent in the 2R R2 chamber 522(or more) is dissolved in this step. For example, in some embodiments, the rotational speed of the device 500 is increased to deflate the trapped air in the pneumatic chamber 515 (and / or other pneumatic chambers connected to the 2R R1 / detection chamber 520(or more) and 1R / detection chamber 518(or more)), forcing the mixture into the 2R R2 chamber 522(or more) and dissolving the lyophilized reagent in the 2R R2 chamber 522(or more).

[0085] Referring to Figure 5I, mixing and detection processes according to some exemplary embodiments of the present disclosure are shown. In some embodiments, in this process, a mixture of lyophilized reagent and dilution buffer is mixed and / or optical measurements are performed. For example, in some embodiments, the rotation speed of device 500 is increased or decreased to expand and contract the confined air in the pneumatic chamber 515 (and / or other pneumatic chambers connected to the 2R R1 / detection chambers 520(or more) and 1R / detection chambers 518(or more)) and force the mixture to reciprocate between the 2R R2 chamber 522, 2R R1 / detection chamber 520, 1R / detection chamber 518, and preparative chamber 524. Increasing / decreasing the rotation speed of device 500 can be repeated as desired, as programmed, or until sufficient / thorough mixing is achieved. Once mixing is complete (for example, once sufficient mixing is achieved), the reaction mixture can be returned to the 2R R2 chamber 522, the 2R R1 / detection chamber 520, and the 1R / detection chamber 518, where optical measurements can be performed as desired.

[0086] In some embodiments, the workflow is completed at this point. The rotation of device 600 can be stopped and device 100 can be discarded.

[0087] Referring to Figure 6, an exemplary workflow 600 (e.g., workflow) is shown according to some exemplary embodiments of the present disclosure. Workflow 600 can be run on any device disclosed herein (e.g., device 500). Workflow 600 can also be automated.

[0088] In some embodiments, workflow 600 includes a process 602 for loading a buffer (e.g., water) into the device and a process 606 for loading a sample (e.g., whole blood) into the device. In some embodiments, processes 602 and 606 are the same as or similar to those disclosed herein with respect to Figure 5B.

[0089] In some embodiments, workflow 600 includes a process 604 for weighing a buffer and a process 608 for separating a sample (e.g., separating whole blood into plasma and cell fractions). In some embodiments, processes 604 and 608 are the same as or similar to those disclosed herein with respect to Figure 5C.

[0090] In some embodiments, workflow 600 includes a process 610 for measuring a sample or components of a sample. For example, in embodiments where the sample is whole blood separated into plasma and cell fractions, process 610 measures the plasma. In some embodiments, process 610 is the same as or similar to that disclosed herein with respect to Figure 5D.

[0091] In some embodiments, workflow 600 includes process 612 for mixing the metered buffer with the metered sample (e.g., metered plasma). In some embodiments, process 612 is the same as or similar to that disclosed herein with respect to Figures 5E-1 and 5E-2.

[0092] In some embodiments, workflow 600 includes process 614 for dispensing a mixture of a metered buffer and a metered sample (e.g., metered plasma). In some embodiments, process 614 is the same as or similar to that disclosed herein with respect to Figures 5F-1 and 5F-2.

[0093] In some embodiments, workflow 600 includes process 616 for resuspending the dried R1 beads(s), e.g., 2R R1 / detection chamber 520(s), and process 616 for dissolving the lyophilized reagents in 1R / detection chamber 518(s). In some embodiments, process 616 is the same as or similar to that disclosed herein with respect to Figures 5F-1 and 5F-2.

[0094] In some embodiments, the workflow includes process 618 of incubating a mixture of metered buffer, metered plasma, and dissolved lyophilized reagent. In some embodiments, process 618 may be carried out under controlled conditions (e.g., at a controlled temperature and / or over a predetermined period of time).

[0095] In some embodiments, workflow 600 includes a process 620 for resuspending the dried R2 beads(s), for example, a process 620 for dissolving the lyophilized reagent in a 2R R2 chamber(s) 522(s). In some embodiments, process 620 is the same as or similar to that disclosed herein with respect to Figure 5H.

[0096] In some embodiments, workflow 600 includes process 622 of incubating a mixture of metered buffer, metered plasma, and dissolved lyophilized reagent. Similar to process 618, in some embodiments, process 622 may be carried out under controlled conditions (e.g., at a controlled temperature and / or over a predetermined period of time).

[0097] In some embodiments, workflow 600 may include one or more measurements, which may be formed at any suitable stage as desired. For example, measurements may be performed to measure absorbance before, during, or after dissolution of the lyophilized reagent. Measurements may be the same as or similar to those disclosed herein with respect to Figures 5G and 5I. In some embodiments, workflow 600 may include a measurement process 624 after resuspending the dried R1 beads(s), a measurement process 626 after the first incubation and before resuspending the dried R2 beads(s), a measurement process 628 after resuspending the dried R2 beads(s) and before the second incubation, a measurement process 630 after the second incubation, or any combination thereof.

[0098] The devices and methods of this disclosure offer numerous advantages. For example, the devices of this disclosure are valve-free and more compact. They are independent of coating / surface tension and the total number of reaction structures and reactions. They are also insensitive to sample / biological variations and can be easily prototyped (independent of materials, surface roughness, etc.). The devices and methods of this disclosure enable well-controlled mixing strategies, readout during mixing cycles, and simultaneous execution of multiple reaction methodologies.

[0099] The devices and methods disclosed herein can be used in a variety of applications, including but not limited to clinical chemistry, immunoassay, and hematology. Examples of clinical chemistry, immunoassay, and / or hematology applications are disclosed in WO2018 / 119437, WO2018 / 140719, WO2022 / 029731, and WO2022 / 029732, the contents of which are incorporated herein by reference in their entirety. The devices and methods disclosed herein may be operated or performed by systems similar to those disclosed in U.S. Patent Application No. 17 / 371,746, the contents of which are incorporated herein by reference in their entirety.

[0100] Examples of subject matter as clauses

[0101] Various examples of the aspects of this disclosure are described for convenience as numbered clauses (1, 2, 3, etc.). These are provided as examples and do not limit the subject art.

[0102] Clause 1. A device comprising a rotating shaft and a reaction chamber, wherein the reaction chamber has an inlet for receiving fluid and a first well that serves as both a reagent well and a readout well, wherein (i) when the device is fabricated, a first reagent is placed in the first well, (ii) the radial position of the fluid meniscus in the reaction chamber depends at least in part on the rotational speed of the device, and (iii) the first well has substantially flat walls perpendicular to the rotating shaft to allow reproducible light transmission. In some embodiments, the connection between the inlet and the first well is designed to ensure that a portion of the liquid in the first well can return to the inlet.

[0103] Clause 2. The device described in Clause 1, wherein the first reagent is lyophilized.

[0104] Clause 3. A device as described in any of the preceding clauses, wherein the reagent described in Clause 1 above is in the form of beads.

[0105] Clause 4. The device according to any of the preceding clauses, wherein the reaction chamber is non-permeable to facilitate homogeneous mixing of the first reagent and the fluid.

[0106] Clause 5. The device according to Clause 4, wherein the reaction chamber comprises an outlet connected to an air chamber.

[0107] Clause 6. The device according to any of the preceding clauses, further comprising an upstream chamber located radially inward of the reaction chamber and connected to the inlet of the reaction chamber.

[0108] Clause 7. The device described in any of the preceding clauses, wherein the first well is connected to an air chamber.

[0109] Clause 8. The device according to any one of Clauses 1 to 4, wherein the reaction chamber further comprises a second well and an intermediate chamber between the first well and the second well, and when the device is fabricated, the second reagent is placed in the second well. In some embodiments, the reaction structure or chamber is non-aerated so that the meniscus level in the preparative chamber is variable during operation, etc.

[0110] Clause 9. The device described in Clause 8, wherein the second reagent is different from the first reagent.

[0111] Clause 10. The device according to any one of Clauses 8 to 9, wherein the inlet is located in the first well and the second well is located radially inward of the first well.

[0112] Clause 11. The device according to Clause 10, wherein the outlet is located in the second well.

[0113] Clause 12. The device according to any one of Clauses 8 to 11, wherein the reaction chamber, the upstream chamber, the air chamber, or a combination thereof is configured to allow initial filling of the first well with the fluid without wetting the second well.

[0114] Clause 13. A device comprising a rotating shaft, a plurality of reaction structures, and one or more connecting siphons, wherein the plurality of reaction structures are arranged circumferentially over at least a portion of the device, and each of the plurality of reaction structures comprises a preparative chamber having an outlet and a reaction chamber having an inlet connected to the outlet of the preparative chamber, the reaction chamber being located radially outward of the preparative chamber, the one or more connecting siphons being radially horizontal to one another and radially inward of the outlets of the preparative chambers of the plurality of reaction structures, and each of the one or more connecting siphons connecting the preparative chambers of corresponding adjacent reaction structures in the plurality of reaction structures.

[0115] Clause 14. The device according to Clause 13, wherein, for each of the plurality of reaction structures, the reaction chamber comprises a first well.

[0116] Clause 15. The device according to Clause 14, wherein, for each of the plurality of reaction structures, when the device is fabricated, a first reagent is placed in the first well.

[0117] Clause 16. The device according to any one of Clauses 14 to 15, wherein, for each of the plurality of reaction structures, the first well serves as both a reagent well and a readout well.

[0118] Clause 17. The device according to any one of Clauses 13 to 16, wherein the first wells of the plurality of reaction structures are radially horizontal to each other.

[0119] Clause 18. The device according to any one of Clauses 13 to 17, wherein, for each reaction structure in at least a subset of the plurality of reaction structures, the reaction chamber comprises a second well and an intermediate chamber between the first well and the second well.

[0120] Clause 19. The device according to Clause 18, wherein, for each reaction structure in at least the subset of the plurality of reaction structures, a second reagent is placed in the second well when the device is fabricated.

[0121] Clause 20. The device according to any one of Clauses 18 to 19, wherein the second wells of at least the subset of the plurality of reaction structures are radially horizontal to each other.

[0122] Clause 21. The device according to any one of Clauses 18 to 20, wherein the inlet of the reaction chamber is located in the first well and the second well is located radially inward of the first well.

[0123] Clause 22. The device according to any one of Clauses 13 to 21, wherein each of the plurality of reaction structures comprises an air chamber, and the reaction chamber of each of the reaction structures has an outlet connected to the air chamber.

[0124] Clause 23. The device according to any one of Clauses 13 to 22, further comprising an overflow chamber and an overflow channel connecting the preparative chamber of the last reaction structure among the plurality of reaction structures to the overflow chamber.

[0125] Clause 24. The device according to Clause 23, wherein the overflow channel comprises a siphon portion, the siphon portion comprising (optionally or additionally) an inlet radially horizontal to the inlets and outlets of each connecting siphon in the one or more siphons, and a apex horizontal to the apex of each connecting siphon in the one or more siphons, or radially outward from the apex.

[0126] Clause 25. The device according to Clause 24, wherein the overflow channel further comprises a U-channel having an outlet connected to the overflow chamber. In some embodiments, the outlet of the U-channel is radially outward of the siphon inlet. In some embodiments, the outer top of the U-channel is outside the channel outlet.

[0127] Clause 26. A method of using the device described in any of the preceding clauses to mix one or more fluids, resuspend reagents, detect a mixture or reaction, or a combination thereof.

[0128] Clause 27. A method for obtaining (A) a device, the device comprising a rotating shaft, a plurality of reaction structures, and one or more connecting siphons, wherein the plurality of reaction structures are arranged circumferentially over at least a portion of the device, and each of the plurality of reaction structures comprises a preparative chamber having an outlet, and a reaction chamber having an inlet connected to the outlet of the preparative chamber, wherein the reaction chamber is located radially outward of the preparative chamber, and the one or more connecting siphons are radially horizontal to each other, and the plurality of reaction structures are radially inward of the outlet of the preparative chamber, and the one or more connecting siphons The method comprises: (A) obtaining a device such that each connecting siphon connects the preparative chambers of corresponding adjacent reaction structures among the plurality of reaction structures; (B) rotating the device at a certain speed to fill a first portion of the reaction chamber of each reaction structure among the plurality of reaction structures with fluid; (C) increasing the speed to discharge the fluid from each corresponding connecting siphon among the one or more connecting siphons; and (D) decreasing the speed to create a gas volume trapped inside each corresponding connecting siphon among the one or more connecting siphons, thereby interrupting fluid communication between the plurality of reaction structures. In some embodiments, the reaction chambers are non-aerated.

[0129] The method of the preceding paragraph, wherein by raising (C), at least a portion of the fluid contained in the preparative chamber of each of the plurality of reaction structures is moved out of the preparative chamber of each of the plurality of reaction structures, such that the fluid is located outside either the inlet or the outlet of each corresponding connecting siphon in the one or more connecting siphons, thereby discharging the fluid from each corresponding connecting siphon in the one or more connecting siphons.

[0130] The method according to any one of the claims 27 to 28, wherein by lowering (D), at least a portion of the fluid contained in the preparative chamber of each of the plurality of reaction structures is moved into the preparative chamber of each of the plurality of reaction structures, thereby wetting the inlet and outlet of each of the corresponding connecting siphons in the one or more connecting siphons with the gas volume confined inside each corresponding connecting siphon.

[0131] Clause 30. The method according to any one of Clauses 27 to 29, wherein the first portion of the reaction chamber of each of the plurality of reaction structures comprises a first well having a first reagent, and the method further comprises (E) raising (C) and lowering (D) one or more times to promote mixing of the fluid and the first reagent.

[0132] Clause 31.(F) The method according to Clause 30, further comprising detecting light transmitted through the first well or the wall of the first well of the reaction chamber for each of the plurality of reaction structures.

[0133] The method according to any one of the Clauses 32.(G) increasing the rate to fill the second portion of the reaction chamber of each reaction structure in at least a subset of the plurality of reaction structures, and (H) performing the decreasing (D).

[0134] Clause 33. The method according to Clause 32, wherein the second portion of the reaction chamber of each reaction structure in at least the subset of the plurality of reaction structures comprises a second well having a second reagent, and the method further comprises (I) repeating the raising (G) and the running (H) one or more times to promote mixing of the fluid and the second reagent.

[0135] Clause 34.(J) The method according to Clause 33, further comprising detecting light transmitted through the first well or the wall of the first well of the reaction chamber for each reaction structure in at least the subset of the plurality of reaction structures.

[0136] Clause 35. A system that operates a device described in any of the preceding clauses, or performs a method described in any of the preceding clauses.

[0137] Terminology and References The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the claims. Where used in the description of embodiments and in the appended claims, the singular forms "a," "an," and "the" are intended to include the plural form unless the context clearly indicates otherwise. Terms such as "left" or "right," "up" or "down," "underside" or "upper side," "internal" or "external," "inside" or "outside," etc., will be understood to be used to describe features of exemplary embodiments with reference to the location of such features as shown in the figures. Terms such as "first," "second," etc., may be used herein to describe various elements, but it will be understood that these elements should not be limited by these terms. These terms are used merely to distinguish one element from another. For example, as long as "first element" and "second element" are consistently renamed, the first element may be referred to as the second element, and similarly, the second element may be referred to as the first element, without changing the meaning of the description.

[0138] As used herein, the term "and / or" means and encompasses any possible combination of one or more of the related enumerated items. As used herein, the terms "include," "includes," "including," "comprise," "comprises," and / or "comprising" identify the presence of the described features, components, processes, operations, elements, and / or elements, but do not exclude the presence or addition of one or more other features, components, processes, operations, elements, elements, and / or groups thereof.

[0139] In this specification, the terms “about” or “approximately” are used to provide literal support not only for the exact number preceding the term, but also for a number that is close to or nearly close to the number preceding the term. When determining whether a number is close to or approximates a specifically mentioned number, a close or approximate non-enumerated number may be a number that, in the context in which it is presented, provides substantial equivalence to the specifically mentioned number. All numbers and ranges disclosed herein, whether “about” is used with them or not, should be understood as approximations and approximate ranges. Furthermore, the term “about” as used herein should be understood as referring to a value that, in relation to a number, is ±0.01% (inclusive), ±0.1% (inclusive), ±0.5% (inclusive), ±1% (inclusive), ±2% (inclusive), ±3% (inclusive), ±5% (inclusive), ±10% (inclusive), or ±15% (inclusive) of that number. In addition, where a numerical range is disclosed herein, any number that falls within that range should also be understood as specifically disclosed.

[0140] As used herein, the term "if" is optionally interpreted, depending on the context, to mean "when," "upon," "in response to a decision," "in response to detection," or "according to the decision." Similarly, as used herein, the phrases "when a decision is made" or "[the described condition or event] is detected" are optionally interpreted, depending on the context, to mean "when a decision is made," "in response to a decision," "when "[the described condition or event] is detected," "in response to the detection of "[the described condition or event]," or "according to the decision that "[the described condition or event] has been detected."

[0141] When a reference number is given the notation "i-th", the reference number refers to a general component, set, or embodiment. For example, "unit i" refers to the i-th unit among multiple units.

[0142] All references cited herein are incorporated herein by whole reference to the same extent as individual publications or patents or patent applications are specifically and individually indicated to be incorporated by whole for all purposes.

Claims

1. It is a device, The axis of rotation and Includes a reaction chamber, The reaction chamber comprises an inlet for receiving fluid and a first well that serves as both a reagent well and a readout well, wherein (i) when the device is fabricated, a first reagent is placed in the first well; (ii) the radial position of the fluid meniscus in the reaction chamber depends at least in part on the rotational speed of the device; and (iii) the first well has substantially flat walls perpendicular to the axis of rotation to allow reproducible light transmission.

2. The device according to claim 1, wherein the reaction chamber is non-aerated to facilitate homogeneous mixing of the first reagent and the fluid.

3. The device according to claim 2, wherein the reaction chamber is provided with an outlet connected to an air chamber.

4. The device according to claim 3, wherein the first well is connected to the air chamber.

5. The device according to claim 3, wherein the reaction chamber further comprises a second well and an intermediate chamber between the first well and the second well, and when the device is fabricated, a second reagent is placed in the second well.

6. The device according to claim 5, wherein the inlet is located in the first well and the second well is located radially inward of the first well.

7. The device according to claim 6, wherein the outlet is located in the second well.

8. It is a device, The axis of rotation and Multiple reaction structures, Includes one or more connected siphons, The plurality of reaction structures are arranged circumferentially over at least a portion of the device, and each of the plurality of reaction structures comprises a preparative chamber having an outlet and a reaction chamber having an inlet connected to the outlet of the preparative chamber, the reaction chamber being located radially outward of the preparative chamber, The device wherein one or more connecting siphons are radially horizontal to one another and are located radially inward from the outlets of the preparative chambers of the plurality of reaction structures, and each of the one or more connecting siphons connects the preparative chambers of corresponding adjacent reaction structures in the plurality of reaction structures.

9. The device according to claim 8, wherein, for each of the plurality of reaction structures, the reaction chamber comprises a first well, and a first reagent is placed in the first well.

10. The device according to claim 9, wherein, for each of the plurality of reaction structures, the corresponding first well serves as both a reagent well and a readout well.

11. The device according to claim 8, wherein the first wells of the plurality of reaction structures are radially horizontal to each other.

12. The device according to claim 9, wherein, for each reaction structure in at least a subset of the plurality of reaction structures, the reaction chamber comprises a second well and an intermediate chamber between the first well and the second well, and a second reagent is disposed in the second well.

13. The device according to claim 12, wherein the second wells of at least the subset of the plurality of reaction structures are radially horizontal to each other.

14. The device according to claim 12, wherein the inlet of the reaction chamber is located in the first well, and the second well is located radially inward of the first well.

15. The device according to claim 8, wherein each of the plurality of reaction structures is provided with an air chamber, and the reaction chamber of each of the reaction structures has an outlet connected to the air chamber.

16. Overflow chamber and The device according to claim 8, further comprising: an overflow channel connecting the preparative chamber of the last reaction structure among the plurality of reaction structures to the overflow chamber.

17. The overflow channel comprises a siphon portion, and the siphon portion is The inlet and outlet of each connecting siphon in the one or more siphons, and the radially horizontal inlet, The device according to claim 16, comprising a top horizontal to the top of each connecting siphon in the one or more siphons, or a top radially outward from the top.

18. The device according to claim 17, wherein the overflow channel further comprises a U-channel having an outlet connected to the overflow chamber.

19. It is a method, (A) To obtain a device, the device is It includes a rotating shaft, multiple reaction structures, and one or more connected siphons, The plurality of reaction structures are arranged circumferentially over at least a portion of the device, and each of the plurality of reaction structures comprises a preparative chamber having an outlet and a reaction chamber having an inlet connected to the outlet of the preparative chamber, the reaction chamber being located radially outward of the preparative chamber, The one or more connecting siphons are radially horizontal to one another, located radially inward from the outlets of the preparative chambers of the plurality of reaction structures, and each of the one or more connecting siphons connects the preparative chambers of corresponding adjacent reaction structures in the plurality of reaction structures. To obtain the aforementioned (A) device, (B) Rotating the device at a certain speed in order to fill the first portion of the reaction chamber of each of the plurality of reaction structures with fluid, (C) In order to discharge the fluid from each corresponding connecting siphon in the one or more connecting siphons, (D) The method comprising reducing the speed to create a gas volume confined inside each corresponding connecting siphon in the one or more connecting siphons, thereby interrupting the fluid communication between the plurality of reaction structures.

20. The method according to claim 19, wherein raising (C) causes at least a portion of the fluid contained in the preparative chamber of each of the plurality of reaction structures to be moved out of the preparative chamber of each of the plurality of reaction structures so that the fluid is located outside either the inlet or the outlet of each corresponding connecting siphon in the one or more connecting siphons, thereby discharging the fluid from each corresponding connecting siphon in the one or more connecting siphons.

21. The method according to claim 19, wherein by lowering (D), at least a portion of the fluid contained in the preparative chamber of each of the plurality of reaction structures is moved to the preparative chamber of each of the plurality of reaction structures, thereby wetting the inlet and outlet of each of the corresponding connecting siphons in the one or more connecting siphons with the gas volume confined inside each corresponding connecting siphon.

22. The first portion of the reaction chamber of each of the plurality of reaction structures comprises a first well containing a first reagent, The method according to claim 19, further comprising (E) repeating the raising (C) and lowering (D) one or more times to promote mixing of the fluid and the first reagent.

23. (F) The method according to claim 22, further comprising detecting light transmitted through the first well or the wall of the first well of the reaction chamber for each of the plurality of reaction structures.

24. (G) Increasing the rate to fill the second portion of the reaction chamber of each reaction structure in at least a subset of the plurality of reaction structures, The method according to claim 19, further comprising (H) performing the lowering (D).

25. The second portion of the reaction chamber of each reaction structure in at least the subset of the plurality of reaction structures comprises a second well having a second reagent, The method according to claim 24, further comprising (I) repeating the raising (G) and the execution (H) one or more times to promote mixing of the fluid and the second reagent.

26. (J) The method of claim 25, further comprising detecting light transmitted through the first well or the wall of the first well of the reaction chamber for each reaction structure in at least the subset of the plurality of reaction structures.

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