Nursing point concentration analyzer

Abstract

The analyzer system includes a motor, a dock coupled to the motor, and a cartridge held in the dock. The cartridge includes a fluid circuit configured to receive a sample, isolate a target analyte of the sample, and collect a first marker in an amount proportional to an amount of the target analyte in the sample. The fluid circuit includes a sample port configured to receive a sample, a mixing chamber in fluid communication with the sample port, and a fluid inlet in fluid communication with the mixing chamber. The fluid circuit includes an isolation path extending from the fluid inlet to the mixing chamber. The system also includes a fluid delivery line configured to be coupled to the fluid inlet to deliver the fluid through the fluid inlet to the cartridge and urge the fluid along the isolated path toward the mixing chamber.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 488,677, filed on March 6, 2023, 63 / 488,681, filed on March 6, 2023, and 63 / 591,546, filed on October 19, 2023, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to automated sample processing, measurement, and analysis of samples to separate, label, detect, and quantify specific target analytes that may be present at very low concentrations. Background Art

[0004] Numerous studies and advances in understanding the root causes and progression of disease have demonstrated that detecting infectious agents or injuries at an early stage, combined with appropriate treatment, substantially improves clinical outcomes. Many conditions that once required expensive symptom-based measures such as anatomical imaging (which require trained specialists to administer and interpret) can now be diagnosed at the cellular and molecular levels through the presence and / or concentration of specific biomarkers. These biomarkers include upregulated or downregulated proteins, nucleic acids, or other molecules that are highly specific for a disease condition or infection.

[0005] It is often desirable to diagnose certain conditions at the point of care, where the timing and administration of the correct treatment are crucial to patient outcomes. This is particularly true in acute care settings such as trauma centers, where patients may have experienced acute myocardial infarction (AMI), acute decompensated heart failure, pulmonary embolism, sepsis, or other conditions that require a prompt response. In non-acute settings, rapid turnaround times are also desirable, especially in the case of highly infectious diseases such as Clostridium difficile infection, where isolation may be necessary. However, even in a doctor's office or retail clinic, determining whether a condition is viral or bacterial before administering antibiotics is highly beneficial.

[0006] In some disease conditions, the concentration of the biomarker or analyte of interest is relatively high, and simple, low-cost lateral flow devices can be used for sample processing and readout. The cost of these devices and consumable components that interact with the sample is very low and can be used quickly at the point of care with relatively little or no training. However, even when using an objective reader system to measure the strips, lateral flow-based tests tend to have low accuracy and difficulty obtaining high-quality quantitative measurements. In addition, depending on the stage of the disease or infection, the concentration of the target analyte is often too low to be detected using lateral flow in blood, urine, saliva, or other sample types.

[0007] In these cases, sample processing and readout are more complex. It often requires precise metering to assess concentration, high efficiency to avoid loss of the target analyte, and centrifugation as a major step in the purification process. In addition to centrifugation, additional purification steps often include incubation with reagents containing binding partners or molecules with complementary sequences or structures to bind to the target analyte biomarker. These binding partners can be substrates, such as micro- or nanoparticles with complementary molecules on their surfaces, or molecules conjugated to transduction markers, or both. Once binding occurs, additional process steps must be taken to wash and further isolate the target analyte, suspending it in the original buffer solution or placing it on a clean surface before measurement. To perform these processing steps, multiple devices including centrifuges, mixers, incubators, precision pipettes, and thermal cyclers are used, with multiple disposable tips, tubes, plates, and other sample containers typically used to transfer and meter samples between processing steps. Once processing is complete, the processed samples are measured using highly sensitive and precise instruments to determine the presence and / or abundance of the target analyte.

[0008] While analysis of low-concentration biomarkers can take several forms, typically sample processing and measurement have the following key features:

[0009] 1. Separation step, used to perform the first separation of the target analyte from other sample components

[0010] 2. Introduction of Binding Ligands and Reagents

[0011] 3. Mixing and incubation to label the target analyte and bind it to the substrate

[0012] 4. Introduction of buffer and steps to wash away unbound markers and other contaminants

[0013] 5. Sterile containers for precise metering and sample containment during processing

[0014] 6. Efficient and accurate sample transfer

[0015] 7. Provides a measurement method with high sensitivity and accuracy to determine the presence and abundance of target analytes.

[0016] Currently, the processing and measurement of low-concentration biomarkers must be performed by trained staff or using highly specialized equipment at a centralized location. As a result, the turnaround time from sample collection to results is long, the instrument costs are high, and measurements cannot be performed at the point of care.

[0017] Accordingly, the present inventors have recognized that there is a need for an improved technology that can address the key features listed above for low-concentration biomarker processing and measurement. The technology should be suitable for point-of-care settings, have minimal consumables, accurate metering, rapid turnaround time, and have a sensitivity that overcomes the limitations of existing technologies.

[0018] U.S. Patent No. 8,264,684 and U.S. Patent Application Publication No. 2016 / 0178520 describe systems that previously achieved extremely sensitive detection, each of which is incorporated herein by reference. The present disclosure provides further developments in this area. Summary of the Invention

[0019] Disclosed herein are analyzer systems, cartridges, and methods for detecting a target analyte in a sample. Advantageously, embodiments of the analyzer system use a compact cartridge to process and analyze the sample, which allows the analyzer to have a reduced size so that it can be provided at the point of care.

[0020] Thus, in a first aspect, the present disclosure provides an analyzer system for detecting the presence of a target analyte in a sample, the analyzer system comprising:

[0021] Motor;

[0022] a dock coupled to the motor so as to be rotated by actuation of the motor;

[0023] A cartridge retained in a dock and comprising a fluid circuit configured to receive a sample, isolate a target analyte of the sample, and collect an amount of a first marker proportional to the amount of the target analyte in the sample, the fluid circuit comprising:

[0024] a sample port configured to receive a sample,

[0025] a mixing chamber in fluid communication with the sample port and configured to mix at least a portion of the sample so as to bind the target analyte to the first label, and

[0026] a fluid inlet in fluid communication with the mixing chamber and configured to receive a wash buffer and an elution buffer,

[0027] wherein the fluid circuit includes an isolated path extending from the fluid inlet to the mixing chamber;

[0028] a fluid delivery line configured to couple to the fluid inlet so as to deliver fluid to the cartridge through the fluid inlet and to urge the fluid along the isolated path toward the mixing chamber;

[0029] a first magnet fixed to the stage, the first magnet being movable relative to the cartridge and configured to move the paramagnetic beads within the cartridge;

[0030] a first electromagnetic radiation source configured to provide electromagnetic radiation to form a detection space within the detection chamber of the cartridge;

[0031] a first detector configured to detect electromagnetic radiation emitted by the marker in the detection space if the marker is present in the detection space; and

[0032] A controller is configured to identify the presence of a target analyte in the sample based on the electromagnetic radiation detected by the first detector.

[0033] In another aspect, the present disclosure provides a method comprising:

[0034] receiving the cartridge in an analyzer system such that the cartridge is coupled to a motor of the analyzer system;

[0035] The cartridge is rotated using a motor so that a volume of sample moves toward a mixing chamber in the cartridge;

[0036] A volume of sample is mixed in a mixing chamber by moving the cartridge to bind the target analyte and marker to the paramagnetic capture beads;

[0037] introducing a series of fluids from a primed fluid delivery line into the cartridge through a fluid inlet, the series of fluids including a wash buffer and an elution buffer;

[0038] Propelling a series of fluids along isolated paths in a first direction from the fluid inlet to the mixing chamber;

[0039] Using a magnet, the paramagnetic capture beads are moved out of the mixing chamber along an isolated path in a second direction toward the fluid inlet.

[0040]

[0011] Aspects of the present invention, as well as other aspects, advantages and alternatives, will become apparent to those of ordinary skill in the art from a reading of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are included to provide a further understanding of the methods and apparatuses of the present disclosure and are incorporated into and constitute a part of this specification. The drawings are not necessarily drawn to scale, and the dimensions of various elements may be distorted for clarity. The accompanying drawings illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles and operation of the present disclosure.

[0042] Figure 1 is a schematic perspective side view of a high sensitivity analyzer according to an embodiment of the present disclosure;

[0043] Figure 2 yes Figure 1 A schematic perspective top view of an analyzer;

[0044] Figure 3 is Figure 1 A schematic perspective view of the optical system of a processing quality control camera used in an analyzer;

[0045] Figure 4 yes Figure 1 A schematic perspective view of a portion of an analyzer comprising a centrifuge, an objective lens, and an objective lens radial stage and a Z stage;

[0046] Figure 5 yes Figure 1 a schematic perspective side view of a portion of an analyzer including a manifold and a cartridge;

[0047] Figure 6 yes Figure 5 a schematic perspective bottom view of a manifold;

[0048] Figure 7 Including objective lens Figure 1 A schematic side view of a portion of an analyzer;

[0049] Figure 8 yes Figure 1 Schematic diagram of the fluid system of the analyzer;

[0050] Figure 9 is a schematic diagram of a pump, priming fluid lines, and pump flush lines and valves according to an embodiment of the present disclosure;

[0051] Figure 10 is a schematic top view of a cartridge including several fluid circuits according to an embodiment of the present disclosure;

[0052] Figure 11 In the first example of the method according to the embodiment of the present disclosure Figure 10 A schematic top view of the fluid circuit of the cartridge;

[0053] Figure 12 In the second example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0054] Figure 13 In the third example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0055] Figure 14 In the fourth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0056] Figure 15 In the fifth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0057] Figure 16 In the sixth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0058] Figure 17 In the seventh example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0059] Figure 18 In the eighth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0060] Figure 19 In the ninth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0061] Figure 20 In the tenth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0062] Figure 21 In the eleventh example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0063] Figure 21 In the twelfth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0064] Figure 22 In the thirteenth example of the method according to the embodiment of the present disclosure Figure 11 A schematic top view of a fluid circuit;

[0065] Figure 23 is a schematic top view of a fluid circuit according to another embodiment of the present disclosure;

[0066] Figure 24 yes Figure 1 A schematic side view of a portion of an analyzer including a magnetic stage;

[0067] Figure 25 is a schematic side view of various steps in a washing operation using a magnet according to an embodiment of the present disclosure;

[0068] Figure 26 is a schematic side view of various steps in another washing operation using a magnet according to an embodiment of the present disclosure;

[0069] Figure 27 shows data from a reading process performed by an analyzer according to an embodiment of the present disclosure;

[0070] Figure 28 is a schematic top view of a fluid circuit according to another embodiment of the present disclosure; and

[0071] Figure 29 is a schematic top view of a fluid circuit according to yet another embodiment of the present disclosure.

[0072] Figure 30 is a schematic top view of a fluid circuit according to another embodiment of the present disclosure.

[0073] Figure 31 is a schematic top view of a fluid circuit according to yet another embodiment of the present disclosure.

[0074] Figure 32 is included Figure 31 Schematic top view of a box with several fluid circuits.

[0075] Figure 33 Shown include Figure 32 A system of cartridges and magnetic carriers.

[0076] Figure 34 is a schematic top view of a box according to another embodiment of the present disclosure.

[0077] Figure 35 is a schematic top view of a box according to yet another embodiment of the present disclosure. DETAILED DESCRIPTION

[0078] The following detailed description presents an overview of example embodiments of the methods and systems according to the present invention. This overview is followed by a further description of various example embodiments incorporating the methods, systems, and apparatus of the present invention.

[0079] Overview of Example Embodiments

[0080] The present invention relates to a sample processing and analysis system for isolating target analytes and determining their concentrations. The systems and methods described herein use a cartridge in the form of a single consumable disk that is coupled to a centrifuge. However, aspects of the present disclosure may also be implemented using other containers and sample processing configurations. Furthermore, while the following examples use a cartridge with a fluid circuit having a specific configuration ( Figures 10 to 22 ), but other fluid circuit configurations are possible (e.g. Figure 23 、 Figures 28 to 31 ), as further described below.

[0081] The box can include various chambers and passages for receiving and processing samples. As the sample is processed, analytes and markers can be directed through the box so as to pass through various different zones in the box. These zones can be defined by structural features, such as a narrow passage between two larger chambers, or can be different areas within the box that can contain fluids. For example, an elongated chamber formed by passages can provide several functional zones as the components of the sample move along the length of the chamber.

[0082] Separation and metering

[0083] Figure 10 A cartridge 150 is shown having three fluid circuits 151 that can be used for all sample processing, metering, and containing the resulting processed sample during measurement. Each of the fluid circuits 151 can receive a separate sample for processing and analysis. Figure 11 A more detailed view of one of the fluid circuits 151 is shown in FIG. Figures 12 to 22 Shown in Figure 10 The sequence of steps for processing a sample in one of the fluid circuits 151 of the cartridge 150 is shown. In the embodiment shown, processing involves high-speed rotation of the disc to spin down the dense elements contained in the original sample. Figure 11 As shown, the fluid circuit 151 includes a sample port 153 for receiving a sample, an inlet 154 for receiving various solutions used to process the sample, and a vent 155. Given the use of one inlet 154 and one vent 155, the circuit 151 includes a single flow path 156 along which fluid introduced through the inlet 154 travels toward the vent 155 during processing. As explained in more detail below, in some embodiments, this path can be isolated from at least the mixing chamber 175 to the input port 154, which simplifies processing.

[0084] In such Figure 13 During the initial centrifugation step shown, the sample 200 is transferred from the sample chamber 158 to the inner and outer separation regions 161, 162. The cartridge 150 is then spun at a higher speed (e.g., 7000 rpm) to separate the dense elements into the outer separation region 162, as shown. Figure 14 As shown. The resulting supernatant in the internal separation region 161 is then transferred to a mixing chamber 175 containing a reagent consisting of the binding ligand. The volumes of the internal separation chamber 161 and mixing chamber 175, as well as the method of supernatant transfer, are used to meter the amount of sample used in the process to maintain accuracy. The spin-down process and transfer can be imaged and analyzed by a process quality control camera during processing to ensure proper separation and metering.

[0085] Reagents and binding ligands

[0086] According to embodiments of the present disclosure, various binding ligands can be used. For example, the first substance can be a paramagnetic bead substrate functionalized with a molecule having a binding site specific for the target analyte. The second substance of the binding ligand can include a label conjugated to a molecule having a binding site specific for a separate but different portion of the target analyte. The third and fourth substances of the binding ligand can be used as control detection, which include a second group of functionalized paramagnetic bead substrates and a second group of fluorescent markers that emit light at a wavelength different from the first group of fluorescent markers. The third substance and the fourth substance are designed to bind to each other. Since these substances undergo the same detection process and the amount of the third substance is known a priori, it can be used as a control detection to monitor the efficacy of sample processing and measurement.

[0087] In some embodiments, the binding ligand can be pre-loaded into the cartridge. For example, the binding ligand can be Figure 15 and Figure 16 The lyophilized particles 157 may be stored in a mixing chamber in the form of dry reagents or lyophilized particles 157 identified in the lyophilized particles 157. The lyophilized particles 157 may contain a variety of binding ligand substances.

[0088] In other embodiments, the cartridge can be configured to receive a binding ligand during processing. For example, Figure 23 Shown is an embodiment of a fluid circuit 351 comprising a mouth 352 for receiving a binding ligand. The mouth 352 allows a liquid reagent 457 to be introduced into the fluid circuit 351 before or during an analytical procedure. In the illustrated embodiment, the mouth 352 is radially inside a mixing chamber 375 so that the rotation of the associated box drives the liquid reagent 457 into the mixing chamber 375. This embodiment allowing the introduction of liquid reagents can enable the general use of the box of the present disclosure. The box can receive the liquid reagent selected by the user through the mouth 352, rather than the dedicated freeze-dried particles for storage and detection. Accordingly, such an embodiment enables the user to input the application-specific binding ligand contained in the liquid reagent 457 when in use.

[0089] Mixing and incubation

[0090] Once the supernatant and reagents are in the mixing chamber, a mixing process will occur where the disc 150 will be rotated slowly but at a variable rpm. Specifically, the disc will be rotationally accelerated and decelerated at a controlled rate to execute a preferred motion trajectory during rotation. Figure 16As shown, mixing balls 176, such as brass or glass, having a density higher than that of the sample, may also be incorporated into the mixing chamber. Advantageously, the acceleration and deceleration of the cartridge will induce the balls to move through the supernatant to promote the dissolution and distribution of the dry reagents and ensure uniform mixing of all reagents and target analytes. The mixing chamber geometry may be configured along a substantially constant radius from the center of rotation. In addition, the chamber may include various features to further promote mixing and incubation and ensure that the mixture remains in the mixing chamber during the mixing process. The result of the mixing process will promote a uniform suspension to facilitate the binding of the target analyte to the paramagnetic capture beads and the marker molecules. To ensure accuracy, the mixing process may be performed within a controlled time.

[0091] The flow path 156 passes through the mixing chamber 175 via two channels 173 and 174, each extending radially inwardly from the mixing chamber 175. One or both of these channels may contain capillary barriers 178, 179 in the form of diverging channel widths, such as Figure 16 174, 181, 182. The passages 174 are arranged to hold the sample in the mixing chamber 175 ...

[0092] Once the mixing and incubation process is complete, the cartridge 150 and manifold 108 ( Figure 5 ) can be rotated into alignment so that the fluid lines 111 on the manifold 108 ( Figure 5 ) can be connected to the inlet 154 on the box ( Figure 11 ) engagement. The manifold may be coupled to a motor 110 for precise rotational motion. The manifold may also be on a movable arm 109, such as Figure 1 As shown, the manifold is lowered onto and raised from the cassette. Once the manifold is aligned and in contact with the cassette, the manifold motor enables precise rotational motion of the cassette 150 about the axis of the centrifuge motor. At this point, the centrifuge motor can be powered off, and its bearings and shaft can be used as a precise rotation stage for the cassette.

[0093] After the manifold 108 is registered on the cartridge 150, one or more magnets are moved into position above the mixing chamber. The magnets can be positioned above and below the cartridge on the Z stage with the axis of motion perpendicular to the flat surface of the cartridge, as shown in FIG. Figure 24As shown. This enables the magnet 145 to be moved closer to the box 150, thereby increasing its effective pull on the paramagnetic capture beads, while the other magnet 146 is moved away from the box 150, thereby reducing its effect. The magnet Z stage 147 is also connected to the radial stage 148. The radial stage allows the magnet to be moved closer to or away from the axis of rotation of the box. As discussed later, the various channels and chambers on the box are nominally arranged radially or circumferentially. The various Z and radial stages combined with the manifold motor enable the magnets to be placed in any desired position relative to the chambers and channels contained within the box 150.

[0094] like Figure 18 As shown, after the one or more magnets are introduced, the magnet Z stage and radial stage are controlled together with the partial cartridge rotation to perform a predetermined sequence of moves to pull all the paramagnetic capture beads, now bound to the target analyte and control marker, out of suspension. The magnet 145 on the bottom side of the cartridge is brought into proximity with the surface of the cartridge in a preferred position to pull the beads into suspension. Figure 18 The beads are then pulled into a compact mass 177. The bead mass can be imaged and analyzed by a process quality control camera to ensure that the beads have been properly pulled from the suspension.

[0095] Introduce wash buffer and wash

[0096] At this point, fluid is introduced from manifold 108 into fluid circuit 151 through inlet 154. In some embodiments, fluid can be introduced from a preloaded line containing a precise volume of the fluid to be introduced. As described below, by preloading the line with the fluid required for the process, a single pump can be used to quickly and efficiently direct all fluids through fluid circuit 151.

[0097] With the magnet holding the analyte beads in place, the pump pushes wash buffer 270 into the fluid circuit 151 through the inlet 154 and fills the circumferential channel 182, radial channel 181, elbow 180, post-mixing chamber channel 174, mixing chamber 175, and pre-mixing chamber channel 173 with wash buffer. Figure 19 As shown. This pushes the sample fluid and unbound marker out of the mixing chamber 175, thereby initiating the wash process. Wash buffer flowing through the analyte-containing mass washes the unbound marker out of the mass, and continues to do so as the wash buffer flows through the mass and out of the mixing chamber 175 toward the waste chamber 166. Aside from capillary barriers 178 and 179, channels 172, 173, 180, 181, and 182 have a generally uniform cross-sectional area with few interruptions or abrupt corners. This geometry facilitates filling with buffer without leaving air pockets that could interfere with subsequent washing, elution, and reading processes.

[0098] Once the initial wash buffer fill is complete, further wash sequences can begin. This can be accomplished with a single magnet by dragging the pellet back and forth in the mixing chamber or into the post-mixing chamber channel 174, around the elbow 180 (e.g., Figure 20 ) and downwardly through radial passage 181 to circumferential passage 182 (as Figure 21 While being dragged through channels 174, 180, and 181, the pellet can be continuously exposed to clean wash buffer to wash away unbound and non-specifically bound markers. If desired, further washes can be performed using a second magnet, as described in more detail below.

[0099] Elution of analyte and label from paramagnetic beads

[0100] To initiate elution, the beads may be moved to a predetermined area, such as in the circumferential channel 182. Figure 21 As shown, this area is clean and not exposed to any detection components. At this time, the pump 118 pumps the pre-air separator 273 between the wash buffer 270 and the elution buffer 271 through the inlet 154, into the circumferential channel 182, and across the bead cluster to a predetermined position outside the cluster in the circumferential channel 182, as shown. Figure 22 A second air separator 273 between the bolus and the deionized water 272 will be on the side of the bolus closer to the inlet 154, thereby providing a region of elution buffer in which the target analyte and label are eluted for the final reading of the sample to count the number of target analyte molecules present.

[0101] Once the elution buffer is in place, a sequence of magnet movements similar to the wash sequence can be initiated to pull the bead mass up and down through the circumferential channel 182, thereby exposing the individual beads to the elution buffer, which will cleave the bond between the analyte and the paramagnetic bead, leaving the analyte and marker suspended in a slug of elution buffer between the air separators, e.g. Figure 22 As the magnet moves, the paramagnetic beads will follow the path of the magnetic field. Eventually, the cluster can reassemble in the channel and be dragged away from the elution zone, leaving a space between the cluster and the elution zone containing the label bound to the analyte of interest, as shown in Figure 2. Figure 22 shown.

[0102] Measurement

[0103] During the measurement step, the confocal laser-based optical system focuses within the elution chamber, for example, at a point in the elution chamber that is away from the walls, upper surface, and lower surface of the chamber. Measurement and detection of the analyte occurs in the elution chamber formed by the circumferential channel 182, which thus serves as both an elution chamber and a detection chamber.

[0104] The cartridge itself can be made of ultra-low autofluorescence materials, and the elution buffer, pump materials, valves, fluid lines, etc. can be selected so that they do not shed or leach materials that might autofluoresce if carried into the elution chamber. By rotating the cartridge back and forth at a predetermined rpm via a manifold motor, a small detection volume is scanned across the liquid in the elution chamber. The detection volume is defined by the lateral extent of the laser spot and the lateral extent of the cone of light forming the laser spot. The detection volume is further defined along the optical axis by the size of the confocal aperture, which is positioned conjugate with the field in the optical system. As those skilled in the art will appreciate, the confocal architecture is used to remove light from locations far from the focal plane. The farther from the focal plane and the smaller the confocal aperture, the more light from distant locations is attenuated. In imaging applications, this reduction of out-of-focus light reduces noise and provides clear image slices. Light from locations far from the focal plane (or image slice) does not represent structure in the image slice and is therefore noise. The same noise reduction process can be applied in the present invention; however, in this case, the confocal system is not used for imaging. As the laser spot scans through the fluid, it may encounter fluorescent markers from the target analyte. Upon encountering, the laser excites fluorescence from the marker, and single photons are emitted from the marker and directed by the optical system to the detector where they are counted. On the way to the focal plane and not in the focal plane, the laser may encounter elements that autofluoresce, including glass and bonding materials, which comprise the optical system, windows on the box, the back side of the box that forms the elution chamber, or the elution buffer itself. Any fluorescence from those components is noise because it is not from the marker of the target analyte. The confocal architecture attenuates those signals by preferentially allowing signals from the target analyte marker in or near the focal plane. Therefore, when the laser passes through the target marker, the photon flow received and counted by the detector increases above the background photon level, as shown in FIG. Figure 27 As shown and described in further detail below, a processing algorithm detects the elevated photon counts and classifies them as molecules of interest. In this way, individual molecules from the target analyte can be counted to determine the concentration of the target analyte in the original sample.

[0105] The present disclosure achieves significant advantages over the prior art for accurately detecting and quantifying the amount of a target analyte in a sample with a low concentration of the target analyte. In addition, the methods and systems of the present disclosure have features suitable for deployment in a point-of-care environment. Other aspects of the present disclosure relate to methods for sample processing and analysis to isolate a target analyte and determine its concentration. These methods implement steps generally consistent with the sample processing and measurement described above.

[0106] Example Embodiments

[0107] Examples and systems are described herein. It should be understood that the words "example" and "exemplary" are used herein to mean "serving as an example, instance, or illustration." Any embodiment or feature described herein as "example" or "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments or features. In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the subject matter presented herein.

[0108] The example embodiments described herein are not meant to be limiting. It will be readily understood that the various aspects of the present disclosure, as generally described herein and illustrated in the accompanying drawings, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0109] Unless otherwise indicated, the terms "first," "second," etc. are used herein merely as labels and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Furthermore, reference to an item, for example, "second," does not require or preclude the presence of an item, for example, "first," or lower-numbered, and / or an item, for example, "third," or higher-numbered.

[0110] Reference herein to "one embodiment," "an embodiment," "an example," or "an example" means that one or more features, structures, or characteristics described in connection with the example are included in at least one implementation. The phrases "one embodiment" or "an example" in various places in the specification may or may not refer to the same example.

[0111] As used herein, a system, device, equipment, structure, article, element, component, or hardware that is “configured to” perform a particular function is actually capable of performing the specified function without any changes, rather than merely having the potential to perform the specified function after further modification. In other words, a system, device, structure, article, element, component, or hardware that is “configured to” perform a particular function is specifically selected, created, implemented, utilized, programmed, and / or designed for the purpose of performing the particular function. As used herein, “configured to” means an existing characteristic of a system, device, structure, article, element, component, or hardware that enables the system, device, structure, article, element, component, or hardware to perform the particular function without further modification. For the purposes of this disclosure, a system, device, structure, article, element, component, or hardware that is described as “configured to” perform a particular function may additionally or alternatively be described as “adapted to” and / or “operable to” perform that function.

[0112] In the following description, numerous specific details are set forth to provide a thorough understanding of the disclosed concepts, which can be practiced without some or all of these details. In other cases, details of known devices and / or processes have been omitted to avoid unnecessarily obscuring the present disclosure. Although some concepts will be described in conjunction with specific examples, it should be understood that these examples are not intended to be limiting.

[0113] Example Analyzer System

[0114] In one aspect, the present disclosure provides an analyzer system such as Figure 1 The analyzer system shown in FIG. 1 includes an analyzer 100 and a cartridge 150. The cartridge 150 is configured to receive a sample and includes a plurality of chambers for separating target analytes of the sample and collecting an amount of a first marker proportional to the amount of the target analyte in the sample. The analyzer 100 includes an optical system 120. For clarity, the components of the optical system 120 are shown in FIG. Figure 2 150 . As shown, optical system 120 includes an electromagnetic radiation source 121 configured to provide electromagnetic radiation to form a detection space within the detection chamber of cartridge 150. Optical system 120 also includes a detector 122 configured to detect electromagnetic radiation emitted by the first marker in the detection space if the first marker is present in the detection space. Other components of optical system 120 are described in more detail below.

[0115] The analyzer 100 also includes a controller 140, which Figure 1 1. Controller 140 includes a non-transitory computer-readable medium having stored thereon program instructions for executing the steps performed by analyzer 100 and identifying the presence of a target analyte in a sample based on electromagnetic radiation detected by detector 122. Controller 140 includes a processor 141, a memory 142, and a network interface 143.

[0116] The processor 141 of the controller 140 comprises a computer processing element, such as a central processing unit (CPU), an integrated circuit that performs processor operations, a digital signal processor (DSP), or a network processor. In some embodiments, the processor includes register memory for temporarily storing instructions being executed and corresponding data, as well as cache memory for temporarily storing executed instructions. Memory 142 is computer-usable memory, such as random access memory (RAM), read-only memory (ROM), or non-volatile memory such as flash memory, a solid-state drive, or a hard disk drive. In some embodiments, memory 142 stores program instructions that are executable by the controller 140 to perform the methods and operations disclosed herein. A network interface 143 provides digital communication between the controller 140 and other computing systems or devices. In some embodiments, the network interface operates via a physical wired connection, such as an Ethernet connection. In other embodiments, the network interface communicates via a wireless connection, such as IEEE 802.11 (Wi-Fi) or Bluetooth. Other communication protocols are also possible.

[0117] In some embodiments, the analyzer 100 includes at least one motor configured to rotate the cartridge to manipulate any sample placed in the cartridge and align the cartridge with components of the analyzer. In some embodiments, the motor is a centrifuge drive motor, and in other embodiments, the motor is a positioning motor. Furthermore, in some embodiments, the analyzer includes both a centrifuge and a positioning motor. For example, Figure 1 The illustrated analyzer 100 includes a centrifuge 101 and a positioning motor 110 .

[0118] In the analyzer 100, a centrifuge 101 is coupled to the cartridge 150 to spin the cartridge at a speed of at least 100 rpm. Details of the centrifuge 101 are described in Figure 4 As shown, the centrifuge drive motor 103 is configured to be coupled to the cartridge using the dock 102. Figure 4 The dock shown in FIG includes a three-point kinematic mount 104. The cartridge 150 has corresponding countersunk slots 152 to register and retain the cartridge 150 on the dock 102. Additionally, the manifold 108 ( Figure 6 ) may include a bearing 113 on a spring-loaded plunger 114 to hold the disk 150 against the dock when the disk 150 is spun by the centrifuge motor 103. Accordingly, when the cartridge 150 is inserted into the analyzer 100, the cartridge 150 can be securely held in the dock 102.

[0119] The dock 102 is connected to a centrifuge drive motor 103 for rotating the dock 102 and the cartridge 150 attached to the dock 102. In addition, the centrifuge drive motor 103 may include an electronic drive phase sensor 106 and a marker wheel 107 for high-speed, precise control of the centrifuge 101 during operation. In some embodiments, the dock 102 is directly driven by the centrifuge drive motor 103, while in other embodiments, a power transmission system (such as a gearbox or belt drive) may be used to couple the dock 102 to the centrifuge drive motor 103. As explained below, the dock 102 may also be driven by a manifold 108 (see Figure 1 A specific embodiment of the operation of the centrifuge 101 is described in more detail below.

[0120] In some embodiments, the analyzer includes a manifold 108 having one or more ports, each port configured to couple to a corresponding port of the cartridge. Figure 5 A depiction of the manifold 108 coupled to the cassette 150 is shown in FIG. Figure 6 1. A bottom view of manifold 108 is shown in FIG. 1 to illustrate fluid line port 111 and bearing 113 for holding cassette 150 to dock. To transfer fluid to the cassette, manifold 108 includes a fluid line 115 connected to port 111.

[0121] The fluid line ports 111 of the manifold 108 are registered to the cartridge 150 at ports 154 and can include seals that cover corresponding ports of the cartridge 150 to isolate fluid transfer between the manifold 108 and the cartridge 150. For example, the fluid line ports 111 can include O-rings or other features to form a seal between the manifold and the cartridge port surrounding the inlet of the cartridge 150. In some embodiments, the inlet of the cartridge 150 is already open when the cartridge 150 is placed in the analyzer. In other embodiments, the manifold is configured to pierce the cartridge 150 to open the inlet of the cartridge 150.

[0122] In some embodiments, the manifold 108 is disposed on a movable arm 109 ( Figure 1 This allows the manifold 108 to be separated from the cartridge 150 when inserting or removing the cartridge 150 from the analyzer. Figure 6 As shown, when the centrifuge 101 rotates the cartridge 150 at high speed, the bearing 113 on the plunger holds the cartridge 150 on the dock. The coupling of the manifold 108 to the cartridge 150 for rotation via the manifold can be achieved by an alignment structure. For example, Figure 6As shown, the manifold 108 may include pins 112 to secure the manifold to the cassette. When the cassette 150 is rotated by the manifold 108, the manifold moves downward onto the cassette 150 to engage the pins 112 with the mounting holes of the cassette 150. In other embodiments, the pins of the dock 102 may pass through the mounting holes 152 of the cassette 150 and into receiving holes in the manifold 108. Such a structure provides a secure connection between the manifold 108, the cassette 150, and the dock 102. As will be appreciated by those skilled in the art, other mounting structures are also possible.

[0123] In some embodiments, the analyzer 100 includes a positioning motor 110 coupled to the cartridge 150. In some embodiments, the positioning motor 110 can be coupled to the manifold 108, which is coupled to the cartridge 150. The positioning motor 110 can be configured to pivot the cartridge 150 so as to align the circumferential channel 182 (see FIG. Figure 11 ) and from the first electromagnetic radiation source 121 ( Figure 2 ) through objective lens 123 ( Figure 1 ) electromagnetic radiation alignment. Furthermore, in conjunction with one or more magnets or through sample fluid dynamics, the positioning motor 110 can be further used to circulate the target analyte through the chambers of the cartridge 150, as described in more detail below. The positioning motor 110 can be a stepper motor or another actuator with specific positioning control. For example, in some embodiments, the position of the positioning motor 110 can be specified to within 2° of rotation, or within 1° of rotation, or in increments of less than 1°. Specific examples of embodiments using the positioning motor 110 are described in more detail below.

[0124] In some embodiments, the positioning motor 110 is directly coupled to the manifold 108, while in other embodiments, a power transmission system, such as a gearbox or belt drive, may be provided between the positioning motor 110 and the manifold 108. Figure 1 In the illustrated analyzer 100, the positioning motor 110 is coupled to the cartridge 150 via the manifold 108. Specifically, the manifold 108 is disposed on a shaft of the positioning motor 110. Accordingly, the manifold 108 and the cartridge 150 can be moved synchronously while maintaining a closed fluid connection therebetween.

[0125] In some embodiments, the analyzer 100 includes an optical system 120 ( Figure 2 ), which directs electromagnetic radiation from the first electromagnetic radiation source 121 to the circumferential channel 182 of the cartridge 150 and then collects the electromagnetic radiation emitted by the marker to the first detector 122. The optical system 120 may include one or more mirrors and lenses to manipulate the electromagnetic radiation and direct the electromagnetic radiation to and from the detection space. In addition, the optical system may include an objective lens 123, such as Figure 7150 , for focusing electromagnetic radiation from a first electromagnetic radiation source onto a detection volume in a cartridge 150 . In some embodiments, the objective lens 123 is connected to a movable stage 124 that allows the objective lens to be moved relative to the cartridge 150 .

[0126] In some embodiments, the optical system 120 is a confocal system. For example, the electromagnetic radiation source 121 is imaged as a spot in the focal plane of the objective lens 123 within the circumferential channel 182. Light emitted from the markers in the circumferential channel 182 excited by the electromagnetic radiation source 121 is collected by the objective lens 123 and directed by the optical system 120 onto the confocal aperture 125 in the optical system 120, as shown in FIG. Figure 2 As shown. The confocal aperture 125 is then imaged onto the detector 122. The confocal arrangement preferably passes light from the marker in the focal plane of the objective lens 123 while excluding light from outside the focal plane. In this way, the arrangement increases the signal-to-noise ratio by transmitting the signal from the marker while excluding light from the liquid suspension, the box and elements in the optical system that does not originate from the marker. As is known to those skilled in the art, the arrangement can also use a dichroic filter 126 to reflect laser light and pass light emitted by the marker to allow only light from the marker to reach the detector while inhibiting the laser light from reaching the detector. In addition, if more than one radiation source is used to detect additional markers, one or more additional dichroic filters 126 can be used to reflect laser light and marker electromagnetic radiation from a first electromagnetic radiation source and marker while passing electromagnetic radiation from a second electromagnetic radiation source and a second marker, as Figure 2 shown. Figure 2 A three-channel optical system is shown with a dichroic filter 126 on each laser and a second dichroic filter (or mirror) on each collection channel.

[0127] In some embodiments, all components of analyzer 100 are housed in a common housing. The common housing can be small enough to fit on a work surface. For example, in some embodiments, the common housing is no larger than 1 meter in any direction. Furthermore, in some embodiments, the common housing fits within a 30-inch x 30-inch x 30-inch cube.

[0128] In some embodiments, the controller 140 includes a network interface 143 for receiving control information from a user and for outputting analysis data to the user. For example, in some embodiments, the analyzer communicates with the user through software on an external device (such as a smartphone, tablet, laptop, or desktop computer). The analyzer receives information from the user of the external device and outputs information to the user of the external device by communicating with the external device via the network interface. Such communication can be through a wireless or wired connection, such as a USB or other bus. In some embodiments, the analyzer 100 may include input and / or output devices for communicating directly with the user, such as a keyboard for receiving input and a display for outputting information. In addition, in some embodiments, the display may include a touch screen for both outputting information and receiving information from the user. In some embodiments, the analyzer includes a network interface, input, and display.

[0129] In some embodiments, the methods of the present disclosure include directing a portion of a sample through regions of cartridge 150, including chambers and channels, without cartridge 150 including any valves. Additionally, in some embodiments, cartridge 150 lacks any valves.

[0130] In some embodiments, the liquid within the cassette 150 is at least partially moved through the cassette using a pump and valve coupled to an inlet of the cassette, as described in more detail below. For example, in the illustrated embodiment, the pump 118 is used to pre-fill the priming line 116 with the various fluids in the desired amounts and in the desired order, and then pump the various fluids sequentially into the cassette 150. Figure 8 An example of such a pump arrangement is shown in .

[0131] Figure 8 The pump 118 is shown connected to an input port 131 of a dispensing valve 119. The various dispensing ports on the valve are connected to a vent port 133, a waste container port 134, a deionized water port 135, a wash buffer port 136, an elution buffer port 137, and a manifold port 138. To prepare for injecting buffer into the cartridge, the dispensing valve 119 is moved to a position between the dispensing ports to block the input port 131. When the input port 131 is blocked, a solenoid valve 130 located between the pump 118 and a waste chamber 139 opens, allowing the pump 118 to empty any contents and dump any contents into the waste chamber 139 via a waste line 132.

[0132] After pump 118 is emptied, solenoid valve 130 closes and dispense valve 119 connects input port 131 to deionized water port 135. Pump 118 draws deionized water into perfusion line 116 between dispense valve input port 131 and pump 118. Dispense valve 119 then connects input port 131 to vent port 133, and pump 118 draws in a predetermined amount of air. Dispense valve 119 then connects input port 131 to elution buffer port 137, and pump 118 draws in a predetermined amount of elution buffer into perfusion line 116. Once again, dispense valve 119 moves to connect input port 131 to vent port 133, and pump 118 draws in a predetermined amount of air. Now, dispense valve 119 connects input port 131 to wash buffer port 136, and pump 118 draws in a predetermined amount of wash buffer. At this time, the perfusion line 116 between the pump 118 and the dispensing valve 119 is perfused with the wash buffer 270, the elution buffer 271 and the deionized water 272. Figure 9 As shown. Elution buffer 271 is encapsulated in the perfusion line 116 along with air 273 to prevent any mixing between the wash buffer, deionized water, and elution buffer. It should be understood that Figure 8 and Figure 9 Not to scale.

[0133] It will be appreciated by those skilled in the art that the order in which the dispensing valves, pumps, and solenoid valves are actuated can ensure that the pump 118 is filled with deionized water with little to no air when the deionized water is initially loaded. Furthermore, it will be appreciated by those skilled in the art that the air spaces 273 between the buffers in the lines can be made very small, but large enough to prevent mixing of the buffers. These steps will ensure that the buffers are accurately dispensed into the cartridge by minimizing any spring action of air in the lines or pumps. Furthermore, it will be appreciated by those skilled in the art that executing this sequence of events can help ensure that only deionized water 272 is present in the pump. This can be advantageous because different buffers can be used depending on which assay the present invention is processing. In some cases, certain buffers may degrade the pump seals and leach contaminants into the buffer and fluid lines, where these contaminants may enter the cartridge, fluoresce during the reading process, and generate noise, which may compromise sensitivity.

[0134] Once the line 116 has been primed with buffer, the dispensing valve connects the input port 131 to the manifold port 138, and the pump 118 pushes the primed fluid to the end of the fluid delivery line 115 in the manifold. At this point, the fluid system is ready to dispense controlled volumes of fluid into the cartridge at the appropriate time for detection processing. While the depicted embodiment shows a single valve operating to fill the priming line with various fluids, in some embodiments, multiple valves can be used to fill the priming line. Similarly, embodiments of the present disclosure can include various pumps and valves that work together to inject the desired fluid into the cartridge rather than using a priming line.

[0135] In another aspect, the present disclosure provides a fluid circuit for separating a target analyte of a sample and collecting an amount of a first marker that is proportional to the concentration of the target analyte in the sample.

[0136] In some embodiments, the box 150 is planar and one or more fluid circuits of the box are located in a single plane. For example, in some embodiments, the box 150 is a circular flat disk and the chambers, passages and channels of the box are positioned circumferentially around the box. As used herein, the term circumferential refers to an angular or circumferential direction as opposed to a radial or axial direction. Unless otherwise specified, the term circumferential is not intended to mean extending around the entire circumference of the box, but rather to mean a circumferential direction in a plane of rotation. In some embodiments, at least one set of chambers and channels of a fluid circuit can be connected sequentially along the circumference around a portion of the box. In other embodiments, the box has a flat linear configuration having a single fluid circuit, or several fluid circuits arranged in one or more rows.

[0137] In some embodiments, the cartridge may include a flat base and a molded body disposed on the base, wherein the body includes an open path extending therethrough that defines the chambers and channels of cartridge 150. In some embodiments, the body may be a single, integral piece. Thus, for example, in some embodiments, the side walls and interconnecting channels of all chambers of the cartridge may be formed from a single, integral piece forming the body. Furthermore, in some embodiments, the body also forms the upper walls of the chambers and channels. In other embodiments, the upper wall of the cartridge is formed by a lid that opposes the base and is attached to the body. As an example, the body may be a single molded piece of 5 mm thick cycloolefin polymer, and the base may be a 188 micron thick layer of cycloolefin polymer. Laser welding or ultrasonic welding may be used to bond the layers to the body to provide a bond as strong as if the materials were bonded together. In some embodiments, the base of cartridge 150 extends over and encloses the chambers and microfluidic channels of the cartridge, but it may also include ports, as described above, to receive fluid or allow discharge from the cartridge.

[0138] In some embodiments, the box is configured to receive samples in the range of 50 microliters to 1 milliliter. For example, in some embodiments, the box is configured to receive samples in the range of 100 to 300 microliters. In particular, the box can include a metering chamber for receiving the sample.

[0139] In some embodiments, the cartridge includes reagents stored within at least one chamber or passageway within the cartridge. For example, in some embodiments, the cartridge includes reagents that are stabilized and dried prior to insertion of the cartridge into the analyzer. For example, the reagents can be lyophilized or dried onto the surface of one or more chambers of the cartridge. Alternatively, they can be in the form of lyophilized particles placed within one or more chambers or cartridges.

[0140] Although the cartridge is shown and described herein as a disk that rotates within an analyzer, in other embodiments, the cartridge is not a disk. Furthermore, some aspects of the present disclosure are performed without the use of a cartridge at all. For example, in some embodiments, aspects of the present disclosure are performed in discrete, separate elements that form distinct chambers.

[0141] Processing quality control camera

[0142] In some embodiments, the analyzer 100 includes a process quality control camera for monitoring the movement of substances through the box 150. For example, the process quality control camera can be installed above the box 150 to observe the substances within the box 150. In some embodiments, the process quality control camera is configured to output only the characterization of light detected in the visible wavelength spectrum, that is, the camera cannot detect infrared or ultraviolet light. In some embodiments, the controller 140 is configured to analyze the images from the process quality control camera to confirm that the sample processing occurs as expected or to detect any unexpected situations. For example, the controller 140 can be configured to detect the presence of unwanted bubbles in the detection box. Other example embodiments using the process quality control camera are described below.

[0143] In some embodiments, the analyzer includes a strobe positioned to illuminate the field of view of the process quality control camera. For example, the strobe can be configured to activate at a frequency corresponding to the rotational speed of the cartridge 150 so as to monitor a specific area of ​​the cartridge 150 as it rotates. In particular, in some embodiments, the strobe can be used while the centrifuge 101 is rotating the cartridge 150.

[0144] Optical quality control cameras

[0145] In some embodiments, analyzer 100 includes an optical quality control camera for monitoring the performance of optical system 120. For example, the optical quality control camera can use mirrors on a slide to intercept the optical path before and after the confocal stop to image the laser light at the confocal stop, ensuring that the electromagnetic radiation has the appropriate intensity, is focused at the correct location, and / or has the correct intensity distribution. To image the laser light at the confocal stop, the objective lens can be positioned so that the electromagnetic radiation source is imaged onto the surface of a window on cartridge 150. When this occurs, due to the difference in refractive index between the window and the medium on the other side of the window, a portion of the radiation will be reflected from the window back toward the objective lens. This radiation will be imaged by the optical system onto the confocal stop. The window on the cartridge can be sized to have the correct thickness to simulate the thickness of the detection chamber window and the height of the fluid layer between the window and the focused spot of the electromagnetic radiation. The image of the electromagnetic radiation at the confocal stop can be analyzed by controller 140. Controller 140 can be configured to analyze the image from the optical quality control camera to verify that the electromagnetic spot has the correct size, shape, intensity, and location relative to the confocal stop, ensuring that there are no anomalies in the optical system. The measured size, shape, intensity, and position can be compared to known and acceptable values ​​for these parameters. If the measured value is outside of or near the limits of accepted values, the controller can notify the user of the analyzer or prevent use of the analyzer.

[0146] Example Method

[0147] Figures 12 to 22 An example fluid circuit and method utilizing various embodiments of the present disclosure are shown, wherein the sample is blood. In other embodiments, the chambers of the cartridge and the methods used can be adapted for use with other sample types. For example, the analyzer, methods, and cartridges of the present disclosure can be adapted for use with other biological fluids, such as urine, diluted feces, or oral fluid. Other types of samples are also possible. Furthermore, the sample can be pure or diluted.

[0148] Loading and sample separation

[0149] like Figure 12As shown, the fluid circuit of the cartridge 150 is initially loaded with a sample 200 in an inlet chamber 158. The inlet chamber 158 includes a sample port 153 for receiving the sample 200 prior to analysis. In some embodiments, the sample 200 is received in the cartridge 150, for example by a medical professional or a robot using a syringe, prior to injection into the analyzer 100. In other embodiments, the inlet chamber 158 is loaded with the sample 200 after the cartridge 150 is received in the analyzer 100. As described above, in some embodiments, the sample port 153 can be sealed prior to injecting the sample 200, and the seal can be pierced or removed to enable injection of the sample 200. In other embodiments, the sample port 153 can be a simple opening that can be used to receive the sample 200 without being "opened." In some embodiments, the sample port 153 can be sealed after the sample has been input. In other embodiments, the manifold 108 includes a seal to cover the port when the manifold is in contact with the cartridge. In some embodiments, the inlet chamber 158 is a metering chamber configured to receive a specific amount of sample, while in other embodiments, the inlet chamber 158 is oversized and can accommodate more sample than is used in the analysis. Figures 11 to 22 The inlet chamber 158 in the illustrated example is configured to receive approximately 200 μL of liquid.

[0150] Once the sample 200 is loaded into the inlet chamber 158, as Figure 12 As shown, and the box 150 is inserted into the analyzer 100, the box 150 is connected to the centrifuge 101, as shown Figure 1 101 can rotate the cartridge 150. As explained in more detail below, the geometries of the chambers and passages within the cartridge 150 are designed to affect the transfer of fluid through the cartridge 150. To facilitate understanding of these geometries, the following description refers to cylindrical / polar directions. In particular, use of the terms "inside," "inwardly," "outwardly," "outwardly," and similar descriptors refers to radially inner and radially outer directions relative to the center of rotation of the cartridge, which is generally located near the geometric center of the cartridge. The description also refers to a first circumferential direction and a second circumferential direction, which relate to the directions in which the cartridge is configured to be rotated by the centrifuge, wherein the cartridge is configured to rotate in the first circumferential direction. For example, a region at a first circumferential end of a chamber will pass through a stationary reference position before a region at a second circumferential end of the same chamber. In Figures 12 to 22 In the embodiment shown in , the first circumferential direction is clockwise, however, other embodiments of the cartridge may be configured to rotate in the opposite direction such that in these embodiments, the first circumferential direction is counterclockwise.

[0151] With the cartridge 150 loaded in the analyzer 100, the centrifuge 101 is activated to spin the cartridge 150 so as to move the sample 200 from the inlet chamber 158 through the opening 159 into the separation region 160, as shown. Figure 13As shown. Due to "centrifugal force," an inertial phenomenon that causes objects to move outward when rotated, the rotation of the cartridge 150 causes the sample 200 to move radially outward. If the sample volume is larger than the amount required for analysis, any excess sample can flow out of the separation region 160 through the overflow channel 165. In some embodiments, the inlet chamber 158 can be offset from the center of the cartridge 150 to facilitate transfer of the sample to the separation region 160. In other embodiments, the inlet chamber 158 is located at the center of the cartridge 150 so that once loaded with sample, rotation of the disc-shaped cartridge 150 will keep the sample and any other liquid received in the cartridge 150 away from the sample port 153. Furthermore, in some embodiments, the sample port 153 can be centered on the cartridge 150. In some embodiments, to move the sample from the inlet chamber 158 to the separation region 160, the cartridge can be rotated from 0 rpm to 1000 rpm, for example, at a rate of 2000 rpm / s, and maintained at this speed for several seconds, e.g., 2-10 seconds. Thus, sample transfer can occur very quickly. The rotation rates and accelerations provided are exemplary and the actual rate selected will depend on the sample being processed and may vary from 100 rpm to 10,000 rpm in rotation with acceleration varying between 100 rpm / s and 8,000 rpm / s.

[0152] In some embodiments, the separation region can include an inner separation chamber 161 and an outer separation chamber 162 that are configured to hold the different components of the sample after they are separated. In some embodiments, the center of the inner separation chamber can be located at 19 mm from the center of rotation, and the center of the outer separation chamber can be located at 28 mm from the center of rotation. When the centrifuge rotates the box 150, the denser components of the sample are pushed radially outward into the outer separation chamber 162, while the less dense components move radially inward into the inner separation chamber 161. In some embodiments, the inner separation chamber 161 and the outer separation chamber 162 of the separation region 160 are separated by a constricted neck 163 that is, for example, located at 22 mm from the center of rotation. The constricted neck 163 has a smaller cross-sectional area than either of the chambers. For example, in some embodiments, the constricted neck 163 can have a cross-sectional area of ​​3 mm. 2 The inner separation area 162 has a cross-sectional area of ​​12 mm 2 The average cross-sectional area of ​​the outer separation region 162 is 30 mm 2 In this exemplary embodiment, the neck 163 is sized to readily allow denser components to move downwardly, while less dense components quickly move upwardly through the neck 163. However, as discussed below, when the cartridge decelerates rapidly, the converging neck 163 restricts movement of denser components into the inner separation region 161.

[0153] In order to generate an accurate concentration value for the sample for further processing, it may be beneficial to know the exact volume of the sample. If the sample fails to fill the separation region 160 and is inadvertently wasted, or if the separation region is sized to accept more than the sample volume, it may be difficult to obtain an accurate concentration value. Therefore, in some embodiments, the cartridge 150 may include various features for metering an exact amount of fluid into the separation region 160.

[0154] For example, some embodiments of the cartridge 150 may include one or more features to avoid trapping air in the cartridge, particularly during transfer of the sample from the inlet chamber 158 to subsequent chambers. If air is trapped in the separation region 160 as the sample is loaded into the separation region 160, some of the sample may flow through the overflow channel 165 prematurely, and accurate metering of the sample into the separation region 160 may be unsuccessful. Accordingly, it is beneficial to avoid the formation of trapped air in the cartridge during loading.

[0155] In some embodiments, the opening 159 is coupled to a first circumferential end of the inner separation chamber 161. As the sample moves outward from the inlet chamber 158 through the opening 159 and into the separation region 160, the rotation and / or acceleration of the cartridge 150 in the first circumferential direction by the centrifuge 101 may cause the sample to flow in a second circumferential direction. Accordingly, if the opening 159 is coupled to the middle of the inner separation chamber 161, additional precautions may be required to avoid the formation of trapped air at the corner at the first circumferential end toward the inside of the inner separation chamber 161. However, if the opening 159 is coupled to the first circumferential end of the inner separation chamber 161, as Figures 12 to 22 As shown in the box 150, it is avoided to include an inner corner that is further in the first circumferential direction than the opening 159. Likewise, air that may be trapped in such a corner is also avoided.

[0156] In addition, in some embodiments, the size and depth of the opening 159 can be contracted compared to the separation region 160. Such contraction can slow the flow of sample into the separation region 160, thereby allowing air to be cleared from the separation region 160 as it fills. In addition, the contracted size and depth can also help avoid the formation of liquid sheets on the cross-section of the separation region 160, which may also form trapped air. For example, in one embodiment, the depth of the inlet opening 159 can be 0.5 mm, while the depth of the inner separation chamber 161 is 2 mm. Accordingly, the sample flow flowing from the inlet opening 159 into the inner separation chamber 161 will not span the entire depth of the inner separation chamber 161, thereby allowing air to flow around the flow and out of the separation region 160.

[0157] Furthermore, in some embodiments, the cross-sectional area of ​​the inlet opening 159 can be narrower than the cross-sectional area of ​​the constricted neck 163 between the inner separation chamber 161 and the outer separation chamber 162. For example, the inlet opening 159 can have a cross-sectional area of ​​0.5 mm. 2 The cross-sectional area of ​​the constricted neck 163 is 3 mm 2 Accordingly, the volumetric flow rate of the sample entering the separation region 160 is less likely to impact the constricted neck 163 and trap air in the outer separation chamber.

[0158] To prevent air from being trapped in the outer separation chamber 162, in some embodiments, the inner edge 164 of the outer separation chamber 162 extends at an inwardly protruding angle as the inner edge 164 approaches the constricted neck 163 that separates the inner separation chamber 161 from the outer separation chamber 162. Accordingly, when the outer separation chamber 162 is filled with sample due to rotation of the cartridge, the air in the outer separation chamber 162 will "float" inwardly to the inner edge 164 and then along the inner edge 164 to the constricted neck 163. The air will then pass through the constricted neck 163, through the inner separation chamber 161, and out of the separation region 160.

[0159] In some embodiments, the controller 140 is configured to capture an image of the separation region 160 or a portion thereof using a process quality control camera after filling the separation region 160. The controller can be further configured to analyze the image to confirm that the volume of any bubbles within the separation region 160 is free of any bubbles or that the volume of air in the separation region is below a predetermined threshold. For example, the controller can be configured to calculate the shape of any bubbles within the separation region 160 and calculate the total volume of air within the separation region 160. If the calculated air volume is above a predetermined threshold, the controller can be configured to interrupt the analysis or disqualify the result at the end of the process. Similarly, the controller can be configured to continue the analysis if the calculated air volume is below a predetermined threshold or is zero.

[0160] In some embodiments, the separation region 160 and surrounding channels can include one or more features for accurate metering of samples and controlled separation of sample components. For example, in some embodiments, an overflow channel 165 can be positioned to enable accurate metering of the amount of sample 200 entering the separation region 160. If the amount of sample 200 received in the cartridge 150 exceeds the amount required for analysis, the excess sample will be discharged through the overflow channel 165. In some embodiments, the overflow channel 165 leads to a waste chamber 166, in which excess liquid can be stored.

[0161] Due to the rotation of the box 150 and the centrifugal force on the sample, the separation area 160 is filled from the outer end toward the inner end. Accordingly, positioning the opening of the overflow channel 165 at a specific radial position in the inner separation chamber 161 determines the amount of sample that can be loaded into the separation area 160. For example, when the centrifuge 101 rotates the box 150, the sample will move toward the outer end of the outer separation chamber 162 and produce a fill line that moves inward as the separation area 160 fills. Once the fill line reaches the radial position of the overflow channel 165, for example, at a radial distance of 17 mm, any additional volume of sample entering the separation area 160 will leave the separation area 160 through the overflow channel 165. Therefore, the amount of sample to be analyzed can be accurately measured based on the radial position of the overflow channel 165.

[0162] Separation of sample components

[0163] like Figure 14 As shown, after sample has been loaded in separation area 160, centrifuge 101 can continue to rotate box 150, so that sample 200 is separated into different components.For example, centrifuge 101 can rotate box 150, so that the denser component of sending sample outwards, and radially inwardly leave less dense component.In certain embodiments, the speed of centrifuge 101 can be increased to separate the component of sample 200.For example, in one embodiment, after loading sample, centrifuge 101 can accelerate box 150 to the speed of 1000 rpm with the acceleration of 2000 rpm / s.When reaching 1000 rpm, centrifuge 101 can further accelerate box 150 to the speed of 7000 rpm with 5000 rpm / s, and keep 90 seconds to separate component under this speed.In another embodiment, centrifuge 101 can skip initial transfer rotational speed, and directly progress to the separation speed of 10000 rpm from 0 rpm with the acceleration of 2000 rpm / s. The separation step can occur at a rotation speed of 1000 rpm to 20000 rpm, depending on the sample being analyzed, the radius of the separation chamber from the center of rotation, and the strength of the cartridge 150 against breakage. The duration of the separation can be in the range of 10 seconds to 5 minutes.

[0164] In some embodiments, the sample 200 may be whole blood, and continued rotation of the cartridge 150 may separate the red blood cells 202 from the plasma 201 , as shown in FIG. Figure 14 For example, in the separation region 160 of the illustrated embodiment, the inner separation chamber 161 can serve as a plasma compartment, and the outer separation chamber 162 can serve as a red blood cell trap. In response to the high-speed rotation of the cartridge 150, the higher-density red blood cells 202 are pushed radially outward, while the lower-density plasma 201 moves radially inward into the plasma compartment 161.

[0165] The angled inner edge 164 of the outer separation chamber 162 can help separate the components of the sample in a similar manner because it promotes the removal of air from the outer separation chamber 162, as described above. When the centrifuge 101 spins the cartridge 150, the higher density components will move outward, while the lower density components will move inward. Accordingly, similar to the flow path of air in the outer separation chamber 162 during the filling process, the light components of the sample will move inward and then follow the angled inner edge 164 of the outer separation chamber 162 until they reach the constricted neck 163 and pass through the inner separation chamber 161.

[0166] In some embodiments, the controller 140 can be configured to capture an image of the separation region or a portion thereof using a process quality control camera after the separation process. The controller 140 can also be configured to analyze the image to determine the fill level of the denser components of the sample in the separation region 160. In some embodiments, the controller 140 is configured to confirm that some of the denser components of the sample have moved outward from a predetermined fill level. The controller can also be configured to continue analysis in response to such confirmation.

[0167] For example, if the sample is whole blood, controller 140 can be configured to analyze the image to determine the red blood cell (RBC) fill level in the separation area. If the RBC fill level is outside a predetermined radius, controller 140 can be configured to continue analysis. On the other hand, if the RBC fill level is within the predetermined radius, controller 140 can be configured to send a control signal to centrifuge 101 to continue spinning the cartridge to further separate the components of the blood sample. For example, images can be captured and analyzed within a 90-second separation time. If the RBC level is within a threshold distance from the center of rotation, such as 22 mm, controller 140 can be configured to send a control signal to spin for an additional 30 seconds before capturing additional images and reassessing the RBC level. In some embodiments, the duration or speed of this additional control signal can be based on the identified RBC fill level. Alternatively, controller 140 can be configured to interrupt analysis or disqualify the results at the end of processing. In some embodiments, the method is configured to divert a portion of the sample excluding RBCs. The inclusion of RBCs would add hemoglobin to the plasma, which could affect the analysis. Accordingly, identifying the RBC fill level allows the quality of the plasma diverted for further analysis to be determined.

[0168] Likewise, in some embodiments, an image of the separation region 160 after the separation process can be analyzed by the controller to determine the clarity of the plasma in the inner separation chamber. Additionally, the controller 140 can be configured to perform the analysis in response to confirming that the plasma meets a threshold clarity.

[0169] Furthermore, in some embodiments, the controller 140 can be configured to analyze an image of the separated blood sample to determine the hematocrit level of the blood based on the radial distance of the red blood cell line and the rotation time. Those skilled in the art will readily appreciate that, for a given chamber geometry, rotation rate, and rotation time, blood with a lower hematocrit level will exhibit a separation line at a larger radius than blood with a higher hematocrit level. For a given cartridge geometry and rotation parameters, different hematocrit levels can be run and evaluated to determine a calibration table stored in the controller 140. When running a sample of unknown hematocrit, after a predetermined rotation time, the separation line can be compared to a value stored in the controller to determine the hematocrit level of the sample being run. Furthermore, the controller 140 can be configured to continue analysis in response to confirming that the hematocrit level is below a predetermined threshold.

[0170] Transfer of supernatant

[0171] like Figure 15 As shown, a portion of the sample 200 can be removed from the separation region 160 by a siphon 167 extending from the separation region 160. The siphon 167 can be a 1 mm cross-sectional area. 2 The siphon 167 is in the form of a microfluidic channel that opens into a second chamber, such as a mixing chamber 175. The siphon 167 may include a first segment 168 extending from the separation region 160, a peak 169, and a second segment 170 extending from the peak 169 to the mixing chamber 175. The first segment 168 of the siphon 167 extends from a siphon inlet 171 away from the inner separation chamber 161 toward the peak 169 in a direction having a radially inward component. Furthermore, the second segment 170 extends from the peak 169 to a siphon outlet 172 at the mixing chamber 175. The siphon outlet 172 is further radially outward than the siphon inlet 171 of the siphon 167. For example, the siphon inlet 171 may be at a radial position of 21 mm from the center of rotation, while the siphon peak may be at 16 mm from the center of rotation, and the siphon outlet 172 may be at a radial distance of 30 mm from the center of rotation. Other radial distances may be selected to suit the needs of the application, so long as the siphon outlet 172 is at a greater radial distance than the siphon inlet 171 and the peak 169 is at a lesser radial distance than both the siphon inlet 171 and the siphon outlet 171. Thus, the peak 169 is the radially innermost point of the siphon 167, and the siphon outlet 172 is radially outward compared to the siphon inlet 171. Accordingly, because the rotation of the centrifuge generally drives the sample radially outward, once a portion of the sample passes the peak 169, the siphon 167 will drive a portion of the sample from the inner separation chamber 161 to the mixing chamber 175.

[0172] In some embodiments, the siphon tube can be primed, i.e., a portion of the sample can be forced past the peak to initiate siphoning by capillary action. In other words, capillary forces can draw the sample into the first section 168 of the siphon tube 167 and over the peak 169 until the siphoning action draws more fluid from the inner separation chamber 161. The cross-sectional area of ​​the siphon tube 167 can be smaller, e.g., about 0.1 mm 2 to about 0.3 mm 2 , or about 0.2 mm 2 In other embodiments, the siphon tube 167 can be primed using a pump that draws the sample into the siphon tube 167 until the sample passes the peak.

[0173] In addition, in some embodiments, the siphon can be filled by acceleration. For example, in one embodiment, after the box 150 completes the separation step at 7000 rpm, it is slowed down to 3000 rpm by the centrifuge 101 at a deceleration rate of 2000 rpm / s to prepare for the siphon step. When the box 150 rotates in the first circumferential direction, inertia will cause the sample to be pushed to continue moving in this direction. Accordingly, if the box 150 is quickly decelerated from 3000 rpm to 0 rpm at 8000 rpm / s, for example, inertia will cause the sample 200 to continue moving in the first circumferential direction, and the sample will flow through the first section 168 of the siphon 167 and pass through the peak 169 due to the first section 168 of the siphon 167 extending along the first circumferential direction. The peak 169 is radially outside the filling level of the separation area 160. At this time, the centrifuge 101 can reverse the direction of rotation to -1000 rpm and maintain this speed at an acceleration of 2000 rpm / s. Centrifugal force will cause the fluid in channel 170 to move radially outward toward siphon outlet 172, which is radially outside of siphon inlet 171. The separation region 160 will continue to empty until the fill level is radially outward of (or "drops below") the connection of the first section 168 of the siphon 167 to the inner separation chamber 161. This method of priming and siphoning is significantly faster than capillary action and / or pump-based priming and siphoning because the entire process can occur in a few seconds. In some embodiments, the peak 169 is radially inside the overflow channel 165, which prevents sample from flowing through the siphon 167 as the separation region 160 is filled. Other rotational speeds and accelerations can be used as long as the acceleration is sufficient to force the fluid over the siphon peak 169 and the cartridge 150 continues to rotate to pull the fluid out of the separation region 160.

[0174] As described above, the first section 168 of the siphon 167 extends radially inward in the first circumferential direction. In addition, in some embodiments, the shape of the first section 168 of the siphon 167 is specifically formed to facilitate priming of the siphon 167. For example, in some embodiments, the portion of the first section 168 at the end connected to the inner separation chamber 161 is substantially parallel to the first circumferential direction, for example, within 10 degrees of parallel. As the first section 168 extends toward the peak 169, it gradually bends inward. As described above, when the box 150 decelerates, the sample is pushed along the first circumferential direction. Accordingly, with the first portion of the first section 168 substantially aligned with the first circumferential direction, the sample flows into the siphon 167 with a large momentum. As a result of this momentum, the sample is able to reach and flow through the peak 169, thereby priming the siphon 167.

[0175] In some embodiments, the connection location between the first section 168 of the siphon tube 167 and the inner separation chamber 161 is selected to transfer a metered amount of sample through the siphon tube 167. For example, Figure 13 In the embodiment depicted in FIG, the siphon 167 will transfer a precise amount of sample, for example, 50 microliters, based on the radial distance between the opening of the overflow channel 165 and the opening of the first section 168 of the siphon 167. As the sample is transferred through the siphon 167, the fill level in the inner separation chamber 161 will decrease (i.e., move radially outward) and be replaced by air from the opening 159 of the inlet chamber 158 or the overflow channel 165. Once the interface between the sample and the air reaches the first section 168 of the siphon 167, no additional amount of sample will be drawn from the inner separation chamber 161. Accordingly, the location at which the first section 168 opens into the inner separation chamber 161 can be used to define the metered amount of sample transferred to the downstream chamber.

[0176] The location of the opening 171 of the first section 168 of the siphon tube 167 into the inner separation chamber 161 can also be selected to restrict the transfer of only certain components of the sample through the siphon tube 167. For example, in embodiments where the sample is whole blood and the separation chambers 162, 161 are used to separate red blood cells from plasma, the opening 171 of the first section 168 can be positioned radially inward from the separated red blood cells. Inadvertent inclusion of red blood cells in the sample transferred to the mixing chamber can lead to hemoglobin contamination during the mixing process. Accordingly, it is advantageous to position the opening of the first section 168 to avoid including red blood cells in the sample transferred through the siphon tube 167. Thus, in the case where the outer separation chamber 162 is a red blood cell trap configured to receive red blood cells after the separation process, the opening 171 of the first section 168 can be positioned radially inward from the red blood cell trap and positioned within the plasma container. Likewise, the volume of the outer separation chamber 162 can be selected based on typical red blood cell volumes, such as a 52% hematocrit level, to ensure that the volume of the red blood cell trap can accommodate the volume of red blood cells present in most whole blood samples.

[0177] In some embodiments, the outer separation chamber 162 extends in a first circumferential direction away from the constricted neck 163. Accordingly, as the cartridge 150 decelerates and less dense components of the sample are pushed through the siphon 167, denser components are likewise pushed toward the closed end of the outer separation chamber 162 and away from the constricted neck 163 and the siphon inlet 171. For example, in embodiments using whole blood, as plasma above the constricted neck 163 is displaced through the siphon 167, red blood cells are pushed toward the closed end of the red blood cell trap formed by the outer separation chamber 162.

[0178] As discussed, a large deceleration rate can be used to prime the siphon. As the cartridge decelerates, the dense components in the outer separation chamber move toward the closed end and away from the converging neck 163. However, there may be a density gradient in the outer separation chamber, where the fluid density is higher toward the more radially outward portion of the outer separation chamber 162. In this case, there may be some backflow at the top of the outer separation chamber, where the separated components at the top of the chamber move toward the converging neck 163. If these components move far enough toward the neck 163, they can be carried upward into the upper separation chamber 161 and siphoned from the outer separation chamber 162 into the mixing chamber 175.

[0179] In some embodiments, the cartridge includes a post 191 within the outer separation chamber 162. The post 191 is formed by an attachment structure that extends through the outer separation chamber 162 and secures the opposing sides of the cartridge together. For example, the post 191 can be formed by a raised protrusion in the body of the cartridge that is attached to the lid of the cartridge to provide support between the body and the lid within the outer separation chamber 162. This support can help prevent separation between the lid and the body when the cartridge rotates, particularly when sample components are separated at high rpm and the pressure within the outer separation chamber 162 increases.

[0180] Sample mixing

[0181] The plasma 201 moves from the separation region 160 to the mixing chamber 175, which may have a reagent 157 therein, such as Figure 16 For example, the mixing chamber 175 may include lyophilized paramagnetic capture beads, a detection marker, a control analyte, and a control marker. Once in the mixing chamber 175, the plasma is mixed with the reagents by rapid acceleration and deceleration of the cartridge 150 while continuing to rotate in a first circumferential direction, as shown. Figure 17 shown.

[0182] In some embodiments, mixing of the plasma with the reagent is facilitated by mixing balls 176 disposed in the mixing chamber 175. As the cartridge 150 rotates in a first circumferential direction, acceleration and deceleration of the cartridge 150 causes the mixing balls 176 to move back and forth through the mixing chamber 175, bouncing off its walls. For example, in one embodiment, the centrifuge 101 can move the cartridge 150 at a rotational speed between 200 rpm and 500 rpm, accelerating and decelerating at 1500 rpm / s. This corresponds to a mixing frequency of 5 Hz. The turbulent motion of the mixing balls 176 initially rehydrates and releases the paramagnetic capture beads, the detection marker, the control analyte, and the control marker into the plasma. The mixing balls 176 also help facilitate the mixing of the target analyte with the paramagnetic capture beads 177 ( Figure 18 ) and the detection marker. After the mixing step, the target analyte and the detection marker can be attached together and attached to the paramagnetic capture beads dispersed throughout the plasma. In some embodiments, the rehydration of the reagents and the incubation of the target analyte occur in less than 20 minutes, for example, less than 10 minutes, or less than 5 minutes.

[0183] In some embodiments, mixing chamber 175 has geometric features that enhance the mixing ability of mixing ball 176 by changing the direction of mixing ball 176. For example, in some embodiments, the outer surface of mixing chamber 175 includes a rough or textured surface to promote the bouncing of mixing ball when mixing ball rolls back and forth. Similarly, in some embodiments, the outer surface of mixing chamber 175 may include radially inward protrusions so that when mixing ball passes over the protrusions, it is made to "jump". In addition, in some other embodiments, the end of mixing chamber 175 is tilted in a radially inward direction to push mixing ball inward at the end of mixing chamber and make mixing ball reverse and return through mixing chamber near the radial inside of mixing chamber. For example, both ends may have such an inclined plane to realize the figure 8 pattern of mixing ball when box rotates back and forth.

[0184] The term "mixing ball" is used herein to refer to the movement of the feature, rather than to any particular shape. Thus, the mixing ball 176 can be spherical in some embodiments, but have another shape in other embodiments. As an example, the mixing ball 176 can be elliptical, cubic, or star-shaped. In some embodiments, the mixing ball is non-magnetic. As used herein, the term non-magnetic includes those materials that are neither magnetic nor paramagnetic. Additionally, in some embodiments, the surface of the mixing ball includes a substance with low reactivity. For example, in some embodiments, the mixing ball 176 can include brass, glass, or Teflon. Plastics, ceramics, or other hard materials with a density higher than that of the sample can also be used for the mixing ball. In other embodiments, particularly in embodiments where paramagnetic capture beads are not used, the mixing ball 176 can include a ferromagnetic material, such as steel. Similarly, in some embodiments, the mixing ball is coated with a substance with low reactivity.

[0185] In some embodiments, the mixing chamber 175 and surrounding channels include one or more features to retain the sample in the mixing chamber during the mixing process. Figure 17 As shown, in fluid circuit 151, both front mixing chamber channel 173 and rear mixing chamber channel 174 extend radially inward from mixing chamber 175. Accordingly, when cartridge 150 is spun by centrifuge 101, the centrifugal force pushes the sample outward and into mixing chamber 175.

[0186] Likewise, to prevent the sample from moving out of the mixing chamber by capillary action, at least one of the channels 173, 174 directly connected to the mixing chamber 175 may include a capillary barrier 178, 179. Figure 16In the box 150 shown, the front mixing chamber channel 173 and the rear mixing chamber channel 174 include corresponding capillary barriers 178, 179. Each of the capillary barriers 178, 179 is formed by a section of the corresponding channel 173, 174 that expands in a direction pointing away from the mixing chamber 175. As the sample moves away from the mixing chamber 175, the expanded cross-sectional area of ​​the capillary barriers 178, 179 results in reduced capillary forces. The use of capillary barriers 178, 179 reduces the effects of capillary action and retains the incubated fluid in the chamber after mixing and incubation of the paramagnetic capture beads, detection markers, control analytes and control markers. This allows the magnet 145 time to pull the paramagnetic beads out of the suspension without the incubated fluid leaving the chamber 175, as discussed in more detail below. Figure 12-22 In the embodiment shown, the capillary partitions are in the form of diamonds. In other embodiments, other shapes that expand as they protrude away from the mixing chamber 175 are also possible.

[0187] Furthermore, using two capillary barriers can help balance the forces on the sample to retain it within mixing chamber 175. For example, mixing chamber 175 can be filled to a point where the fill line lies within capillary barriers 178 and 179 on either side of mixing chamber 175. Accordingly, if the sample moves toward one side of the mixing chamber, causing the fill line in one of the channels to move radially inward toward the widened portion of the corresponding capillary barrier (e.g., 178), the capillary forces within that channel will decrease. Simultaneously, the fill line in the channel on the opposite side of mixing chamber 175 will move radially outward into the smaller cross-sectional area of ​​the opposing capillary barrier (e.g., 179), where the capillary forces will be stronger. Thus, the capillary forces on the sample from both capillary barriers will help retain the sample within the mixing chamber. To help promote this balancing effect, in some embodiments, the two capillary barriers 178 and 179 are located at the same radial position.

[0188] Capillary barriers 178, 179 can also be used as a reservoir to keep a portion of the sample during the early stages of the mixing process. In certain embodiments, reagents can be stored in the box 150 in a stable and dry form. For example, the reagent can be freeze-dried before the analytical method of the present disclosure. In this case, the mixing of the plasma and the freeze-dried reagent that occurs in the mixing chamber 175 may cause the release of trapped air during the freeze-drying process. Again, due to the centrifugal force caused by the rotation of the box, as the mixing process occurs, this air will move radially inward and leave the sample. Therefore, the total volume occupied by the sample when the sample first arrives at the mixing chamber is greater than the total volume occupied by the sample later in the mixing process when the air has been released. Capillary barriers 178, 179 can be used as a reservoir to keep a portion of the sample until the air has been released and allowed to escape from the sample.

[0189] In some embodiments, the controller 140 can be configured to capture an image of the mixing chamber 175 or a portion thereof using a process quality control camera after transfer from the separation region 160. The controller 140 can also be configured to analyze the image to determine the fill level of the mixing chamber 175. Knowing the precise volume of the sample being analyzed can be used to determine the accurate concentration of the target analyte. Accordingly, the controller 140 can be configured to continue the analysis in response to determining that the volume in the mixing chamber 175 exceeds a threshold. In addition, the controller 140 can be configured to use the volume of the sample being analyzed to normalize the data generated by the analysis.

[0190] In some embodiments, the volume of the portion of the sample transferred to the mixing chamber 175 is greater than the volume of the mixing chamber, such that portions of the sample remain in the front mixing chamber channel 173 and the rear mixing chamber channel 174. Therefore, the controller 140 can be configured to identify the meniscus lines of the sample in both channels from images captured by the process quality control camera and calculate the volume based on the positions of these meniscus lines.

[0191] Figure 23 Another embodiment of a fluid circuit 351 according to the present disclosure is shown. Fluid circuit 351 includes a port 352 and a channel 355 coupled to a mixing chamber 375. Port 352 and channel 355 enable the introduction of a liquid reagent 457 into fluid circuit 351 (and associated cartridge) during use. This can occur, for example, when a sample is loaded into sample chamber 358. Rotation of the cartridge, on which fluid circuit 351 is included, causes liquid reagent 457 to move radially outward into mixing chamber 375 due to "centrifugal force." This can occur while the sample is being transferred to separation chambers 361 and 362. As previously described, liquid reagent 457 can remain in mixing chamber 375 throughout the centrifugation process. After centrifugation is complete and the supernatant is transferred to mixing chamber 375, the supernatant can be mixed with the binding ligand contained within liquid reagent 457 in a manner similar to the binding ligand contained in the lyophilized particles described above.

[0192] Although Figure 23 The illustrated embodiment shows the liquid reagent port 352 and passage 355 exiting the right side of the mixing chamber 375, where the reagent passage intersects the siphon passage, but in other embodiments, the liquid reagent passage and port can be positioned elsewhere. For example, in some embodiments, the liquid reagent passage can be directly connected to the mixing chamber between the inlet and outlet passages of the mixing chamber, with the liquid reagent port located radially inward.

[0193] Sample magnetic motion

[0194] In some embodiments, the analyzer 100 may include one or more magnets 145 configured to move paramagnetic capture beads, as described in more detail below. Figure 24As shown in the cross-sectional portion of the analyzer 100 shown, each of the magnets can be coupled to a movable stage 147, 148. The magnets 145, 146 can be positioned above or below the cartridge to enable the paramagnetic capture beads 177 to be moved from the exterior of the cartridge 150. The linear movement of the magnets 145 in the radial and axial directions combined with the rotation of the cartridge 150 caused by the manifold positioning motor 110 allows the magnets 145 to be positioned on any portion of the cartridge 150 without requiring the magnets 145 to be moved in a circumferential direction. Thus, in some embodiments, the stage 148 can be enabled to move the magnets 145, 146 forward and backward in the radial direction of the cartridge 150 using the radial magnet stage 148, and to move the magnets 145, 146 in the axial direction toward and away from the cartridge 150 using the axial magnet stage 147 to introduce or remove the magnetic attraction of the paramagnetic capture beads 177. In other embodiments, the movable stage can be operable to move in three dimensions so as to move over any portion of the cartridge 150 without rotating the cartridge 150. In some embodiments, the magnet can be an electromagnet, while in other embodiments, the magnet can be a permanent magnet. Furthermore, in some embodiments, the electromagnet can be activated using AC current to further facilitate manipulation of paramagnetic beads.

[0195] Once the contents of the mixing chamber 175 are fully mixed and the target analyte is attached to the dispersed paramagnetic capture beads 177 (e.g. Figure 17 As shown, paramagnetic capture beads 177 can be immobilized in a portion of the mixing chamber 175 by a magnet or other means. With the paramagnetic capture beads 177 immobilized in the mixing chamber 175, wash buffer 270 can be pumped through the mixing chamber 175 to remove plasma 201 therefrom, as shown. Figure 19 As wash buffer 270 from transfer line 115 is pumped into the cartridge, it travels along path 156 to mixing chamber 175, pushing plasma 201 and any other contaminants not bound to paramagnetic beads 177 out of mixing chamber 175 and toward the waste chamber. As wash buffer 270 continues to flow over paramagnetic beads 177 and the walls of the cartridge, unwanted contaminants are washed away. Through repeated washing, the concentration of such contaminants can be reduced to undetectable or manageable levels.

[0196] To facilitate movement of the paramagnetic capture beads 177 through the cartridge 150, magnets 145, 146 may be introduced. With the magnet 145 positioned adjacent the mixing chamber 175, the cartridge 150 may be rotated back and forth over the magnet 145 to aggregate the paramagnetic capture beads 177, such as Figure 18As shown. In some embodiments, controller 140 is configured to capture an image of the bead cluster after collecting the paramagnetic capture beads using magnet 145. This can occur in the mixing chamber or at some other location in the circuit. Furthermore, in some embodiments, controller 140 is configured to measure the size of the paramagnetic bead cluster and, if the size of the bead cluster is within a predetermined range, proceed with the analysis. Otherwise, controller 140 may identify an error and interrupt the analysis or disqualify the result at the end of the process.

[0197] In some embodiments, magnets 145, 146 can be introduced prior to the introduction of the wash buffer. In such embodiments, the clump of paramagnetic capture beads 177 can be held in a specific location within the mixing chamber 175 to prevent dispersion of the clump during the removal of plasma 201 from the mixing chamber 175. For example, the clump can be located in a corner of the mixing chamber 175 during the removal of plasma.

[0198] In some embodiments, wash buffer 270 is introduced from fluid delivery line 115 into cassette 150 via manifold 108 using pump 118, as shown in FIG. Figure 8 For example, in some embodiments, pump 118 moves a portion of the contents of prefilled priming line 116 into cassette 150 via manifold 108. Because path 156 ( Figure 11 ) is isolated between the inlet 154 and the mixing chamber 175, so simply injecting the contents of the perfusion line 116 into the cassette 150 will push the wash buffer 270 through the circumferential channel 182, through the radial channel 181, around the elbow 180, through the post-mixing chamber channel 174, and into the mixing chamber 175. As used herein, the term "isolated" means that there are no separate branches extending from the path or openings in the path for fluid to escape. Accordingly, fluid pumped into the cassette via the inlet 154 will ultimately reach the mixing chamber 175. In the illustrated embodiment, the isolated path continues from the mixing chamber 175 to the separation region 160. However, in other embodiments, the fluid circuit may include other branches connected to the mixing chamber. Moreover, aspects of the present disclosure may be utilized without such an isolated path.

[0199] In some embodiments, the controller 140 can be configured to capture an image of at least a portion of the mixing chamber 175 after the mixing chamber 175 is filled with the wash buffer 270. Furthermore, the controller 140 can be configured to analyze the image of the mixing chamber 175 to confirm the absence of air within the mixing chamber 175 or to confirm that the volume of any bubbles within the mixing chamber is below a predetermined threshold. For example, the controller 140 can be configured to calculate the shape of any bubbles within the mixing chamber 175 and to calculate the total volume of air within the mixing chamber 175. If the calculated air volume is above a predetermined threshold, the controller can be configured to pump more fluid or interrupt the analysis. Similarly, the controller can be configured to continue the analysis if the calculated air volume is below a predetermined threshold or is zero.

[0200] In the case where the paramagnetic capture beads 177 are collected in a clump in the mixing chamber 175, as Figure 19 As shown, the magnet 145 can be moved by the movable stage 148 in conjunction with the rotation of the cartridge 150 to carry the cluster of paramagnetic capture beads 177 around the elbow 180 and into the radial channel 181, which can serve as another washing area for the cartridge 150. In some embodiments, the controller 140 can be configured to capture an image of at least a portion of the radial channel 181 after the cluster of paramagnetic capture beads 171 has been transferred to the radial channel 181 to verify that the transfer has occurred. In addition, in some embodiments, the controller 140 is configured to measure the size of the cluster of paramagnetic beads in the radial channel 181 and, if the size of the cluster of beads in the radial channel 181 is within a predetermined range, perform the analysis. Otherwise, the controller 140 can identify an error and interrupt the analysis or disqualify the result at the end of the process.

[0201] Once the paramagnetic capture beads 177 are disposed in the radial channel 181, the movable stage 148 can be moved back and forth to effectively wash the paramagnetic capture beads 177, thereby removing appreciable levels of contaminants from the sample other than the target analyte, the detection marker, and any controls used in the system, e.g. Figure 20 In some embodiments, the used wash buffer can be repeatedly swept out of the radial channel 181 and a new volume of wash buffer 270 can be added to the radial channel 181 before the wash step is repeated. The wash step can be performed several times, for example three times or more.

[0202] In some embodiments, a second magnet 146 can be introduced during the washing step to disperse and reaggregate the paramagnetic capture beads 177 during a series of steps of the washing operation. In particular, the magnet 145 and the second magnet 146 can be positioned on opposite sides of the cartridge 150 to disperse and reaggregate the paramagnetic capture beads 177 as they move along the radial channel 181. Spreading out the paramagnetic capture beads 177 allows them to be washed more efficiently with the wash buffer than if the beads were held together. Accordingly, the time and number of cycles required for the washing step can be reduced compared to conventional washing methods.

[0203] Figure 25 and 26 Two exemplary embodiments of washing operations according to the invention are shown. Figure 25 A washing operation is shown in which the two magnets 145, 146 move in a zigzag pattern relative to the radial channel 181. In particular, Figure 25 Five discrete positions P1-P5 that the first magnet 145 and the second magnet 146 occupy during the sawtooth washing operation are shown. In position P1, the second magnet 146 is away from the box 150, while the first magnet 145 is adjacent to the box 150, which causes the paramagnetic capture beads to form a cluster adjacent to the first magnet 145. The magnets 145, 146 are then moved in the axial direction so that the second magnet 146 is pulled near the box 150, while the first magnet 145 is moved away from the box 150. In response to this movement, the movable stage 148 can also be moved so that the magnets 145, 146 are also repositioned laterally along the radial channel 181. When the first magnet 145 moves away from the paramagnetic capture beads 177, the cluster is dispersed into the washing solution so that unnecessary components of the plasma can be separated from the paramagnetic capture beads 177 and washed. The dispersion of the paramagnetic capture beads 177 is shown in FIG. Figure 25 When the second magnet 146 approaches radial channel 181, the paramagnetic capture beads 177 are pulled out of suspension and reassembled into a compact mass. The dispersion and reaggregation steps can then be repeated in the opposite direction as the magnets 145, 146 move from position P2 to position P3. Similarly, this process can continue in a zigzag pattern for several additional steps.

[0204] Figure 26 Another embodiment of a washing operation is shown in which two magnets 145, 146 move in a square wave or trapezoidal pattern relative to the radial channel 181. In particular, Figure 26The nine discrete positions P1-P9 occupied by the first and second magnets 145, 146 during the sawtooth wash operation are shown. Again, in position P1, the second magnet 146 is positioned away from the cartridge 150, while the first magnet 145 is positioned adjacent to the cartridge 150, causing the paramagnetic capture beads to form a cluster adjacent to the second magnet 146. The movable stage 148 is then moved, causing the magnets 145, 146 to move relative to the radial channel 181. Advantageously, the stage 148 can be moved at a sufficient speed to spread the paramagnetic capture beads 177 along the surface of the radial channel, thereby dispersing the paramagnetic capture beads in the wash buffer along the surface of the radial channel. The magnets 145, 146 are then moved to position P3, causing the second magnet 146 to be drawn toward the cartridge 150, while the first magnet 145 is moved away from the cartridge 150. Again, as the first magnet 145 moves away from the paramagnetic capture beads 177, the cluster is dispersed into the wash solution, allowing unwanted components of the plasma to be separated from the paramagnetic capture beads 177 and washed. Likewise, when the second magnet 146 approaches the cartridge 150, as shown at position P3, the paramagnetic capture beads 177 are pulled out of suspension and against the wash chamber wall.

[0205] Although Figure 25 and Figure 26 The embodiment of the wash operation shown in includes reaggregation of the paramagnetic capture beads into a compact mass, but in other embodiments, the paramagnetic capture beads can be directed through the channel during operation without strictly agglomerating. For example, during the operation step, the beads can remain relatively dispersed in the wash fluid but be moved back and forth along the length of the radial channel by the magnet.

[0206] As described above, in some embodiments, the magnet 145 and the second magnet 146 are positioned on opposite sides of the cartridge 150, for example, above and below the cartridge 150. In other embodiments, the magnets 145, 146 are disposed on the same side of the cartridge 150, but on opposite sides of the radial channel relative to the circumferential direction. Furthermore, in some embodiments, the magnets 145, 146 spread the paramagnetic capture beads 177 along the length of the radial channel 181. Furthermore, in some embodiments, the distance between the first magnet 145 and the second magnet 146 is varied using the Z stage 147 during the wash step. This relative movement of the magnets 145, 146 can facilitate the disruption of clusters of paramagnetic capture beads 177, thereby enhancing the wash operation.

[0207] After the washing operation, the paramagnetic capture beads 177 can be gathered again with the first magnet 145 and moved to the circumferential channel 182. The pump 118 can then be activated to further push the fluid from the perfusion line 116 along the path 156 extending from the inlet 154 to the mixing chamber 175. Specifically, the pump 118 is operated until the first air bead 273 is pushed through the mass of paramagnetic capture beads 177, so that the paramagnetic capture beads 177 are immersed in the elution buffer 271, as shown. Figure 21While the paramagnetic capture beads 177 are held by one or more magnets 145, 146, the cartridge 150 can be rotated back and forth to move the paramagnetic capture beads 177 through the circumferential channel 182 and elution buffer 271, which removes the bonds between the paramagnetic capture beads 177 and the target analyte and between the marker and the target analyte. This leaves a pure suspension of the fluorescent dye conjugate in the elution buffer 271 within the circumferential channel 182, as shown. Figure 22 shown.

[0208] To enhance the elution of target analytes and markers, a magnetic elution procedure similar to the washing procedure described above can be used. For example, the magnets 145, 146 can be moved relative to the circumferential channel 182 in a specific pattern, such as Figure 25 and Figure 26 The magnetic elution procedure is enhanced by manipulating the paramagnetic beads in a controlled manner similar to the washing procedure.

[0209] While the described embodiment uses radial channels 181 as wash zones and circumferential channels 182 as elution zones, in other embodiments, the zones where washing and elution occur may have other configurations. Furthermore, in some embodiments, washing and elution may occur in portions of the same channel. For example, in some embodiments, washing may continue into the region of the circumferential channel near the radial channels, while elution occurs in the portion of the circumferential channel closer to the inlet 154. However, it may be advantageous to maintain distinct wash and elution zones along the path 156 from the inlet to the mixing chamber 175, with the elution zone closer to the inlet 154 than the wash zone. In situations where fluid flows in a single direction along the path 156 through the fluid circuit 151, having the elution zone closer to the inlet may help ensure that elution occurs at a location where the spent wash buffer has not yet traveled.

[0210] After the elution process has been performed, the paramagnetic capture beads 177 can be moved to the outside of the circumferential channel 182, or to one end of the circumferential channel 182, as shown in FIG. Figure 22 As shown, the optical system 120 is not disturbed. The optical system 120 of the analyzer 100 can then be activated to analyze the solution in the circumferential channel to determine the presence or concentration of the target analyte in a volume of fluid in the circumferential channel, as described above. Accordingly, at least a portion of the circumferential channel 182 can be used as an elution zone, in which the elution buffer removes the label from the paramagnetic beads, as well as a detection zone.

[0211] Figure 27From left to right, the signal from the elution buffer is shown, where elution has not yet occurred. In this area, the signal in each signal interval is generally less than 30 photons. Near the middle of the figure, the area where the marker has been eluted is clearly visible as a series of intervals with signals above 30 photons. To the right of the elution zone, another area is visible in which no marker is eluted, and the signal is also generally less than 30 photons / interval. To the right of this area is an area where the bead mass is dragged after the elution process. Because the bead base fluoresces and strongly scatters light, very strong signals far above 150 photons are observed in this area. Finally, to the right of the mass area is another area of ​​clean elution buffer, in which no elution occurs.

[0212] The entire purification process briefly described herein is designed to produce a suspension containing only the isolated target analyte and the label that was once bound to the target analyte. The paramagnetic capture beads used to capture the target analyte are a source of noise in the read process. After an elution sequence in which the target analyte bond is cleaved, the dissociated paramagnetic capture beads can be pulled to a preferred position in the elution chamber just described. Figure 27 The reading process shown in can cover multiple areas in the elution channel, including the original elution buffer area, the elution area, and the bead area. These individual segments can be analyzed to identify and count single molecules from the fluorescent marker that is carried to the elution area by binding to the target analyte.

[0213] Although Figure 10-22 The fluid circuit shown in includes various different structures for processing samples using the system described herein, but the methods described herein can be performed using other fluid circuits. For example, in some embodiments, a fluid circuit can be formed that does not have any separation chamber or transfer structure. For example, the fluid circuit can include a more direct route from the sample port to the mixing chamber. In addition, in some embodiments, the fluid circuit can be configured to have no separate waste chamber. For example, the flow path of the fluid delivered to the box can be from the fluid inlet toward the sample port, wherein the sample chamber also serves as a waste chamber.

[0214] For example, Figure 28Another embodiment of a fluidic circuit 551 according to the present disclosure is shown, in which the initial centrifugation step can be omitted. Compared to circuit 151 described above, separation chambers 161 and 162 have been eliminated. In embodiments utilizing circuit 551, sample is injected into sample chamber 558 via sample port 553. When the cartridge is rotated as described above, the sample is moved into mixing chamber 575 due to centrifugal force, eliminating the need for a priming step. In this embodiment, sample volume input can be controlled, allowing the overflow channel and overflow chamber to be omitted. Circuit 551 can be used to perform the same mixing, washing, elution, and reading processes as previously described for circuit 151. Buffer is introduced into inlet 554 in a manner similar to that previously described for circuit 151. When wash buffer is introduced, the sample is pushed out of mixing chamber 575 and into sample chamber 558, which also serves as a waste chamber. In a later step, elution buffer is also introduced, pushing wash buffer into chambers 575 and 558. In some embodiments, different portions of channel 582 can be used for washing, elution, and reading. As described above with respect to loop 151, analytes generally move toward inlet 554, and buffer and waste move toward sample port 553. In this way, analytes are generally exposed to clean, uncontaminated portions of the cartridge and clean buffer. In the embodiment of loop 551, reading occurs in a radial direction along channel 582. However, in other embodiments, such a channel may have an elbow region where the channel changes direction and ultimately curves circumferentially below the mixing chamber and ultimately upward toward the center of rotation.

[0215] Accordingly, loop 551 illustrates the flexibility of the concepts described in this disclosure for use with different sample types and how different configurations can use various different aspects of the disclosure.

[0216] Figure 29 Another embodiment of a fluid circuit 751 according to the present disclosure is shown, in which the initial centrifugation step can be omitted. Figure 23 As shown, fluid circuit 751 enables liquid reagent 857 to be used. Fluid circuit 751 includes port 752 and channel 755, so that in addition to the sample, liquid reagent 857 can be input into circuit 751. Channel 755 keeps liquid reagent 857 from mixing with the sample until the disk including fluid circuit 751 thereon begins to rotate. In various embodiments, the chamber size can be adjusted to accommodate different ratios of liquid reagent and sample. As previously described, once the disk rotates, centrifugal force moves the sample and liquid reagent 857 radially outward and into mixing chamber 775. Circuit 751 can be used to perform the same mixing, washing, elution and reading processes as previously described with respect to circuit 151.

[0217] Figure 30Shown is another embodiment of a fluid circuit 951 according to the present disclosure, in which centrifugal sample separation is omitted, and liquid reagent is introduced into the fluid circuit 951 through a reagent port 952. Reagent port 952 and sample port 953 are both arranged in the sample chamber 938, but they are separated from each other by a separator 956. Separator 956 keeps the liquid reagent from mixing with the sample until the box forming the fluid circuit 951 starts to rotate. In various embodiments, the position of the separator 956 can be adjusted to adapt to the different ratios of liquid reagent and sample. Alternatively, in some embodiments, the fluid circuit may include reagent and sample port without a separator. In addition, in some embodiments, the sample chamber may include a single port suitable for receiving both sample and reagent.

[0218] As previously described, once the cartridge is spun, centrifugal force moves the sample and liquid reagents radially outward, driving these components into the mixing chamber 975. The same mixing, washing, elution, and reading processes as previously described with respect to the circuit 151 can be performed using the circuit 951.

[0219] Similar to fluid circuit 151, fluid circuit 951 includes a series of channels leading from a mixing chamber 975 along isolated paths to an inlet. A post-mixing chamber channel 974 extends radially inward from mixing chamber 975, allowing fluid to be driven into mixing chamber 975 as the cartridge rotates. This path continues along post-mixing chamber channel 974 to elbow 980, where it reverses direction along radial channel 981 through mixing chamber 975. The path then curves around mixing chamber 975 along working channel 982. On the opposite side of mixing chamber 975, the path returns radially inward to inlet 954. Each of these channels can be used for washing and / or elution as target analytes are moved from mixing chamber 975 toward inlet 954 via a fresh volume of wash buffer and / or elution buffer. Sample chamber 938 of circuit 951 also includes a vent 955, which allows sample chamber 938 to also serve as a waste chamber for spent wash buffer as fluid continues to enter fluid circuit 951 through inlet 954. The vent 955 allows fluid to fill the sample chamber 938 while preventing fluid from flowing into the manifold via the reagent port 952 or the sample port 953 .

[0220] Each of the channels between the mixing chamber 975 and the inlet 954 can also be used as a reading chamber. The working channel 982 is particularly suitable for use as a reading chamber on a circular box. The working channel 982 extends circumferentially around the box at a constant radius from the center of rotation. Therefore, guiding electromagnetic radiation along a portion of the working channel 982 can be achieved by simply rotating the box when the source is activated. Alternatively, in some embodiments, the box can be a straight box that is configured to move along the axis of the box during processing and reading. In such an embodiment, the working channel can be straight, rather than curved along the circumference.

[0221] For example, Figure 31 Another embodiment of a fluid circuit 1151 suitable for use with a linear cartridge is shown. Similar to fluid circuit 951, fluid circuit 1151 includes a sample chamber 1138 having a reagent port 1152 and a sample port 1153 separated by a divider 1156. Fluid circuit 1151 extends from sample chamber 1138 along an isolated path through mixing chamber 1175 toward inlet 1154. Sample chamber 1138 also includes a vent 1155 that allows fluid pumped into fluid circuit 1151 through inlet 1154 to collect in sample chamber 1138 after flowing through the circuit.

[0222] Fluid circuit 1151 extends from mixing chamber 1175 around elbow 1180 to working channel 1182, which can again be used as a wash, elution, and / or reading chamber. In contrast to working channel 982 of fluid circuit 951, working channel 1182 has a straight configuration. Accordingly, sliding lateral movement of the cartridge forming fluid circuit 1151 can be used to introduce electromagnetic radiation along the entire length of working channel 1182.

[0223] In another aspect, the present disclosure provides a cartridge comprising a plurality of fluid circuits according to the present disclosure. For example, Figure 10 A cassette 150 is shown comprising three fluid circuits 151 having Figure 11-22 The configuration shown and discussed herein. Cartridge 150 allows three samples to be processed on a single cartridge, such that three samples can be loaded in a single operation. Figure 32 Another embodiment of a cassette 1150 comprising multiple fluid circuits is shown. Specifically, Figure 32 The box 1150 shown in FIG is formed as a straight line box and includes a Figure 31 There are six fluid circuits in the form of fluid circuit 1151 shown in .

[0224] The fluid circuits 1151 of the cartridge 1150 each have the same configuration and are arranged in evenly spaced rows. To move the sample from the sample chamber to the mixing chamber, the cartridge is rotated so that centrifugal force drives the fluid toward the mixing chamber, as with the other cartridges described above. However, instead of rotating the cartridge in the plane of the fluid circuit, the cartridge is rotated about an axis 1160 that extends parallel to the plane of the circuit. Because each of the fluid circuits 1151 is spaced the same distance from the axis 1160, the centrifugal force on each sample is the same. Furthermore, because the fluid circuits 1151 are aligned along rows, the samples can be processed simultaneously. To mix the sample and reagents within the fluid circuits 1151 of the cartridge 1150, the cartridge 1150 is translated back and forth along the axis 1160. Furthermore, as Figure 33As shown, a carrier 1144 supporting six evenly spaced magnets 1145 can be moved relative to the cartridge 1150 and simultaneously transport all six magnets 1145 along the path of the fluid circuit in the same manner. In this way, the paramagnetic beads in each fluid circuit can be simultaneously drawn along the path of the fluid circuit. A second carrier can be used on the opposite side of the cartridge for washing or elution, as described above.

[0225] 1350. The embodiment of a linear box can include a row of fluid circuits greater or less than the six circuits shown in box 1150. In addition, in some embodiments, a linear box can include multiple rows. For example, diagram 1350 includes a first row 1352 of fluid circuits on one side of the box and a second row 1353 of fluid circuits on the opposite side of the box. The fluid circuits all have the same configuration and are formed in the same plane. In order to balance the centrifugal force on the sample in each fluid circuit, the box can be rotated about a central axis 1360 during the initial stage of moving the fluid into the mixing chamber.

[0226] While the foregoing examples of cassettes with multiple fluid circuits illustrate complex circuits in a rotating cassette and simple fluid circuits in a linear cassette, both cassette types can be used with various configurations of fluid circuits. For example, Figure 30 The fluid circuit 1151 has a wedge shape so that several instances of the fluid circuit 1151 can be assembled on a single rotary box. Figure 35 A linear cassette 1550 is shown that includes four similar fluid circuits 1551 configured to separate components of a sample. The fluid circuits 1551 include similar Figures 11 to 22 The separation region and overflow channel of the fluid circuit 151. The fluid is drawn from the separation region to the mixing chamber by a siphon activated by capillary action, where the sample and reagents are mixed before washing, elution and reading, as explained above.

[0227] In some embodiments, a fluid processing cartridge with multiple fluid circuits includes a set of fluid circuits, each of which has a working channel extending along a common linear or circular path. This configuration allows for identification of target analytes in the working channel of each fluid circuit using a single read operation. For example, where the working channels of multiple fluid circuits extend along a single circular path of a rotating cartridge, such as cartridge 150, the cartridge can simply be rotated to obtain a reading along each working channel. Similarly, where the working channels of multiple fluid circuits extend along a straight line through a linear cartridge (e.g., cartridge 1150), the cartridge can be moved laterally to obtain a reading along each working channel.

[0228] While each of the illustrated fluid circuits is configured so as to facilitate the binding of the target analyte, the label, and the paramagnetic beads together in a mixing chamber in a single step, in other embodiments, this binding may occur in more than one step. For example, in some embodiments, the fluid circuit may include a pre-mixing chamber in which the target analyte binds to the paramagnetic beads. The paramagnetic beads may then be moved to a mixing chamber in which the label binds to the target analyte.

[0229] In another aspect of the present disclosure, the optical system 120 of the analyzer 100 includes a second electromagnetic radiation source 128 and a second detector 129 for multiplexing. In some embodiments, the analyzer second electromagnetic radiation source 128 and the second detector 129 can be used to determine the presence of a second target analyte in the sample. In other embodiments, the second electromagnetic radiation source 128 and the second detector 129 can be used to measure the concentration of a control analyte in the cartridge 150. For example, the cartridge 150 can include an accurate and known amount of a control analyte. Accordingly, the measured concentration of the control analyte can be used as a comparator for the target analyte. This measured concentration can then be used to adjust the detected concentration of the target analyte.

[0230] For example, if the measured concentration of the control analyte is only 95% of the actual known concentration of the control analyte, the controller 140 can use this percentage difference to adapt the detected concentration of the target analyte. For example, the controller 140 can determine that the analyzer 100 also only detects 95% of the target analyte in the sample and adjust the calculated concentration accordingly.

[0231] In some embodiments, the electromagnetic radiation from first electromagnetic radiation source 121 and second electromagnetic radiation source 128 is directed toward the cartridge using the same objective lens. Indeed, in some embodiments, the electromagnetic radiation from both sources is directed toward the same detection volume. In some embodiments, first electromagnetic radiation source 121 and second electromagnetic radiation source 128 emit electromagnetic radiation of different wavelengths (e.g., different colors).

[0232] The present disclosure provides systems and methods for high-sensitivity detection and quantification of one or more target analytes, such as markers of a biological state.

[0233] Singleplex and multiplex assays

[0234] In one aspect, the present disclosure provides systems and methods that can perform "single-plex" detection of a sample to detect and analyze a single type of target analyte in the sample. In other aspects, the present disclosure provides systems and methods that can perform "multiplex" detection of a sample to detect and analyze a plurality of (e.g., two, three or more) different types of target analytes in the sample. Using the multiplex systems and methods described herein can provide faster detection and analysis of multiple target analytes, using reduced sample volumes and reduced reagent volumes than would be required for similar analysis of those target analytes by single-plex detection. In addition, the multiplex systems and methods described herein can allow the analysis of a sample comprising a target analyte to be compared with a control detection of a known concentration.

[0235] In order to detect and analyze multiple different types of target analytes in a sample, a multiplex analyzer system can distinguish one type of target analyte from other types. This can be achieved in part by labeling different target analytes with different markers, which have excitation wavelength bands and / or emission wavelength bands that are different from each other. In some embodiments, different markers have excitation wavelength bands and / or emission wavelength bands that have relatively little or no overlap. In other embodiments, there may be some overlap between the excitation wavelength bands and / or emission wavelength bands of the markers. Multiplexing can also be achieved by implementing more than one fluid circuit on the same box, wherein each fluid circuit is spatially different and carries reagents for different target analytes. For different fluid circuits, different target markers do not necessarily have different excitation and emission wavelengths. Additional circuits can collect samples from the same sample chamber or from different sample chambers.

[0236] Electromagnetic radiation power and range size

[0237] In the optical system, the electromagnetic radiation source 121 can be configured so that the wavelength of the electromagnetic radiation is sufficient to excite the fluorescent marker attached to the target analyte. In some embodiments, the electromagnetic radiation source 121 is a laser that emits light in the visible spectrum. In some embodiments, the laser is a continuous wave laser with a wavelength of 639 nm, 532 nm, 488 nm, 422 nm, or 405 nm. Any continuous wave laser with a wavelength suitable for exciting the fluorescent groups used in the methods and compositions of the present disclosure can be used without departing from the scope of this disclosure. The power setting of the laser is typically between 1 mW and 100 mW. However, those skilled in the art will understand that the laser power can be any setting that achieves the optimal signal-to-noise ratio for the measurement. To this end, the laser power should be set to achieve as many excitation-emission cycles as possible during the residence time of the marker in the detection space. The detector interval time should also be set accordingly. Interval times that are longer than the time it takes to photobleach the marker and / or longer than the residence time of the marker in the detection space will only result in the collection of excess noise. Laser power settings that are too low or too high, or bin time settings that are too long, will not produce the highest possible signal-to-noise ratio.

[0238] When the detection space in the analyzer 100 passes through the labeled target analyte, the photons emitted by the fluorescent particles are recorded by the detector 122 with a time delay indicating the time for the labeled particle to pass through the detection space. The photon intensity is recorded by the detector 122, and the sampling time is divided into intervals, where the interval is a uniform, arbitrary time period with a freely selectable time channel width. The number of signals contained in each interval is evaluated. One or more of several statistical analysis methods are used to determine when a marker or particle is present or when a segment of an interval contains an artifact. Segments of intervals containing artifacts are discarded, and single intervals or segments of intervals containing markers are counted. The number of markers counted indicates the number of target analytes present in the sample.

[0239] Detection volume

[0240] The detection volume can be considered the effective volume of a sample within which target analytes of interest can be detected when present. While various methods exist for calculating the detection volume of a sample, the simplest method for determining the effective volume (V) of the detection volume is to calculate its effective cross-section. Because the detection volume is typically swept through the sample by translating the detection volume through a stationary sample, the volume is typically the result of the cross-sectional area of ​​the detection volume swept over a certain distance during the measurement time. As previously mentioned, the lateral extent of the cross-sectional area of ​​the detection volume (perpendicular to the direction of laser motion relative to the sample and perpendicular to the direction of laser propagation) is limited by the numerical aperture of the laser source imaging the sample space. The longitudinal dimension of the detection volume (along the direction of laser propagation) is determined by the size of the selected confocal aperture. If the sample concentration (C) is known and the number of molecules detected over a period of time (N) is known, the sample volume is the number of molecules detected divided by the sample concentration, or V = N / C (where the sample concentration is expressed in units of molecules per unit volume).

[0241] For example, in some embodiments of the systems described herein, all detected photons are counted and summed within 100-microsecond segments (photon counting intervals). If a molecule of interest is present within a 100-microsecond segment, the detected photon count is typically significantly higher than background. Therefore, the distance the detection volume moves relative to the sample is an appropriate distance for calculating the volume sampled in a single segment (i.e., the detection volume). In this example, if the sample is analyzed for 60 seconds, 600,000 segments are effectively scanned. If the effective volume is divided by the number of segments, the resulting volume is essentially the volume of a single segment, i.e., the detection volume. Mathematically, the volume of a single segment, i.e., the detection volume (Vs), is equal to the number of molecules detected (N) divided by the sample concentration multiplied by the number of segment intervals (C·n, where n represents the number of segment intervals during the time it takes to count N molecules). For illustrative purposes only, consider a run of 600,000 segments of a known standard at a femtomolar concentration, in which 20 molecules of the standard are detected. Correspondingly, the detection volume Vs is equal to N / (C·n) or 20 / (602.214·6E5) or 55.351 μm 3 Therefore, in this example, the detection space volume, which is the effective volume of one sample corresponding to one photon counting interval, is 55.351 μm 3 .

[0242] detector

[0243] In some embodiments, light emitted by a fluorescent marker after exposure to electromagnetic radiation is detected. The emitted light can be, for example, ultraviolet light, visible light, or infrared light. For example, the first detector 122 can capture the amplitude and duration of a burst of photons from a fluorescent group and convert the amplitude and duration of the burst of photons into an electrical signal. Detection equipment such as CCD cameras, video input module cameras, and streak cameras can be used to generate images with continuous signals. Other embodiments use equipment such as bolometers, photodiodes, photodiode arrays, avalanche photodiodes, and photomultipliers that generate sequential signals. Any combination of the aforementioned detectors can be used.

[0244] Molecules used for concentration analysis

[0245] The apparatus, kits, and methods of the present disclosure can be used to sensitively detect and determine the concentrations of a variety of different types of target analytes, such as markers of biological states.

[0246] Examples of molecules or "analytes" that can be detected using the analyzers and associated methods of the present disclosure include: biopolymers such as proteins, nucleic acids, carbohydrates, and small molecules, both organic and inorganic. In particular, the instruments, kits, and methods described herein can be used to detect target analytes of proteins and small molecules in biological samples, as well as to determine the concentration of such molecules in the sample.

[0247] The molecules detected by the present systems and methods may be free or may be part of a complex, such as an antibody-antigen complex, or more generally a protein-protein complex, such as a troponin complex or a prostate specific antigen (PSA) complex.

[0248] In some embodiments, the present disclosure provides compositions and methods for the sensitive detection of biomarkers and the use of such markers in diagnosis, prognosis, and / or determining therapeutic approaches.

[0249] A marker can be, for example, any composition and / or molecule, or complex of compositions and / or molecules, that is associated with a biological state of an organism (e.g., a condition such as a disease or non-disease state). Markers can be, for example, small molecules, polypeptides, nucleic acids (e.g., DNA and RNA), lipids (e.g., phospholipids or micelles), cellular components (e.g., mitochondria or chloroplasts), and the like. Markers contemplated by the present disclosure may be previously known or unknown. For example, in some embodiments, the methods herein can identify novel polypeptides that can be used as markers for a biological state or condition of interest, while in other embodiments, known polypeptides are identified as markers for a biological state or condition of interest. Using the disclosed systems, markers can be observed, such as polypeptides that have high potential for use in determining an organism's biological state but are only present at low concentrations, such as those that "leak" from diseased tissue. Other potentially useful markers or polypeptides can be disease-associated markers or polypeptides, such as those produced in the host environment of a tumor. Any suitable marker that provides information about a biological state can be used in the methods and compositions of the present disclosure. The term "marker" as used herein encompasses any molecule that can be detected in a sample from an organism and whose detection or quantification provides information about the biological state of the organism.

[0250] Biological state includes, but is not limited to, phenotypic state; a condition affecting an organism; developmental state; age; health; pathology; disease detection, treatment, or staging; infection; toxicity; or response to a chemical, environmental, or pharmacological agent (e.g., drug response phenotyping, drug toxicity phenotyping, or drug effectiveness phenotyping).

[0251] As used herein, the term "organism" refers to any organism composed of at least one cell. Organisms can be as simple as single-cell organisms or as complex as mammals. Organisms of the present disclosure are preferably mammals. Such mammals can be, for example, humans or animals such as primates (e.g., monkeys, chimpanzees, etc.), domesticated animals (e.g., dogs, cats, horses, etc.), farm animals (e.g., goats, sheep, pigs, cattle, etc.), or laboratory animals (e.g., mice, rats, etc.). Preferably, the organism is a human.

[0252] markers

[0253] In some embodiments, the present disclosure provides methods and compositions comprising labels for high-sensitivity detection and quantification of molecules (eg, markers).

[0254] Many strategies can be used to label target analytes to enable their detection or differentiation in a mixture of particles. Labels can be attached by any known means, including methods that utilize nonspecific or specific interactions between the label and the target analyte. Labels can provide a detectable signal or affect the mobility of the particle in an electric field. Labeling can be done directly or through binding to a ligand.

[0255] The label may include, but is not limited to, one or more of the following: fluorophores, chromophores, chemiluminescent atoms or compounds, phosphorescent atoms or compounds, electrochemiluminescent atoms or compounds, micro- or nanoparticles, micro- or nanocrystals, nanodiamonds, upconversion phosphors, micro- or nanolasers, electron paramagnetic resonance (EPR) labels, nuclear magnetic resonance (NMR) / magnetic resonance imaging (MRI) sensitive labels, plasmon resonance labels, quantum dots, radionuclides, colloidal metals, viral particles, liposomes, micelles, oligonucleotides, peptides, proteins, enzymes, ribozymes, and aptamers.

[0256] In some embodiments, the label comprises a binding partner for the molecule of interest, wherein the binding partner is attached to a fluorescent group. The compositions and methods of the present disclosure may use highly fluorescent groups. Groups suitable for use in the compositions and methods of the present disclosure are described in more detail below. The fluorescent molecule can be attached to the binding partner by any known means, such as direct conjugation or indirect (e.g., biotin / streptavidin).

[0257] The fluorescent group can be a fluorescent dye molecule. Examples of fluorescent molecules include, but are not limited to, ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXA FLUOR® 647, ALEXA FLUOR® 680, or ALEXA FLUOR® 700, Brilliant Violet™ molecules (BD Biosciences), such as Brilliant Violet 421™, Brilliant Violet 510™, Brilliant Violet 570™, Brilliant Violet 605, and ATTO™ dyes (ATTOTECH GmbH), such as ATTO™ 532. In some embodiments, the dye molecule is the ALEXA FLUOR® 647 dye molecule.

[0258] Binding ligand

[0259] In some embodiments, the binding ligand comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0260] The antibody can be specific for any suitable marker. In some embodiments, the antibody is specific for a marker selected from cytokines, growth factors, tumor markers, inflammatory markers, endocrine markers, autoimmune markers, thyroid markers, cardiovascular markers, diabetes markers, infectious disease markers, neurological markers, respiratory markers, gastrointestinal markers, musculoskeletal markers, skin disease and metabolic markers.

[0261] Any suitable binding ligand having the necessary specificity for the form of the molecule (e.g., marker) to be detected can be used. If the molecule (e.g., marker) has several different forms, a variety of specificities of the binding ligand are possible. Suitable binding ligands are known in the art and include antibodies, aptamers, lectins, and receptors. A useful and common type of binding ligand is an antibody.

[0262] Capture binding ligand and detection binding ligand pairs, such as capture and detection antibody pairs, can be used in embodiments of the present disclosure. Thus, in some embodiments, a heterogeneous assay scheme is used, in which two binding ligands, such as two antibodies, are typically used. One binding ligand is a capture ligand, typically immobilized on a solid support, and the other binding ligand is a detection binding ligand, typically with an attached detectable label. Antibody pairs can be designed and prepared by methods known in the art. The compositions of the present invention include antibody pairs, wherein one member of the antibody pair is a marker as described herein and the other member is a capture antibody.

[0263] In some embodiments, it is useful to use antibodies that cross-react with multiple species as capture antibodies, detection antibodies, or both. Such examples include measuring drug toxicity by measuring, for example, the release of cardiac troponin into the blood as a marker of cardiac damage. Cross-reactive antibodies allow toxicity studies to be conducted in one species (e.g., a non-human species) and the results to be directly transferred to studies or clinical observations in another species (e.g., humans) using the same antibody or antibody pair as the detection reagent, thereby reducing inter-assay variability. Thus, in some embodiments, one or more antibodies that serve as binding partners for a marker of a molecule of interest (e.g., a cardiac troponin, such as cardiac troponin I) can be cross-reactive antibodies. In some embodiments, the antibodies cross-react with markers (e.g., cardiac troponins) from at least two species selected from human, monkey, dog, and mouse. In some embodiments, the antibodies cross-react with markers (e.g., cardiac troponins) from the entire panel consisting of human, monkey, dog, and mouse.

[0264] The above detailed description describes the various features and functions of the disclosed systems, devices, and methods with reference to the accompanying drawings. In the drawings, similar reference numerals generally identify similar components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the scope of the subject matter presented herein. It will be readily understood that the various aspects of the disclosure, as generally described herein and shown in the accompanying drawings, may be arranged, replaced, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0265] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not limitation, with the true scope being indicated by the following claims.

[0266] Example

[0267] Example 1. An analyzer system for detecting the presence of a target analyte in a sample, the analyzer system comprising:

[0268] Motor;

[0269] a dock coupled to the motor so as to be rotated by actuation of the motor;

[0270] a cartridge retained in the dock and comprising a fluid circuit configured to receive a sample, isolate a target analyte of the sample, and collect an amount of a first marker proportional to the amount of the target analyte in the sample, the fluid circuit comprising:

[0271] a sample port configured to receive a sample,

[0272] a mixing chamber in fluid communication with the sample port and configured to mix at least a portion of the sample so as to bind the target analyte to the first label, and

[0273] a fluid inlet in fluid communication with the mixing chamber and configured to receive a wash buffer and an elution buffer,

[0274] wherein the fluid circuit includes an isolated path extending from the fluid inlet to the mixing chamber;

[0275] a fluid delivery line configured to be coupled to the fluid inlet so as to deliver fluid to the cartridge through the fluid inlet and to urge the fluid along the isolated path toward the mixing chamber;

[0276] a first magnet fixed to the stage, the first magnet being movable relative to the cartridge and configured to move paramagnetic beads within the cartridge;

[0277] a first electromagnetic radiation source configured to provide electromagnetic radiation to form a detection space within the detection chamber of the cartridge;

[0278] a first detector configured to detect electromagnetic radiation emitted by the marker in the detection space if the marker is present in the detection space; and

[0279] A controller is configured to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector.

[0280] Embodiment 2. The analyzer system of embodiment 1, further comprising a second magnet arranged to be positioned on a side of the cartridge opposite to the first magnet.

[0281] Embodiment 3. The analyzer system of embodiment 1 or embodiment 2, further comprising a pump configured to push wash buffer and elution buffer through the fluid delivery lines and into the cartridge via the inlet.

[0282] Embodiment 4. The analyzer system of any one of embodiments 1 to 3, further comprising a distribution valve coupled to the fluid delivery line, the wash buffer port, the elution buffer port, and the pump.

[0283] Example 5. The analyzer system according to Example 4 further includes a perfusion line arranged between the dispensing valve and the pump, wherein the dispensing valve is configured to sequentially connect the elution buffer port and the wash buffer port to the perfusion line to load the perfusion line, and then connect the perfusion line to the fluid delivery line.

[0284] Embodiment 6. The analyzer system of any one of embodiments 1 to 5, wherein the cartridge comprises lyophilized reagents in the mixing chamber.

[0285] Embodiment 7. The analyzer system of any one of embodiments 1 to 5, wherein the cartridge comprises a liquid reagent port.

[0286] Embodiment 8. The analyzer system of any one of embodiments 1 to 7, wherein the fluid circuit is one of a plurality of fluid circuits in the cartridge.

[0287] Embodiment 9. The analyzer system of Embodiment 8, wherein each of the fluid circuits has an identical configuration and includes a working channel along the isolation path between the mixing chamber and the inlet.

[0288] Embodiment 10. The analyzer system of Embodiment 9, wherein the working channels extend along a common circular line.

[0289] Embodiment 11. The analyzer system of Embodiment 10, wherein the motor and dock are arranged to rotate the cartridge about an axis perpendicular to the plane of the fluid circuit.

[0290] Example 12. The analyzer system of Example 9, wherein the working channels extend along a common straight line.

[0291] Embodiment 13. The analyzer system of embodiment 12, wherein the motor and dock are arranged to rotate the cartridge about an axis parallel to a plane of the fluid circuit.

[0292] Example 14. A method comprising:

[0293] receiving a cartridge in an analyzer system such that the cartridge is coupled to a motor of the analyzer system;

[0294] rotating the cartridge using the motor to move a volume of sample toward a mixing chamber in the cartridge;

[0295] mixing the volume of sample in the mixing chamber by moving the cartridge to bind the target analyte, label, and paramagnetic capture beads;

[0296] introducing a series of fluids from a primed fluid delivery line into the cartridge through a fluid inlet, the series of fluids comprising a wash buffer and an elution buffer;

[0297] Propelling the series of fluids along isolated paths in a first direction from the fluid inlet to the mixing chamber;

[0298] The paramagnetic capture beads are moved out of the mixing chamber along the isolation path in a second direction toward the fluid inlet using a magnet.

[0299] Example 15. The method according to Example 14, further comprising:

[0300] directing electromagnetic radiation from an electromagnetic radiation source to form a detection space within the cartridge;

[0301] If the fluorescence is present in the detection space, receiving electromagnetic radiation emitted from the detection space in a detector; and

[0302] Using a controller, the presence of the target analyte in the sample is identified based on the electromagnetic radiation detected by the detector.

[0303] Example 16. The method of Example 15, wherein the marker is a fluorescent marker.

[0304] Example 17. The method of any one of Examples 14 to 16, further comprising loading the primed fluid delivery line with the series of fluids by sequentially coupling the fluid delivery line to an elution buffer port and a wash buffer port using a dispensing valve.

[0305] Embodiment 18. The method of any one of embodiments 14 to 17, wherein the cartridge is rotated about an axis perpendicular to the plane of the fluid circuit.

[0306] Embodiment 19. The method of embodiment 18, wherein moving the cartridge to mix the amount of sample comprises further rotating the cartridge.

[0307] Embodiment 20. The method of Embodiment 18 or 19, wherein the fluid circuit is one of a plurality of fluid circuits disposed around the center of the cartridge.

[0308] Embodiment 21. The method of Embodiment 20, wherein each of the fluid circuits has the same configuration and includes a working channel along the isolated path between the corresponding mixing chamber and the inlet.

[0309] Embodiment 22. The method of embodiment 21, wherein the working channels extend along a common circular line disposed at a fixed radius from the center of the cartridge.

[0310] Embodiment 23. The method of any one of embodiments 14 to 17, wherein the cartridge is rotated about an axis parallel to the plane of the fluid circuit.

[0311] Embodiment 24. The method of embodiment 23, wherein moving the cartridge to mix the amount of the sample comprises moving the cartridge in a direction parallel to the plane of the fluid circuit.

[0312] Embodiment 25. The method of Embodiment 23 or 24, wherein the fluid circuit is one of a plurality of fluid circuits arranged in a row across the cartridge.

[0313] Embodiment 26. The method of Embodiment 25, wherein each of the fluid circuits has the same configuration and includes a working channel along the isolated path between the corresponding mixing chamber and inlet.

[0314] Example 27. The method of Example 26, wherein the working channels extend along a common straight line.

Claims

1. An analyzer system for detecting the presence of a target analyte in a sample, the analyzer system comprising: Motor; a dock coupled to the motor so as to be rotated by actuation of the motor; a cartridge retained in the dock and comprising a fluid circuit configured to receive a sample, isolate a target analyte of the sample, and collect an amount of a first marker proportional to an amount of the target analyte in the sample, the fluid circuit comprising: a sample port configured to receive a sample, a mixing chamber in fluid communication with the sample port and configured to mix at least a portion of the sample so as to bind the target analyte to the first label, and a fluid inlet in fluid communication with the mixing chamber and configured to receive a wash buffer and an elution buffer, wherein the fluid circuit includes an isolated path extending from the fluid inlet to the mixing chamber; a fluid delivery line configured to be coupled to the fluid inlet so as to deliver fluid to the cartridge through the fluid inlet and to urge the fluid along the isolated path toward the mixing chamber; a first magnet fixed to the stage, the first magnet being movable relative to the cartridge and configured to move paramagnetic beads within the cartridge; a first electromagnetic radiation source configured to provide electromagnetic radiation to form a detection space within the detection chamber of the cartridge; a first detector configured to detect electromagnetic radiation emitted by the marker in the detection space if the marker is present in the detection space; and A controller is configured to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector. 2 . The analyzer system according to claim 1 , further comprising a distribution valve coupled to the fluid delivery line, the wash buffer port, the elution buffer port, and the pump.

3. The analyzer system according to claim 2, further comprising a priming line provided between the dispensing valve and the pump, wherein The dispensing valve is configured to sequentially connect the elution buffer port and the wash buffer port to the perfusion line to load the perfusion line, and then connect the perfusion line to the fluid delivery line.

4. The analyzer system according to claim 1, wherein The cartridge includes lyophilized reagents in the mixing chamber.

5. The analyzer system according to claim 1, wherein The cartridge includes a liquid reagent port.

6. The analyzer system according to claim 1, wherein The fluid circuit is one of a plurality of fluid circuits in the cassette, and wherein each of the fluid circuits has the same configuration and includes a working channel along the isolated path between the mixing chamber and the inlet.

7. The analyzer system according to claim 6, wherein The working channels extend along a common circular line, and wherein the motor and dock are arranged to rotate the cartridge about an axis perpendicular to a plane of the fluid circuit.

8. The analyzer system according to claim 6, wherein The working channels extend along a common straight line, and wherein the motor and dock are arranged to rotate the cartridge about an axis parallel to a plane of the fluid circuit.

9. A method comprising: receiving a cartridge in an analyzer system such that the cartridge is coupled to a motor of the analyzer system; rotating the cartridge using the motor to move a volume of sample toward a mixing chamber in the cartridge; mixing the volume of sample in the mixing chamber by moving the cartridge to bind the target analyte, label, and paramagnetic capture beads; introducing a series of fluids from a primed fluid delivery line into the cartridge through a fluid inlet, the series of fluids comprising a wash buffer and an elution buffer; Propelling the series of fluids along isolated paths in a first direction from the fluid inlet to the mixing chamber; The paramagnetic capture beads are moved out of the mixing chamber along the isolation path in a second direction toward the fluid inlet using a magnet.

10. The method according to claim 9, further comprising: directing electromagnetic radiation from an electromagnetic radiation source to form a detection space within the cartridge; If fluorescence exists in the detection space, receiving electromagnetic radiation emitted from the detection space in a detector; and Using a controller, the presence of the target analyte in the sample is identified based on the electromagnetic radiation detected by the detector.

11. The method according to claim 10, wherein: The label is a fluorescent label.

12. The method of claim 9, further comprising loading the primed fluid delivery line with the series of fluids by sequentially coupling the fluid delivery line to an elution buffer port and a wash buffer port using a dispensing valve.

13. The method according to claim 9, wherein: The cartridge is rotated about an axis perpendicular to a plane of the fluid circuit, and moving the cartridge to mix the amount of sample includes further rotating the cartridge.

14. The method according to claim 13, wherein: The fluid circuit is one of a plurality of fluid circuits disposed about a center of the cartridge, wherein each of the fluid circuits has an identical configuration and includes a working channel along the isolated path between the corresponding mixing chamber and the inlet, and wherein the working channel extends along a common circular line disposed at a fixed radius from the center of the cartridge.

15. The method according to claim 9, wherein The cartridge rotates about an axis parallel to a plane of the fluid circuit, and moving the cartridge to mix the amount of sample includes moving the cartridge in a direction parallel to the plane of the fluid circuit.

16. The method according to claim 15, wherein The fluid circuit is one of a plurality of fluid circuits arranged in a row across the cartridge, wherein each of the fluid circuits has an identical configuration and includes a working channel along the isolation path between the respective mixing chamber and the inlet, and wherein the working channels extend along a common straight line.

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