Clinical field concentration analyzer

Abstract

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

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 488,677, filed March 6, 2023, 63 / 488,681, filed March 6, 2023, and 63 / 591,546, filed October 19, 2023, each of which is incorporated herein by reference in its entirety.

[0002] The present invention relates generally to automated sample processing, measurement, and analysis of samples to isolate, label, detect, and determine the amount of a specific target analyte that may be present in very low concentrations. [Background technology]

[0003] Numerous studies and advances in understanding the underlying causes and progression of disease have shown that detecting infectious agents or detecting early disorders that lead to appropriate treatment can substantially improve clinical outcomes. Many conditions that once required the use of expensive symptom measurements, such as anatomical imaging, which require trained professionals to administer and interpret, can now be diagnosed at the cellular and molecular level through the presence and / or concentration of specific biomarkers. These biomarkers include up- or down-regulated proteins, nucleic acids, or other molecules that are highly specific to the disease state or infection.

[0004] It is often desirable to diagnose a condition at the point of care, where the timing and administration of correct treatment are critical for patient outcomes. This is especially true in the acute care setting of a trauma center, where patients may be suffering from acute myocardial infarction (AMI), acute decompensated heart failure, pulmonary embolism, sepsis, or other conditions requiring a timely response. In non-emergency settings, rapid turnaround time is also desirable, especially for highly infectious diseases such as Clostridium difficile infection, which may require isolation. However, even in a physician's office or retail clinic, determining whether a condition is viral or bacterial prior to administering antibiotics is extremely beneficial.

[0005] In some disease states, the concentration of biomarkers or analytes of interest is relatively high, allowing simple, low-cost lateral flow devices to be employed for sample processing and readout. These devices and consumable components that interact with the sample are very low-cost and can be used quickly with relatively little or no training at the point of care. However, lateral flow-type tests also tend to suffer from poor accuracy, producing quantitative measurements of marginal quality, even when objective reader systems are used to measure the strips. Also, depending on the stage of 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.

[0006] In these cases, sample processing and readout are more complex. This often requires precise measurement to assess concentration, high efficiency to avoid loss of target analyte, and centrifugation as a primary step in the purification process. Beyond centrifugation, additional purification steps typically involve incubation with reagents containing binding partners or molecules with complementary sequences or structures for binding to the target analyte biomarker. These binding partners may be substrates such as micro- or nanoparticles with complementary molecules on their surface, molecules conjugated to transduction labels, or both. Once binding occurs, additional process steps must then be taken to wash and further isolate the target analyte, suspending it in fresh buffer solution or placing it on a clean surface prior to measurement. Multiple devices, including centrifuges, mixers, incubators, precision pipetters, and thermal cyclers, are used to perform these processing steps, and samples are often transferred and measured between processing steps using multiple disposable tips, tubes, plates, and other sample containers. Once processing is complete, highly sensitive and precise instruments are used to measure the processed sample and determine the presence and / or abundance of the target analyte.

[0007] Analysis of low concentration biomarkers can take several forms, but in general, sample processing and measurement have the following key characteristics: 1. A separation step to perform a first isolation of the target analyte from other sample components 2. Introduction of Binding Partners and Reagents 3. Mixing and incubation to label and bind target analytes to the substrate 4. Introduction of Buffer and Steps to Wash Away Unbound Label and Other Contaminants 5. Sterile containers for precise measurement and sample storage during processing 6. Efficient and precise means of sample transfer 7. A means of measurement that provides high sensitivity and accuracy for determining the presence and abundance of target analytes

[0008] Currently, processing and measurement of low-level biomarkers must be performed by trained staff or with highly specialized equipment in centralized locations. As a result, the turnaround time from sample acquisition to result is long, instrumentation costs are high, and measurements cannot be performed at the point of care.

[0009] Therefore, the inventors have recognized that an improved technique that can address the key characteristics listed above for low concentration biomarker processing and measurement is desirable. The technique should be suitable for point-of-care environments with minimal consumables, precise measurement, rapid turnaround time, and sensitivity that overcomes the limitations of prior art techniques.

[0010] U.S. Patent No. 8,264,684 and U.S. Patent Application Publication No. 2016 / 0178520 (each incorporated herein by reference) describe previous systems that achieved extremely sensitive detection. The present disclosure provides further developments within this field. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 8,264,684 [Patent Document 2] U.S. Patent Application Publication No. 2016 / 0178520 Summary of the Invention [Means for solving the problem]

[0012] Disclosed herein are analyzer systems, cartridges, and methods for the detection of target analytes in a sample. Advantageously, embodiments of the analyzer system use compact cartridges for processing and analyzing samples, allowing the analyzer to be of a reduced size so that it can be provided at the point of care.

[0013] Accordingly, in a first aspect, the present disclosure provides an analyzer system for detecting the presence of a target analyte in a sample, comprising: A motor; a dock coupled to the motor so as to be rotated by actuation of the motor; a cartridge retained within the dock and including a fluidic circuit configured to receive a sample, isolate a target analyte of the sample, and collect an amount of a first label proportional to the amount of the target analyte in the sample, the fluidic 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 to bind the target analyte and the first label; a fluid inlet port in fluid communication with the mixing chamber and configured to receive a wash buffer and an elution buffer; the fluid circuit includes an isolated pathway extending from the fluid inlet port to the mixing chamber; A cartridge and a fluid delivery line configured to be coupled to the fluid inlet port to deliver fluid to the cartridge through the fluid inlet port and force the fluid along an isolated path toward the mixing chamber; a first magnet affixed to the stage, movable relative to the cartridge and configured to move the paramagnetic beads within the cartridge; a first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within the detection chamber of the cartridge; a first detector configured to detect electromagnetic radiation emitted within the interrogation space by the label when the label is present within the interrogation space; a controller configured to identify the presence of a target analyte in the sample based on the electromagnetic radiation detected by the first detector; An analyzer system is provided, comprising:

[0014] In another aspect, the present disclosure provides a method, comprising: receiving the cartridge within the analyzer system such that the cartridge is coupled to a motor of the analyzer system; rotating the cartridge using a motor to move a volume of sample into a mixing chamber within the cartridge; mixing said volume of sample in the mixing chamber by moving the cartridge so that the target analytes and labels bind to the paramagnetic capture beads; introducing a series of fluids from the primed fluid delivery line into the cartridge through the fluid inlet ports, the series of fluids including a wash buffer and an elution buffer; forcing a series of fluids along isolated paths in a first direction from the fluid inlet port to a mixing chamber; using a magnet to move the paramagnetic capture beads out of the mixing chamber along an isolated path in a second direction toward the fluid inlet port; The present invention provides a method comprising:

[0015] This and other aspects, advantages, and alternatives will become apparent to those of ordinary skill in the art upon reading the following detailed description. [Brief explanation of the drawings]

[0016] The accompanying drawings are included to provide a further understanding of the methods and devices of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings are not necessarily to scale, and sizes of various elements may be distorted for clarity. The 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.

[0017] [Figure 1] FIG. 1 is a schematic perspective side view of a high sensitivity analyzer according to an embodiment of the present disclosure.

[0018] [Figure 2] FIG. 2 is a schematic perspective top view of the analyzer of FIG.

[0019] [Figure 3] FIG. 3 is a schematic perspective view of a process quality control camera optical system used in the analyzer of FIG.

[0020] [Figure 4] FIG. 4 is a schematic perspective view of a portion of the analyzer of FIG. 1, including the centrifuge, the objective lens, and the objective lens radial and Z stages.

[0021] [Figure 5] FIG. 5 is a schematic perspective side view of a portion of the analyzer of FIG. 1, including a manifold and a cartridge.

[0022] [Figure 6] FIG. 6 is a schematic perspective bottom view of the manifold of FIG.

[0023] [Figure 7] FIG. 7 is a schematic side view of a portion of the analyzer of FIG. 1, including an objective lens.

[0024] [Figure 8] FIG. 8 is a schematic representation of the fluid system of the analyzer of FIG.

[0025] [Figure 9] FIG. 9 is a schematic diagram of a pump and primed fluid line, along with pump purge lines and valves, according to an embodiment of the present disclosure.

[0026] [Figure 10] FIG. 10 is a schematic top view of a cartridge including several fluid circuits according to an embodiment of the present disclosure.

[0027] [Figure 11] FIG. 11 is a schematic top view of the fluid circuit of the cartridge of FIG. 10 in a first instance of a method according to an embodiment of the present disclosure.

[0028] [Figure 12] FIG. 12 is a schematic top view of the fluid circuit of FIG. 11 in a second instance of a method according to an embodiment of the present disclosure.

[0029] [Figure 13] FIG. 13 is a schematic top view of the fluid circuit of FIG. 11 in a third instance of a method according to an embodiment of the present disclosure.

[0030] [Figure 14] FIG. 14 is a schematic top view of the fluid circuit of FIG. 11 in a fourth instance of a method according to an embodiment of the present disclosure.

[0031] [Figure 15] FIG. 15 is a schematic top view of the fluid circuit of FIG. 11 in a fifth instance of a method according to an embodiment of the present disclosure.

[0032] [Figure 16] FIG. 16 is a schematic top view of the fluid circuit of FIG. 11 in a sixth instance of a method according to an embodiment of the present disclosure.

[0033] [Figure 17] FIG. 17 is a schematic top view of the fluid circuit of FIG. 11 in a seventh instance of a method according to an embodiment of the present disclosure.

[0034] [Figure 18] FIG. 18 is a schematic top view of the fluid circuit of FIG. 11 in an eighth instance of a method according to an embodiment of the present disclosure.

[0035] [Figure 19] FIG. 19 is a schematic top view of the fluid circuit of FIG. 11 in a ninth instance of a method according to an embodiment of the present disclosure.

[0036] [Figure 20] FIG. 20 is a schematic top view of the fluid circuit of FIG. 11 in a tenth instance of a method according to an embodiment of the present disclosure.

[0037] [Figure 21] FIG. 21 is a schematic top view of the fluid circuit of FIG. 11 in an eleventh instance of a method according to an embodiment of the present disclosure.

[0038] [Figure 21] FIG. 21 is a schematic top view of the fluid circuit of FIG. 11 in a twelfth instance of a method according to an embodiment of the present disclosure.

[0039] [Figure 22] FIG. 22 is a schematic top view of the fluid circuit of FIG. 11 in a thirteenth instance of a method according to an embodiment of the present disclosure.

[0040] [Figure 23] FIG. 23 is a schematic top view of a fluid circuit according to another embodiment of the present disclosure.

[0041] [Figure 24] FIG. 24 is a schematic side view of a portion of the analyzer of FIG. 1, including a magnetic stage.

[0042] [Figure 25] FIG. 25 is a schematic side view of various steps in a cleaning operation utilizing a magnet, according to an embodiment of the present disclosure.

[0043] [Figure 26] FIG. 26 is a schematic side view of various steps in another cleaning operation utilizing a magnet, according to an embodiment of the present disclosure.

[0044] [Figure 27] FIG. 27 shows data from a reading process performed by an analyzer according to an embodiment of the present disclosure.

[0045] [Figure 28] FIG. 28 is a schematic top view of a fluid circuit according to another embodiment of the present disclosure.

[0046] [Figure 29] FIG. 29 is a schematic top view of a fluid circuit according to yet another embodiment of the present disclosure.

[0047] [Figure 30] FIG. 30 is a schematic top view of a fluid circuit according to another embodiment of the present disclosure.

[0048] [Figure 31] FIG. 31 is a schematic top view of a fluid circuit according to yet another embodiment of the present disclosure.

[0049] [Figure 32] FIG. 32 is a schematic top view of a cartridge including some of the fluid circuits of FIG.

[0050] [Figure 33] FIG. 33 shows a system including the cartridge of FIG. 32 and a magnetic carrier.

[0051] [Figure 34] FIG. 34 is a schematic top view of a cartridge according to another embodiment of the present disclosure.

[0052] [Figure 35]FIG. 35 is a schematic top view of a cartridge according to yet another embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0053] explanation The following detailed description provides an overview of exemplary embodiments of methods and systems according to the present invention. This overview is followed by further descriptions of various exemplary embodiments of methods, systems, and apparatuses associated with the present invention. Overview of Exemplary Embodiments

[0054] The present invention is directed 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 coupled to a centrifuge. However, aspects of the disclosure may also be achieved using other container and sample processing configurations. Additionally, while the following examples use a cartridge with a fluidic circuit having a particular configuration (FIGS. 10-22), other fluidic circuit configurations are also possible (e.g., FIGs. 23, 28-31), as further described below.

[0055] The cartridge may contain various chambers and passageways for receiving and processing a sample. As the sample is processed, the analytes and labels may be directed through the cartridge to pass through various different zones within the cartridge. These zones may be bounded by structural features, such as a narrow passage between two larger chambers, or may be different areas within the cartridge that may contain fluid. For example, an elongated chamber formed by a channel may provide several functional zones as components of the sample are moved along the length of the chamber. Separation and measurement

[0056] FIG. 10 shows a cartridge 150 with three fluidic circuits 151, which may be used for all sample processing, measurement, and storage of the resulting processed sample during measurement. Each of the fluidic circuits 151 may receive a separate sample for processing and analysis. A more detailed view of one of the fluidic circuits 151 is shown in FIG. 11. FIGS. 12-22 illustrate the sequence of steps in processing a sample within one of the fluidic circuits 151 of the cartridge 150 shown in FIG. 10. In the illustrated embodiment, processing involves high-speed spinning of a disk to spin down high-density elements contained within the original sample. As shown in FIG. 11, the fluidic circuit 151 includes a sample port 153 for receiving the sample, an inlet port 154 for receiving various solutions for processing the sample, and a vent port 155. In light of the use of one inlet port 154 and one vent port 155, circuit 151 includes a single flow path 156 along which fluid introduced through inlet port 154 travels toward vent port 155 during processing. As discussed in more detail below, in some embodiments, this path is isolated at least from mixing chamber 175 to input port 154, which simplifies processing.

[0057] During the initial centrifugation step, the sample 200 is transferred from the sample chamber 158 to the inner and outer separation areas 161, 162, as shown in FIG. 13. The cartridge 150 is then spun at a faster speed, e.g., 7,000 rpm, to separate the high-density components into the outer separation area 162, as shown in FIG. 14. The resulting supernatant in the inner separation area 161 is then transferred to the mixing chamber 175, which contains reagents consisting of binding partners. The volumes of the inner separation chamber 161 and the mixing chamber 175 and the method of transfer of the supernatant act to measure the amount of sample used in processing to maintain accuracy. The spin-down process and transfer may be imaged by a processing quality control camera and analyzed during processing to ensure proper separation and measurement. Reagents and Binding Partners

[0058] Various binding partners may be used in accordance with embodiments of the present disclosure. For example, the first species may be a paramagnetic bead substrate functionalized with a molecule having a binding site specific to the target analyte. The second species of binding partner may include a label conjugated to a molecule with a binding site specific to a separate but distinct portion of the target analyte. The third and fourth species of binding partner may be used as a control assay, comprising a second set of functionalized paramagnetic bead substrates and a second set of fluorophore labels that emit at a different wavelength than the first set of fluorophore labels. The third and fourth species are designed to bind to each other. These species undergo the same assay process, and the amount of the third species is known a priori, so they can serve as a control assay for monitoring the effectiveness of sample processing and measurement.

[0059] In some embodiments, the binding partners may be pre-loaded into the cartridge. For example, the binding partners may be in the form of dried reagents or lyophilized pellets 157, identified in Figures 15 and 16, and stored in the mixing chamber. The lyophilized pellets 157 may contain multiple species of binding partners.

[0060] In other embodiments, the cartridge may be configured to accept a binding partner during processing. For example, FIG. 23 shows an embodiment of a fluidic circuit 351 including a port 352 for accepting a binding partner. The port 352 allows a liquid reagent 457 to be introduced into the fluidic circuit 351 prior to or during an analytical procedure. In the illustrated embodiment, the port 352 is radially interior to the mixing chamber 375 such that rotation of the associated cartridge will drive the liquid reagent 457 into the mixing chamber 375. Such an embodiment that allows for the introduction of a liquid reagent may enable general use of the cartridge of the present disclosure. Rather than storing assay-specific lyophilized pellets, the cartridge may accept a user-selected liquid reagent through the port 352. Thus, such an embodiment allows a user to input an application-specific binding partner to be contained in the liquid reagent 457 at the point of use. Mixing and incubation

[0061] Once the supernatant and reagents enter the mixing chamber, a mixing process will occur in which the disk 150 will spin slowly but at a variable rpm. Specifically, the disk will rotate at a controlled rate, accelerating and decelerating to achieve a desired motion profile during spinning. As shown in FIG. 16, mixing balls 176 made of a material with a higher density than the sample, such as brass or glass, 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, promoting dissolution and distribution of the dried reagents and ensuring a homogeneous mixture of all reagents and target analytes. The mixing chamber geometry may be configured along a substantially constant radius from the center of rotation. Additionally, the chamber may contain various features to further facilitate mixing and incubation and ensure that the mixture remains within the mixing chamber during the mixing process. The result of the mixing process will promote a homogeneous suspension and facilitate binding of the target analytes to the paramagnetic capture beads and labeled molecules. To ensure accuracy, the mixing process can be performed for a controlled amount of time.

[0062] Flow path 156 passes through mixing chamber 175 via two channels 173 and 174, each extending radially inward from mixing chamber 175. One or both of these channels may contain capillary breaks 178, 179 in the form of widened channel widths, as identified in FIG. 16, to prevent capillary action from drawing sample out of the chamber after transfer and while the cartridge is not spinning. During mixing and incubation, these channels keep the sample within the mixing chamber through centrifugal force as the cartridge is rotated and alternately accelerated and decelerated. An elbow 180 at the end of post-mixing chamber channel 174 reverses direction to radial channel 181, which extends outward toward circumferential channel 182. As described below, washing may occur within zones contained within mixing chamber 175 or any of channels 173, 174, 181, 182.

[0063] Once the mixing and incubation process is complete, the cartridge 150 and manifold 108 (FIG. 5) may be rotated into alignment, allowing fluid lines 111 (FIG. 5) on the manifold 108 to connect with inlet ports 154 (FIG. 11) on the cartridge. The manifold may be coupled to a motor 110 for precision rotational movement. The manifold may also be on a movable arm 109, as shown in FIG. 1, to lower and raise the manifold to and from the cartridge. Once the manifold is aligned and in contact with the cartridge, the manifold motor enables precision rotational movement of the cartridge 150 about the axis of the centrifuge motor. At this point, the centrifuge motor may be de-energized, and its bearings and shaft can serve as a precision rotational stage for the cartridge.

[0064] After the manifold 108 is aligned over the cartridge 150, one or more magnets are moved into position across the mixing chamber. The magnets may be positioned above and below the cartridge on a Z-stage, with the axis of motion perpendicular to the cartridge's flat surface, as shown in FIG. 24. This allows magnet 145 to be moved closer to the cartridge 150, increasing its effective attractive force on the paramagnetic capture beads, while the other magnet 146 is moved away from the cartridge 150, decreasing its influence. The magnet Z-stage 147 is also coupled to a radial stage 148. The radial stage allows for movement of the magnet closer to or away from the cartridge's axis of rotation. As discussed later, the various channels and chambers on the cartridge are nominally arranged radially or circumferentially. The various Z- and radial stages, in combination with the manifold motors, allow the magnets to be placed at any desired position relative to the chambers and channels contained within the cartridge 150.

[0065] After the magnet or magnets are introduced, the magnet Z- and radial stages are controlled along with the partial cartridge rotation to perform a predefined sequence of movements, as shown in Figure 18, to pull all paramagnetic capture beads, now bound to the target analyte and control label, out of suspension. The magnet 145 on the bottom side of the cartridge is brought close to the surface of the cartridge at a preferred location, drawing the beads into a tight bolus 177 in Figure 18. The bead bolus may be imaged and analyzed by a process quality control camera to ensure the beads are being properly pulled out of suspension. Wash buffer introduction and washing

[0066] At this point, fluid is introduced into fluid circuit 151 from manifold 108 through inlet port 154. In some embodiments, fluid may be introduced from pre-loaded lines containing precise volumes of fluid being introduced. As explained below, by pre-loading the lines with the fluid needed for processing, a single pump can be used to quickly and efficiently direct all fluid through fluid circuit 151.

[0067] As the magnet holds the analyte bead bolus in place, a pump forces wash buffer 270 through inlet port 154 and into fluid circuit 151, filling 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, as shown in FIG. 19 . This forces sample fluid and unbound label out of mixing chamber 175, thereby initiating the wash process. Wash buffer, flowing across the bolus containing the analyte, washes unbound label in the bolus out of the bolus and continues to do so as it flows across the bolus, out of mixing chamber 175, and toward waste chamber 166. Apart from capillary breaks 178 and 179, channels 172, 173, 180, 181, and 182 generally have consistent cross-sectional areas with few discontinuities or sharp corners. This geometry facilitates filling with buffer following washing without leaving air pockets that can interfere with the elution and reading process.

[0068] Once the initial wash buffer fill is complete, a further wash sequence can begin. This can be done using a single magnet by pulling the bolus back and forth within the mixing chamber, or by pulling the bolus into post-mixing chamber channel 174, around elbow 180 as shown in FIG. 20, and through radial channel 181 to circumferential channel 182 as shown in FIG. 21. During pulling through channels 174, 180, and 181, the bolus is continuously exposed to clean wash buffer, thus allowing unbound and non-specifically bound label to be washed away. If needed, further washing can be implemented through the use of a second magnet, as described in more detail below. Elution of analyte and label from paramagnetic beads

[0069] To initiate elution, the bead bolus may be moved to a predefined zone within circumferential channel 182 that is clean and not exposed to any assay components, for example, as shown in Figure 21. At this point, pump 118 pumps a leading air separator 273 between wash buffer 270 and elution buffer 271 through inlet port 154 into circumferential channel 182 and over the bead bolus to a predefined location within circumferential channel 182 beyond the bolus, as shown in Figure 22. A second air separator 273 between the bolus and DI water 272 will be on the side of the bolus closer to inlet port 154, thereby providing a region of elution buffer that will elute the target analyte and label and quantify the number of target analyte molecules present for final reading of the sample.

[0070] Once the elution buffer is in place, a sequence of magnet movement, similar to a wash sequence, can be initiated to pull the bead bolus up and down across the circumferential channel 182, exposing individual beads to the elution buffer, which will sever the bond between the analyte and the paramagnetic beads, leaving the analyte and label suspended in a slug of elution buffer between the air separators, as shown in FIG. 22. The paramagnetic beads will follow the path of the magnetic field as the magnet is moved. Eventually, the bolus can be recondensed within the channel and pulled away from the elution zone, leaving a space between the bolus and the elution zone containing the label bound to the analyte of interest, as shown in FIG. 22. measurement

[0071] In the measurement step, a confocal laser-based optical system is focused to a point within the elution chamber, e.g., away from the walls, upper, and lower surfaces of the chamber. Analyte measurement and detection occurs within the elution chamber, which is formed by circumferential channel 182, which thus serves as both the elution chamber and the detection chamber.

[0072] The cartridge itself may be made from ultra-low autofluorescence materials, and the elution buffer, pump materials, valves, fluid lines, etc. may be selected so as not to spurt or leach materials that may autofluoresce when delivered into the elution chamber. A small interrogation space is scanned through the liquid in the elution chamber by spinning the cartridge back and forth at a predetermined rpm via the manifold motor. The interrogation space is defined by the lateral extent of the laser spot and the lateral extent of the cone angle of light forming the laser spot. The interrogation space is further defined along the optical axis by the size of the confocal aperture positioned conjugate to the field in the optical system. As those skilled in the art will understand, confocal architectures are used to remove light from locations far from the focal plane. The further away from the focal plane and the smaller the confocal aperture, the more light originating from remote locations is attenuated. In imaging applications, this reduction of out-of-focus light reduces noise and provides sharper image slices. Light originating from locations far from the focal plane (or image slice) does not represent structure within the image slice and is therefore noise. The same process of noise reduction can be employed in the present invention, however, in this case, a confocal system is not used for imaging. As the laser spot scans through the fluid, it may encounter fluorescent labels from the target analytes. In doing so, the laser excites fluorescence from the labels, and individual photons are emitted from the labels and directed by the optical system onto a detector where they are counted. On its way to and beyond the focal plane, the laser light may encounter autofluorescent elements, including the optical system, the window on the cartridge, the glass and binding materials that form the elution chamber, the backside of the elution chamber, or the elution buffer itself. Any fluorescence from these components is noise because it does not originate from the target analyte labels. The confocal architecture attenuates signals from target analyte labels by preferentially allowing those signals within or near the focal plane. As a result, as the laser passes over the target labels, the flow of photons received and counted by the detector increases compared to the background photon level, as shown in FIG. 27 and described in more detail below.A processing algorithm detects and classifies elevated photon counts as molecules of interest. In this manner, individual molecules from the target analyte can be counted to determine the concentration of the target analyte in the original sample.

[0073] The present disclosure allows for substantial advantages over prior art techniques for precisely detecting and quantifying the number of target analytes in samples where the concentration of the target analytes in the sample is low. Furthermore, the methods and systems of the present disclosure have properties that make them suitable for deployment in point-of-care settings. Other aspects of the present disclosure disclosed herein are directed to methods of sample processing and analysis to isolate target analytes and determine their concentrations. These methods generally implement steps consistent with the sample processing and measurement methods described above. Exemplary Embodiments

[0074] Examples and systems are described herein. It should be understood that the terms "example" and "exemplary" are used herein to mean "serving as an example, instance, or illustration." Any embodiment or feature described herein as being "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, like symbols typically identify like components unless the 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.

[0075] The exemplary embodiments described herein are not intended to be limiting, and it should be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0076] 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. Further, reference to, for example, a "second" item does not require or preclude the presence of, for example, a "first" or lower-numbered item, and / or, for example, a "third" or higher-numbered item.

[0077] Reference herein to "one embodiment," "embodiment," "one example," or "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 "one example" in various places within this specification may or may not refer to the same example.

[0078] As used herein, a system, apparatus, device, structure, article, element, component, or hardware that is "configured to" perform a specified function does not merely have the potential to perform the specified function after further modification, but is actually capable of performing the specified function without any alteration. In other words, a system, apparatus, structure, article, element, component, or hardware that is "configured to" perform a specified function is specifically selected, created, implemented, utilized, programmed, and / or designed for the purpose of performing the specified function. As used herein, "configured to" refers to an existing characteristic of a system, apparatus, structure, article, element, component, or hardware that enables the system, apparatus, structure, article, element, component, or hardware to perform the specified function without further modification. For purposes of this disclosure, a system, apparatus, 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.

[0079] In the following description, numerous specific details are set forth to provide a thorough understanding of the disclosed concepts, which may be practiced without some or all of these details. In other instances, details of well-known devices and / or processes are omitted to avoid unnecessarily obscuring the present disclosure. While some concepts will be described in conjunction with specific examples, it will be understood that these examples are not intended to be limiting. Exemplary Analyzer System

[0080] In one aspect, the present disclosure provides an analyzer system, as shown in FIG. 1 , including an analyzer 100 and a cartridge 150. The cartridge 150 is configured to receive a sample and includes multiple chambers for isolating target analytes of the sample and collecting an amount of a first label proportional to the amount of the target analyte in the sample. The analyzer 100 includes an optical system 120. The components of the optical system 120 are shown separate from the rest of the analyzer in FIG. 2 for clarity. As shown, the optical system 120 includes an electromagnetic radiation source 121 configured to provide electromagnetic radiation and form an interrogation space within a detection chamber of the cartridge 150. The optical system 120 also includes a detector 122 configured to detect electromagnetic radiation emitted within the interrogation space by the first label when the first label is present within the interrogation space. Other components of the optical system 120 are also described in more detail below.

[0081] The analyzer 100 also includes a controller 140, represented diagrammatically in Figure 1. The controller 140 includes a non-transitory computer-readable medium having stored thereon program instructions for executing steps performed by the analyzer 100 to identify the presence of a target analyte in a sample based on the electromagnetic radiation detected by the detector 122. The controller 140 includes a processor 141, a memory 142, and a network interface 143.

[0082] The processor 141 of the controller 140 includes 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. The 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, the memory 142 stores program instructions executable by the controller 140 to perform the methods and operations of the present disclosure. The 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.

[0083] In some embodiments, the analyzer 100 includes at least one motor configured to rotate the cartridge to manipulate any samples placed within the cartridge and to align the cartridge with parts 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, the analyzer 100 shown in FIG. 1 includes both a centrifuge 101 and a positioning motor 110.

[0084] In the analyzer 100, the 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 shown more clearly in FIG. 4. As shown, the centrifuge drive motor 103 is configured to couple to the cartridge using a dock 102. The dock shown in FIG. 4 includes a three-point kinematic mount 104. The cartridge 150 has corresponding countersunk slots 152 to align and retain the cartridge 150 on the dock 102. Additionally, the manifold 108 (FIG. 6) may include bearings 113 on the spring-loaded plunger 114 to retain the disk 150 against the dock when the disk 150 is rotated by the centrifuge motor 103. Thus, the cartridge 150 may be securely retained within the dock 102 when inserted into the analyzer 100.

[0085] The dock 102 is connected to a centrifuge drive motor 103 to rotate the dock 102 and the cartridge 150 attached thereto. Additionally, the centrifuge drive motor 103 may include an electronic drive phase sensor 106 and a flag wheel 107 for high-speed, precise control of the centrifuge 101 during operation. In some embodiments, the dock 102 is driven directly by the centrifuge drive motor 103, while in other embodiments, a power transfer system, such as a gearbox or belt drive, may be used to couple the dock 102 to the centrifuge drive motor 103. As discussed below, the dock 102 may also be driven by a manifold 108 (see FIG. 1 ). Specific embodiments of the operation of the centrifuge 101 are described in more detail below.

[0086] In some embodiments, the analyzer includes a manifold 108 with one or more ports, each configured to couple to a respective port on the cartridge. A depiction of the manifold 108 coupled to the cartridge 150 is shown in FIG. 5. Additionally, a bottom view of the manifold 108 is shown in FIG. 6 to illustrate the fluid line ports 111 and bearings 113 for holding the cartridge 150 relative to the dock. To transfer fluid to the cartridge, the manifold 108 includes fluid lines 115 connected to the ports 111.

[0087] Fluid line port 111 of manifold 108 aligns with cartridge 150 at port 154 and may include a seal that covers a corresponding port of cartridge 150 to isolate fluid transfer between manifold 108 and cartridge 150. For example, fluid line port 111 may include an O-ring or other feature to create a seal between the manifold and cartridge port surrounding the inlet port of cartridge 150. In some embodiments, the inlet port of cartridge 150 is already open when cartridge 150 is installed in the analyzer. In other embodiments, the manifold is configured to puncture cartridge 150 to open the inlet port of cartridge 150.

[0088] In some embodiments, the manifold 108 is disposed on a movable arm 109 (shown in FIG. 1 ), which allows the manifold 108 to be decoupled from the cartridge 150 when the cartridge 150 is inserted into or removed from the analyzer. As shown in FIG. 6 , bearings 113 on the plunger hold the cartridge 150 on the dock when the cartridge 150 is spun at high speed by the centrifuge 101. Coupling of the manifold 108 and cartridge 150 for rotation via the manifold may be enabled by a matching structure. For example, as shown in FIG. 6 , the manifold 108 may include pins 112 to secure the manifold to the cartridge. When the cartridge 150 is rotated by the manifold 108, the manifold moves downward onto the cartridge 150, engaging the pins 112 with the cartridge 150 mounting openings. In other embodiments, the pins of the dock 102 may pass through the mounting openings 152 of the cartridge 150 and into receiving holes in the manifold 108. Such a structure provides a secure connection between the manifold 108, the cartridge 150, and the dock 102. Other mounting structures are also possible, as would be understood by one skilled in the art.

[0089] In some embodiments, the analyzer 100 includes a positioning motor 110 coupled to the cartridge 150. In some embodiments, the positioning motor 110 may be coupled to the manifold 108, which couples to the cartridge 150. The positioning motor 110 may be configured to pivot the cartridge 150 to align electromagnetic radiation passing from the first electromagnetic radiation source 121 (FIG. 2) through the objective lens 123 (FIG. 1) with the circumferential channel 182 (see FIG. 11) of the fluid circuit 151 of the cartridge 150. Additionally, the positioning motor 110 may be further used to circulate target analytes through the chambers of the cartridge 150 in cooperation with one or more magnets or by sample hydrodynamics, as described in more detail below. The positioning motor 110 may be a stepper motor or another actuator with specific positioning control. For example, in some embodiments, the location of the positioning motor 110 may be defined within 2° of rotation, or within 1° of rotation, or in less than 1° increments. Specific examples of embodiments using positioning motor 110 are described in more detail below.

[0090] In some embodiments, the positioning motor 110 is directly coupled to the manifold 108, while in other embodiments, a power transfer system, such as a gearbox or belt drive, may be disposed between the positioning motor 110 and the manifold 108. In the analyzer 100 shown in FIG. 1 , the positioning motor 110 is coupled to the cartridge 150 through the manifold 108. In particular, the manifold 108 is disposed on the shaft of the positioning motor 110. Thus, the manifold 108 and the cartridge 150 may move synchronously while maintaining a closed fluid connection therebetween.

[0091] In some embodiments, analyzer 100 includes an optical system 120 (FIG. 2) that directs electromagnetic radiation from a first electromagnetic radiation source 121 into a circumferential channel 182 of cartridge 150 and then collects electromagnetic radiation emitted by the labels onto a first detector 122. Optical system 120 may include one or more mirrors and lenses for steering and directing the electromagnetic radiation into and out of the interrogation space. Additionally, the optical system may include an objective lens 123, as shown in FIG. 7, for focusing the electromagnetic radiation from the first electromagnetic radiation source into the interrogation space within cartridge 150. In some embodiments, objective lens 123 is coupled to a movable stage 124 that allows movement of the objective lens relative to cartridge 150.

[0092] In some embodiments, optical system 120 is a confocal system. For example, electromagnetic radiation source 121 is imaged as a spot in the focal plane of objective lens 123 in circumferential channel 182. Light emitted from labels in circumferential channel 182 excited by electromagnetic radiation source 121 is collected by objective lens 123 and directed by optical system 120 onto a confocal aperture 125 in optical system 120, as shown in FIG. 2 . Confocal aperture 125 is then imaged onto detector 122. The confocal array preferentially passes light from labels within the focal plane of objective lens 123 while excluding light beyond the focal plane. In this way, the array increases the signal-to-noise ratio by passing the signal from the labels while excluding light from the liquid suspension, the cartridge, and elements in the optical system that do not originate from the labels. As known to those skilled in the art, the present arrangement may also use dichroic filters 126 to reflect laser light and pass light emitted by the labels, allowing only light from the labels to reach the detector while prohibiting laser light from reaching the detector. Furthermore, if more than one radiation source is used for detection of additional labels, one or more additional dichroic filters 126 may be used to reflect laser and label electromagnetic radiation from a first electromagnetic radiation source and label while passing electromagnetic radiation from a second electromagnetic radiation source and second label, as shown in Figure 2. Figure 2 shows a three-channel optical system with a dichroic filter 126 on each laser and a second dichroic filter (or mirror) on each collection channel.

[0093] In some embodiments, all of the components of the analyzer 100 are located within a common housing. The common housing can be small in size to fit on a countertop. For example, in some embodiments, the dimensions of the common housing are one meter or less in any direction. Furthermore, in some embodiments, the common housing fits within a 30 inch by 30 inch by 30 inch cube.

[0094] 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 a user through software on an external device, such as a smartphone, table, notebook, computer, or desktop computer. The analyzer receives information from and outputs information to a user of the external device by communicating with the external device through the network interface. Such communication may 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 direct communication with a user, such as a keyboard for receiving input, and a display for outputting information. Further, in some embodiments, the display may include a touchscreen for both outputting information and receiving information from the user. In some embodiments, the analyzer includes a network interface, an input, and a display.

[0095] In some embodiments, the methods of the present disclosure include directing a portion of a sample through a zone of the cartridge 150 that includes a chamber and a channel without the cartridge 150 including any valves. Additionally, in some embodiments, the cartridge 150 does not include any valves.

[0096] In some embodiments, the liquids in cartridge 150 are moved through the cartridge, at least in part, using pumps and valves coupled to the inlet ports of the cartridge, as described in further detail below. For example, in the illustrated embodiment, pump 118 is used to pre-fill priming line 116 with various fluids in desired amounts and in a desired sequence, which are then pumped sequentially into cartridge 150. An embodiment of such a pump configuration is shown in FIG.

[0097] 8, pump 118 is connected to input port 131 of distribution valve 119. Various distribution ports on the valve are connected to air vent port 133, waste container port 134, DI water port 135, wash buffer port 136, elution buffer port 137, and manifold port 138. To prepare for buffer injection into the cartridge, distribution valve 119 moves to a position between the distribution ports and blocks input port 131. While input port 131 is blocked, solenoid valve 130, positioned between pump 118 and waste chamber 139, opens, allowing pump 118 to empty and expel any contents into waste chamber 139 via waste line 132.

[0098] After pump 118 is emptied, solenoid valve 130 closes and distribution valve 119 connects input port 131 to DI water port 135. Pump 118 draws DI water into priming line 116 between distribution valve input port 131 and pump 118. Distribution valve 119 then connects input port 131 to air vent port 133 and pump 118 draws a predefined amount of air. Distribution valve 119 then connects input port 131 to elution buffer port 137 and pump 118 draws a predefined amount of elution buffer into priming line 116. Again, distribution valve 119 moves, connecting input port 131 to air vent port 133 and pump 118 draws a predefined amount of air. Now, distribution valve 119 connects input port 131 to wash buffer port 136, and pump 118 draws in a predefined amount of wash buffer. At this point, priming line 116 between pump 118 and distribution valve 119 is primed with wash buffer 270, elution buffer 271, and DI water 272, as shown in FIG. 9. Elution buffer 271 is encapsulated within priming line 116 along with air 273 to prevent any mixing between the wash buffer, DI water, and elution buffer. It should be understood that FIGS. 8 and 9 are not drawn to scale.

[0099] Those skilled in the art will appreciate that the sequence of dispensing valve, pump, and solenoid valve action can ensure that the pump 118 is filled with DI water with little or no air when the DI water is initially loaded. Furthermore, those skilled in the art will appreciate that the air spaces 273 between the buffers in the lines can be made very small, yet large enough to prevent mixing of the buffers. These steps will ensure accurate dispensing of the buffer into the cartridge by minimizing any spring action of air in the lines or pump. Furthermore, those skilled in the art will appreciate that implementing this sequence of events can help ensure that only DI water 272 is present in the pump. This can be advantageous because different buffers can be used depending on the assay being processed by the present invention. In some cases, certain buffers can degrade pump seals and leach contaminants into the buffers and fluid lines; these contaminants can get into the cartridge, fluoresce during the read process, and generate noise, which can impair sensitivity.

[0100] Once lines 116 are primed with buffer, distribution valves connect input ports 131 to manifold ports 138, and pumps 118 force the primed fluid to the ends of fluid delivery lines 115 within the manifold. At this point, the fluidic system is ready to dispense fluids in controlled volumes at the appropriate times into cartridges for assay processing. While the depicted embodiment shows a single valve operating to fill the priming lines with various fluids, in some embodiments, multiple valves may be used to fill the priming lines. Similarly, embodiments of the present disclosure may have various pumps and valves working together to inject the desired fluids into the cartridge rather than using primed lines.

[0101] In another aspect, the present disclosure provides a fluidic circuit for isolating a target analyte of a sample and collecting a quantity of a first label that is proportional to the concentration of the target analyte in the sample.

[0102] In some embodiments, cartridge 150 is planar, with the cartridge's fluid circuit or circuits lying in a single plane. For example, in some embodiments, cartridge 150 is a round, flat disk, with the cartridge's chambers, passages, and channels positioned circumferentially around the cartridge. As used herein, the term "circumferentially" refers to an angular or circumferential direction, as opposed to a radial or axial direction. Unless otherwise stated, the term "circumferentially" does not mean extending around the entire circumference of the cartridge, but rather is intended to indicate a circumferential direction in a plane of rotation. In some embodiments, at least a group of chambers and channels of a fluid circuit may be sequentially connected circumferentially around a portion of the cartridge. In other embodiments, the cartridge has a flat, linear configuration with a single fluid circuit or several fluid circuits arranged in one or more rows.

[0103] In some embodiments, the cartridge may include a flat base and a molded body disposed over the base, the body including an open passageway extending therethrough that defines the chambers and channels of the cartridge 150. In some embodiments, the body may be a single, integral piece. Thus, for example, in some embodiments, all sidewalls of the chambers and interconnecting channels of the cartridge may be formed by the single, integral piece that forms 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 cover that faces the base and is attached to the body. As an example, the body may be a single, molded piece of cyclic olefin polymer that is 5 mm thick, and the base may be a 188-micron thick laminate of cyclic olefin polymer. The laminate may be bonded to the body using laser welding or ultrasonic welding to provide a bond that is as strong as the materials being bonded together. In some embodiments, the base of cartridge 150 extends across and closes the chambers and microfluidic channels of the cartridge, but may also include ports to receive fluids or allow ventilation from the cartridge, as described above.

[0104] In some embodiments, the cartridge is configured to receive a sample in the range of 50 microliters to 1 milliliter. For example, in some embodiments, the cartridge is configured to receive a sample in the range of 100 to 300 microliters. In particular, the cartridge may include a metering chamber for receiving the sample.

[0105] In some embodiments, the cartridge contains reagents stored in at least one chamber or passage within the cartridge. For example, in some embodiments, the cartridge contains reagents that are stable and are dried before the cartridge is inserted into the analyzer. For example, the reagents may be lyophilized or dried onto the surface of one or more chambers of the cartridge. Or they may be in the form of lyophilized pellets placed within one or more of the chambers or cartridges.

[0106] Although the cartridge is shown and described herein in the form of a disk that spins within the analyzer, in other embodiments, the cartridge is not a disk. Furthermore, some aspects of the present disclosure are implemented without the use of a cartridge at all. For example, in some embodiments, aspects of the present disclosure are implemented within discrete, separate elements that form different chambers. Processing Quality Control Camera

[0107] In some embodiments, analyzer 100 includes a process quality control camera for monitoring the movement of material through cartridge 150. For example, the process quality control camera may be mounted over cartridge 150 to view the material inside cartridge 150. In some embodiments, the process quality control camera is configured to output a representation of only light detected in the visible wavelength spectrum, i.e., the camera is not enabled to detect infrared or ultraviolet light. In some embodiments, controller 140 is configured to analyze images from the process quality control camera to confirm that sample processing is occurring as expected or to detect any unexpected conditions. For example, controller 140 may be configured to detect the presence of undesirable air bubbles in the cartridge. Other exemplary embodiments of using a process quality control camera are described below.

[0108] 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 may be configured to activate at a frequency corresponding to the rotational speed of the cartridge 150 to monitor a particular region of the cartridge 150 as it is rotated. In particular, in some embodiments, the strobe may be used when the centrifuge 101 is spinning the cartridge 150. Optical Quality Control Camera

[0109] In some embodiments, the analyzer 100 includes an optical quality control camera for monitoring the performance of the optical system 120. For example, the optical quality control camera may use mirrors on the slide to block the optical path before and after the confocal aperture images the laser at the confocal aperture to visualize that the electromagnetic radiation has the appropriate intensity, is focused in the correct location, and / or has the correct intensity profile. To image the laser at the confocal aperture, the objective lens may be positioned so that the electromagnetic radiation source is imaged onto the surface of a window on the cartridge 150. When this is done, some of the radiation will reflect off the window back toward the objective lens due to the difference in refractive index between the window and the medium opposite the window. This radiation will be imaged by the optical system onto the confocal aperture. The window on the cartridge may be sized with the correct thickness to simulate the thickness of the window in the detection chamber 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 aperture can be analyzed by the controller 140. The controller 140 may be used to analyze images from the optical quality control camera to verify that the electromagnetic spot is of the correct size, shape, intensity, and position relative to the confocal aperture, ensuring that there are no anomalies in the optical system. The measured size, shape, intensity, and position can be compared to known tolerances for these parameters. If the measurements are outside of tolerance or approaching the limits of tolerance, the controller can notify the user of the analyzer or prevent use of the analyzer. Exemplary Methods

[0110] 12-22 illustrate exemplary fluid circuits and methods utilizing various embodiments of the present disclosure in which the sample is blood. In other embodiments, the cartridges and method chambers used may be suitable for other sample types. For example, the analyzers, methods, and cartridges of the present disclosure may be suitable for use with other biological fluids, such as urine, diluted feces, or oral fluid. Other types of samples are also possible. Additionally, the sample may be pure or diluted. Loading and Sample Separation

[0111] As shown in FIG. 12 , the fluid circuit of the cartridge 150 is initially loaded with a sample 200 in the inlet chamber 158. The inlet chamber 158 includes a sample port 153 that receives the sample 200 prior to analysis. In some embodiments, the sample 200 is received in the cartridge 150 prior to insertion into the analyzer 100, for example, by a medical professional or robot using a syringe. In other embodiments, the inlet chamber 158 is loaded with the sample 200 after the cartridge 150 is received into the analyzer 100. As noted above, in some embodiments, the sample port 153 can be sealed prior to insertion of the sample 200, and the seal can be either punctured or removed to allow insertion of the sample 200. In other embodiments, the sample port 153 can be a simple opening that is available to receive the sample 200 without being “opened.” In some embodiments, the sample port 153 can be sealed after the sample has been entered. In other embodiments, manifold 108 contains a seal to cover the port when the manifold is in contact with the cartridge. In some embodiments, inlet chamber 158 is a metering chamber configured to receive a specific amount of sample, while in other embodiments, inlet chamber 158 is sized larger to accommodate more sample than will be used in the assay. Inlet chamber 158 in the illustrated example of Figures 11-22 is configured to receive approximately 200 μl of liquid.

[0112] Once the sample 200 is loaded into the inlet chamber 158, as shown in FIG. 12, and the cartridge 150 is inserted into the analyzer 100, the cartridge 150 is coupled to the centrifuge 101, as shown in FIG. 1, so that the centrifuge 101 can spin the cartridge 150. As explained in more detail below, the geometries of the chambers and channels within the cartridge 150 are designed to affect the transport of fluids through the cartridge 150. To facilitate understanding of these geometries, the following description will refer to cylindrical / polar directions. In particular, the use of the terms "inward," "inwardly," "outwardly," "outwardly," and similar descriptors refer to radially inward and radially outward directions relative to the center of rotation of the cartridge, which is typically located near the geometric center of the cartridge. The description will also refer to first and second circumferential directions, which relate to the direction in which the cartridge is configured to be spun by the centrifuge, in which the cartridge is configured to be spun in a first circumferential direction. For example, an area at a first circumferential edge of a chamber will pass the stationary reference position before an area at a second circumferential edge of the same chamber. In the embodiments shown in Figures 12-22, the first circumferential direction is clockwise; however, other embodiments of the cartridge may be configured to spin in the opposite direction, such that in these embodiments, the first circumferential direction is counterclockwise.

[0113] Once the cartridge 150 is loaded into the analyzer 100, the centrifuge 101 is activated to rotate the cartridge 150 to move the sample 200 from the inlet chamber 158 through the opening 159 and into the separation area 160, as shown in FIG. 13 . The rotation of the cartridge 150 moves the sample 200 radially outward as a result of “centrifugal force,” i.e., the inertial phenomenon that causes objects to move outward when rotated. If the sample volume exceeds the amount required for analysis, any excess may flow out of the separation area 160 through the overflow channel 165. In some embodiments, the inlet chamber 158 may be offset from the center of the cartridge 150 to facilitate the transfer of the sample to the separation area 160. In other embodiments, the inlet chamber 158 is located at the center of the cartridge 150 so that, once loaded with sample, the rotation of the disk-shaped cartridge 150 will keep the sample and any other liquids received in the cartridge 150 away from the sample port 153. Additionally, in some embodiments, the sample port 153 may be centrally located on the cartridge 150. In some embodiments, to move a sample from the inlet chamber 158 to the separation area 160, the cartridge may be spun from 0 rpm to 1,000 rpm at a rate of, for example, 2,000 rpm / sec and held at that speed for several seconds, for example, 2 to 10 seconds. Thus, sample transfer can occur very quickly. The rotation rates and accelerations provided are exemplary; the actual rates selected depend on the sample being processed and can vary from 100 to 10,000 rpm, with accelerations ranging from 100 rpm / sec to 8,000 rpm / sec.

[0114] In some embodiments, the separation area may include an inner separation chamber 161 and an outer separation chamber 162 configured to retain different components of the sample after separation. In some embodiments, the center of the inner separation chamber may be located 19 mm from the center of rotation, and the center of the outer separation chamber may be located 28 mm from the center of rotation. As the centrifuge spins the cartridge 150, denser components of the sample are forced radially outward into the outer separation chamber 162, while less dense components move radially inward into the inner separation chamber 161. In some embodiments, the inner and outer separation chambers 161, 162 of the separation area 160 are separated by a constriction neck 163 located, for example, 22 mm from the center of rotation. The constriction neck 163 has a smaller cross-sectional area than either of the chambers. For example, in some embodiments, the constriction neck 163 is located 3 mm 2 while the inner isolation area 162 may have a cross-sectional area of ​​12 mm 2 and the outer separation area 162 has an average cross-sectional area of ​​30 mm 2 In this exemplary embodiment, neck 163 is sized to readily allow the denser components to migrate downward, while the less dense components migrate rapidly upward through neck 163. However, as discussed below, constricted neck 163 limits the migration of the denser components into inner separation area 161 when the cartridge is rapidly decelerated.

[0115] It may be beneficial to know the precise volume of the sample to generate an accurate concentration value of the sample for further processing. If the sample is unable to fill the separation area 160 and is unintentionally wasted, or if the separation area is sized to accept more than the sample volume, accurate concentration values ​​may be difficult to obtain. Thus, in some embodiments, cartridge 150 may include various features for metering a precise amount of fluid into separation area 160.

[0116] For example, some embodiments of cartridge 150 may include one or more features to avoid trapping air within the cartridge, particularly during transfer of sample from inlet chamber 158 to subsequent chambers. If air becomes trapped within separation area 160 as sample is loaded therein, a portion of the sample may flow prematurely through overflow channel 165, and accurate metering of the sample into separation area 160 may fail. Therefore, it is beneficial to avoid the formation of trapped air within the cartridge during loading.

[0117] In some embodiments, the opening 159 is coupled to a first circumferential edge of the inner separation chamber 161. As the sample moves outward from the inlet chamber 158 through the opening 159 and into the separation area 160, rotation and / or acceleration of the cartridge 150 in a first circumferential direction by the centrifuge 101 can cause the sample to flow in a second circumferential direction. Thus, if the opening 159 is coupled to the center of the inner separation chamber 161, additional precautions may be necessary to avoid the formation of trapped air in corners at the inward-facing first circumferential edge of the inner separation chamber 161. However, if the opening 159 is coupled to the first circumferential edge of the inner separation chamber 161, as shown in the cartridge 150 of FIGS. 12-22, the inclusion of interior corners farther in the first circumferential direction than the opening 159 is avoided. Similarly, air that may be trapped in such corners is also avoided.

[0118] Additionally, in some embodiments, opening 159 may be reduced in size and depth compared to separation area 160. Such reduction can slow the flow of sample into separation area 160 and allow air to be purged from separation area 160 during filling. Furthermore, the reduced size and depth can also help avoid the formation of a sheet of liquid across the cross section of separation area 160, which can also form trapped air. For example, in one embodiment, the depth of inlet opening 159 may be 0.5 mm, while the depth of inner separation chamber 161 is 2 mm. Thus, the sample stream entering inner separation chamber 161 from inlet opening 159 will not span the entire depth of inner separation chamber 161, allowing air to flow around the stream and exit separation area 160.

[0119] Additionally, in some embodiments, the cross-sectional area of ​​the inlet opening 159 may 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, if the inlet opening 159 is 0.5 mm 2 while the constricted neck 163 may have a cross-sectional area of ​​3 mm 2 Therefore, the volumetric flow rate of the sample into the separation area 160 is unlikely to overwhelm the constricted neck 163 and trap air within the outer separation chamber.

[0120] To prevent air trapping within the outer separation chamber 162, in some embodiments, the inner edge 164 of the outer separation chamber 162 extends at an angle that protrudes inward as the inner edge 164 approaches the constricted neck 163 that separates the inner separation chamber 161 from the outer separation chamber 162. Thus, as the outer separation chamber 162 fills with sample due to rotation of the cartridge, air within the outer separation chamber 162 will "float" inward to the inner edge 164 and then follow 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 area 160.

[0121] In some embodiments, the controller 140 is configured to capture an image of the separation area 160, or a portion thereof, using a process quality control camera after the separation area 160 is filled. The controller may further be configured to analyze the image and verify that the volume of any air bubbles in the separation area 160 is absent or that the volume of air in the separation area is below a predetermined threshold. For example, the controller may be configured to calculate the shape of any air bubbles in the separation area 160 and calculate the overall volume of air in the separation area 160. If the calculated volume of air is above a predetermined threshold, the controller may be configured to abort the analysis or invalidate the results at the end of the process. Similarly, the controller may be configured to continue the analysis if the calculated volume of air is below a predetermined threshold or is zero.

[0122] In some embodiments, the separation area 160 and peripheral channels may include one or more features for precise metering of the sample and controlled separation of the sample's components. For example, in some embodiments, the overflow channel 165 may be positioned to allow precise metering of the amount of sample 200 into the separation area 160. If the amount of sample 200 received within the cartridge 150 exceeds that needed for analysis, the excess will drain through the overflow channel 165. In some embodiments, the overflow channel 165 leads to a waste chamber 166 where excess liquid can be stored.

[0123] Due to the rotation of the cartridge 150 and the centrifugal forces on the sample, the separation area 160 fills from the outer end toward the inner end. Therefore, positioning the opening of the overflow channel 165 at a particular radial position within the inner separation chamber 161 dictates the amount of sample that can be loaded into the separation area 160. For example, as the centrifuge 101 spins the cartridge 150, the sample will move toward the outer end of the outer separation chamber 162, creating 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 that enters the separation area 160 will exit the separation area 160 through the overflow channel 165. Therefore, the amount of sample that will be analyzed can be precisely metered based on the radial position of the overflow channel 165. Separation of sample components

[0124] As shown in FIG. 14 , after the sample is loaded into the separation area 160, the centrifuge 101 may continue to spin the cartridge 150 to separate the sample 200 into its different components. For example, the centrifuge 101 may spin the cartridge 150 to send the sample's denser components outward and leave the less dense components radially inward. In some embodiments, the speed of the centrifuge 101 may be increased to separate the components of the sample 200. For example, in one embodiment, after loading the sample, the centrifuge 101 may accelerate the cartridge 150 to a speed of 1,000 rpm at an acceleration rate of 2,000 rpm / sec. In response to reaching 1,000 rpm, the centrifuge 101 may further accelerate the cartridge 150 to a rate of 7,000 rpm at 5,000 rpm / sec and hold that rate for 90 seconds to separate the components. In another embodiment, the centrifuge 101 may skip the initial transfer spin speed and proceed directly to a separation speed from 0 rpm to 10,000 rpm at an acceleration of 2,000 rpm / sec. The separation step may occur at a spin speed between 1,000 rpm and 20,000 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 to resist crushing. The duration of the separation may be performed in the range of 10 seconds to 5 minutes.

[0125] In some embodiments, the sample 200 may be whole blood, and continued rotation of the cartridge 150 may separate red blood cells 202 from plasma 201, as depicted in Figure 14. For example, in the separation area 160 of the illustrated embodiment, the inner separation chamber 161 may act as a plasma compartment, and the outer separation chamber 162 may act as a red blood cell trap. In response to the high speed rotation of the cartridge 150, the denser red blood cells 202 are forced radially outward, while the less dense plasma 201 moves radially inward into the plasma compartment 161.

[0126] The angled inner edge 164 of the outer separation chamber 162 may assist in separating the components of the sample in a manner similar to that which aided in the removal of air from the outer separation chamber 162, as described above. As the centrifuge 101 spins the cartridge 150, the denser components will migrate outward and the less dense components will migrate inward. Thus, similar to the flow path of air within the outer separation chamber 162 during the filling process, the lighter components of the sample will migrate inward and then follow the angled inner edge 164 of the outer separation chamber 162 until they reach the constricted neck 163 and pass into the inner separation chamber 161.

[0127] In some embodiments, controller 140 may be configured to capture an image of the separation area, or a portion thereof, using a process quality control camera after the separation process. Controller 140 may further be configured to analyze the image and determine the fill level of the denser components of the sample within separation area 160. In some embodiments, controller 140 is configured to confirm that a denser component of the sample has moved outward from a predetermined fill level. The controller may likewise be configured to continue the analysis in response to such confirmation.

[0128] For example, if the sample is whole blood, the controller 140 may be configured to analyze the image and determine the red blood cell fill level within the separation area 140. If the red blood cell fill level is outside a predetermined radius, the controller 140 may be configured to continue the analysis. On the other hand, if the red blood cell fill level is inside the predetermined radius, the controller 140 may be configured to send a control signal to the centrifuge 101 to continue spinning the cartridge to further separate the components of the blood sample. For example, an image may be captured and analyzed at 90 seconds of separation time. If the red blood cell level is within a threshold distance of, for example, 22 mm from the center of rotation, the controller 140 may be configured to capture an additional image and send a control signal to spin for an additional 30 seconds before reassessing the red blood cell level. In some embodiments, the duration or rate of this additional control signal may be based on the identified red blood cell fill level. Alternatively, the controller 140 may be configured to abort the analysis or invalidate the results at the end of processing. In some embodiments, the method is configured to transfer a portion of the sample that excludes red blood cells. The inclusion of red blood cells can add hemoglobin to the plasma, which can affect analysis. Therefore, identifying the red blood cell loading level allows the quality of the plasma transferred for further analysis to be determined.

[0129] Similarly, in some embodiments, an image of the separation area 160 after the separation process may be analyzed by the controller to determine the transparency of the plasma within the inner separation chamber. Further, the controller 140 may be configured to proceed with the analysis in response to confirming that the plasma meets a threshold transparency.

[0130] Still further, in some embodiments, the controller 140 may be configured to analyze an image of the separated blood sample and determine the hematocrit level of the blood based on the radial distance of the red blood cell line and the time of rotation. Those skilled in the art will readily understand that for a given chamber geometry, rotation rate, and rotation time, blood with a lower hematocrit level will exhibit a separation line at a greater radius than blood with a higher hematocrit level. For a given cartridge geometry and spin parameters, different hematocrit levels can be initiated and evaluated to determine a calibration table stored within the controller 140. When a sample of unknown hematocrit is initiated, the separation line can be compared to values ​​stored within the controller to determine the hematocrit level of the sample being initiated after a predetermined spin time. Furthermore, the controller 140 may be configured to proceed with the analysis in response to confirming that the hematocrit level is below a predetermined threshold. Supernatant transfer

[0131] 15, a portion of the sample 200 may be removed from the separation area 160 through a siphon 167 extending from the separation area 160. The siphon 167 extends 1 mm into a second chamber, such as a mixing chamber 175. 2The siphon 167 may be in the form of a microfluidic channel with a cross-sectional area of ​​0.05 mm. The siphon 167 may include a first section 168 extending from the separation area 160, an apex 169, and a second section 170 extending from the apex 169 to a mixing chamber 175. The first section 168 of the siphon 167 extends from a siphon inlet 171 in a direction having a radially inward component, away from the inner separation chamber 161 toward the apex 169. The second section 170 further extends from the apex 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 located at a radial position 21 mm from the center of rotation, while the siphon apex may be located 16 mm from the center of rotation, and the siphon outlet 172 may be located at a radial distance of 30 mm from the center of rotation. Other radial distances may also be selected to suit the needs of the application, so long as siphon outlet 172 is at a greater radial distance than siphon inlet 171 and apex 169 is at a lesser radial distance than both siphon inlet 171 and siphon outlet 172. Thus, apex 169 is the radially innermost point of siphon 167, and siphon outlet 172 is radially outward compared to siphon inlet 171. Therefore, because the rotation of the centrifuge generally drives the sample radially outward, once a portion of the sample has passed over apex 169, siphon 167 will drive a portion of the sample from inner separation chamber 161 to mixing chamber 175.

[0132] In some embodiments, the siphon may be primed, i.e., a portion of the sample may be forced over the apex to begin wicking through capillary action. In other words, capillary forces may draw sample into the first section 168 of the siphon 167 and over the apex 169 until wicking draws additional fluid from the inner separation chamber 161. The cross-sectional area of ​​the siphon 167 may be smaller, for example, about 0.1 mm, to facilitate capillary action. 2 ~about 0.3mm 2, or about 0.2 mm 2 In other embodiments, the siphon 167 may be primed through the use of a pump that draws sample into the siphon 167 until the sample passes the apex.

[0133] Furthermore, in some embodiments, the siphon may be primed by acceleration. For example, in one embodiment, after cartridge 150 completes the separation step at 7,000 rpm, it is decelerated by centrifuge 101 to 3,000 rpm at a deceleration rate of 2,000 rpm / s to prepare for the siphoning step. While cartridge 150 spins in the first circumferential direction, inertia will propel the sample and cause it to continue moving in that direction. Thus, if cartridge 150 is rapidly decelerated, for example, at 8,000 rpm / s from 3,000 rpm to 0 rpm, inertia will cause sample 200 to continue moving in the first circumferential direction, and the sample will flow through first section 168 of siphon 167 and through apex 169, which is radially outward of the fill level of separation area 160, due to its extension along the first circumferential direction. At this point, the centrifuge 101 may reverse its spin direction to -1,000 rpm at an acceleration of 2,000 rpm / s and hold that speed. Centrifugal force will move the fluid in channel 170 radially outward toward siphon outlet 172, which is radially outward of siphon inlet 171. Separation area 160 will continue to drain until the fill level is radially outward of (or "drops below") the connection where first section 168 of siphon 167 opens into inner separation chamber 161. This method of priming and siphoning is significantly faster than capillary action and / or pump-based priming and siphoning, as the entire process can occur in a matter of seconds. In some embodiments, apex 169 is radially inward of overflow channel 165, which prevents sample from flowing through siphon 167 while separation area 160 is filling. Other rotational speeds and accelerations can also be used, so long as the acceleration is sufficient to force fluid over the siphon apex 169 and cause the cartridge 150 to continue spinning and drawing fluid from the separation area 160 .

[0134] As described above, first section 168 of siphon 167 extends radially inward in a first circumferential direction. Furthermore, in some embodiments, the shape of first section 168 of siphon 167 is specifically shaped to facilitate priming of siphon 167. For example, in some embodiments, a portion of first section 168 at the end connected to inner separation chamber 161 is generally parallel to the first circumferential direction, e.g., within 10 degrees of parallel, with the first circumferential direction. First section 168 gradually curves inward as it extends toward apex 169. As described above, in response to deceleration of cartridge 150, the sample is forced in the first circumferential direction. Thus, when the first portion of first section 168 is substantially aligned with the first circumferential direction, the sample flows into siphon 167 with additional momentum. As a result of this momentum, the sample can reach and flow past apex 169, thereby priming siphon 167.

[0135] In some embodiments, the location of the connection between the first section 168 of the siphon 167 and the inner separation chamber 161 is selected to transfer a measured amount of sample through the siphon 167. For example, in the depicted embodiment of FIG. 13 , the siphon 167 will transfer a precise amount of sample, e.g., 50 microliters, based on the distance between the opening of the radial 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 within the inner separation chamber 161 will drop (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 air reaches the first section 168 of the siphon 167, no additional amount of sample will be drawn from the inner separation chamber 161. Thus, the position at which first section 168 opens into inner separation chamber 161 may be used to define the measured amount of sample that is transferred to the downstream chamber.

[0136] The location of opening 171 of first section 168 of siphon 167 into inner separation chamber 161 may also be selected to limit the transfer of only certain components of the sample through siphon 167. For example, in embodiments in which the sample is whole blood and separation chambers 162, 161 are used to separate red blood cells from plasma, opening 171 of first section 168 may be positioned radially inward from the separated red blood cells. Unintentional inclusion of red blood cells in a sample transferred to the mixing chamber can result in hemoglobin contamination during the mixing process. Therefore, it is advantageous to position the opening of first section 168 to avoid inclusion of red blood cells in a sample transferred through siphon 167. Thus, if outer separation chamber 162 is a red blood cell trap configured to receive red blood cells after the separation process, opening 171 of first section 168 may be positioned radially inward from the red blood cell trap and into the plasma container. Similarly, the volume of the outer separation chamber 162 may be selected based on a typical red blood cell volume, e.g., 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.

[0137] In some embodiments, outer separation chamber 162 extends in a first circumferential direction away from constriction neck 163. Thus, as cartridge 150 is decelerated and less dense components of the sample are forced through siphon 167, denser components are similarly forced toward the closed end of outer separation chamber 162, away from constriction neck 163 and siphon inlet 171. For example, in embodiments using whole blood, plasma above constriction neck 163 is transferred through siphon 167, and red blood cells are forced toward the closed end of the red blood cell trap formed by outer separation chamber 162.

[0138] As discussed, a large deceleration may be used to prime the siphon. As the cartridge decelerates, high-density components in the outer separation chamber 162 move toward the closed end and away from the constricted neck 163. However, a density gradient may exist within 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, with separated components at the top of the chamber moving toward the constricted neck 163. If those components move far enough toward the neck 163, they may be transported upward into the upper separation chamber 161 and sucked out of the outer separation chamber 162 into the mixing chamber 175.

[0139] In some embodiments, the cartridge includes posts 191 within the outer separation chamber 162. The posts 191 are formed by mounting structures that extend across the outer separation chamber 162 and secure opposing sides of the cartridge together. For example, the posts 191 may be formed by raised protrusions within the body of the cartridge that are attached to the cover of the cartridge and provide support between the body and cover within the outer separation chamber 162. Such support can help prevent separation between the cover and body as the cartridge is spun, particularly at high rpm, while sample components are being separated and pressure within the outer separation chamber 162 is increased. Sample Mixing

[0140] From the separation area 160, the plasma 201 travels to the mixing chamber 175, which may have reagents 157 therein, as shown in FIG. 16. For example, the mixing chamber 175 may contain lyophilized paramagnetic capture beads 177, a detection label, a control analyte, and a control label. Once in the mixing chamber 175, the plasma is mixed with the reagents by rapid acceleration and deceleration of the cartridge 150, all while continuing to rotate in a first circumferential direction, as shown in FIG.

[0141] In some embodiments, mixing of the plasma with the reagent is facilitated by a mixing ball 176 disposed within the mixing chamber 175. Acceleration and deceleration of the cartridge 150 causes the mixing ball 176 to move back and forth through the mixing chamber 175 and bounce off its walls as it rotates in a first circumferential direction. For example, in one embodiment, the centrifuge 101 may move the cartridge 150 at a rotational speed of 200 rpm to 500 rpm, accelerating and decelerating at 1,500 rpm / s. This corresponds to a mixing frequency of 5 Hz. The turbulent movement of the mixing ball 176 initially rehydrates and releases the paramagnetic capture beads, detection label, control analyte, and control label into the plasma. The mixing ball 176 further serves to facilitate the binding kinetics of the target analyte to the paramagnetic capture beads 177 (FIG. 18) and detection label. After the mixing step, the target analyte and detection label may be attached to the paramagnetic capture beads, which are dispersed together and throughout the plasma. In some embodiments, rehydration of the reagents and incubation of the target analyte occurs in less than 20 minutes, eg, less than 10 minutes or less than 5 minutes.

[0142] The term "mixing ball" is used herein to refer to the movement of this feature, rather than to any particular shape. Thus, in some embodiments, mixing ball 176 may be spherical, but in other embodiments, it may have another shape. By way of example, mixing ball 176 may be oval, cubic, or star-shaped. In some embodiments, the mixing ball is non-magnetic. The term "non-magnetic" as used herein includes those materials that are neither magnetic nor paramagnetic. Additionally, in some embodiments, the surface of the mixing ball comprises a substance having low reactivity. For example, in some embodiments, mixing ball 176 may comprise brass, glass, or Teflon. Plastic, ceramic, or other hard materials with a higher density than the sample may also be used for the mixing ball. In other embodiments, particularly those in which paramagnetic capture beads are not used, mixing ball 176 may comprise a ferromagnetic material, such as steel. Similarly, in some embodiments, the mixing ball is coated with a substance having low reactivity.

[0143] In some embodiments, the mixing chamber 175 and the surrounding channels include one or more features to retain the sample within the mixing chamber during the mixing process. For example, as illustrated in FIG. 17 , in fluid circuit 151, pre-mixing chamber channel 173 and post-mixing chamber channel 174 both extend radially inward from mixing chamber 175. Thus, centrifugal force pushes the sample outward into mixing chamber 175 as cartridge 150 is spun by centrifuge 101.

[0144] Similarly, to prevent sample migration out of the mixing chamber due to capillary action, at least one of the channels 173, 174 directly connected to the mixing chamber 175 may include a capillary break 178, 179. For example, in the cartridge 150 shown in FIG. 16 , the pre-mixing chamber channel 173 and the post-mixing chamber channel 174 both include individual capillary breaks 178, 179. Each of the capillary breaks 178, 179 is formed by a section of the individual channel 173, 174 that expands in a direction away from the mixing chamber 175. The expanding cross-sectional area of ​​the capillary breaks 178, 179 results in reduced capillary forces as the sample migrates away from the mixing chamber 175. The use of the capillary breaks 178, 179 reduces the effect of capillary action, keeping the culture fluid within the chamber after mixing and incubation of the paramagnetic capture beads, detection label, control analyte, and control label. This allows time for the magnet 145 to pull the paramagnetic beads out of suspension without the culture fluid leaving the chamber 175, as discussed in more detail below. In the embodiment shown in Figures 12-22, the capillary break is in the form of a diamond. In other embodiments, other shapes that expand as they protrude away from the mixing chamber 175 are also possible.

[0145] Furthermore, the use of two capillary breaks can help balance the forces on the sample and keep the sample within the mixing chamber 175. For example, the mixing chamber 175 may be filled to such an extent that fill lines are located on either side of the mixing chamber 175 within the capillary breaks 178, 179. Thus, if the sample moves toward one side of the mixing chamber such that the fill line in one of the channels moves radially inward toward the widened section of the respective capillary break (e.g., 178), the capillary forces within that channel will be reduced. At the same time, the fill line in the channel on the opposite side of the mixing chamber 175 should move radially outward and into the smaller cross-sectional area of ​​the opposing capillary break (e.g., 179), where the capillary forces will be stronger. Thus, the capillary forces on the sample from both capillary breaks will urge the sample to remain within the mixing chamber. To help promote this balancing effect, in some embodiments, the two capillary breaks 178, 179 are at the same radial location.

[0146] Furthermore, the use of two capillary breaks can help balance the forces on the sample and retain the sample within the mixing chamber 175. For example, the mixing chamber 175 may be filled to such an extent that fill lines are located on either side of the mixing chamber 175 within the capillary breaks 178, 179. Thus, if the sample moves toward one side of the mixing chamber such that the fill line in one of the channels moves radially inward toward the widened section of the respective capillary break (e.g., 178), the capillary forces within that channel will be reduced. At the same time, the fill line in the channel on the opposite side of the mixing chamber 175 should move radially outward and into the smaller cross-sectional area of ​​the opposing capillary break (e.g., 179), where the capillary forces will be stronger. Thus, the capillary forces on the sample from both capillary breaks will urge the sample to remain within the mixing chamber. To help promote this balancing effect, in some embodiments, the two capillary breaks 178, 179 are at the same radial location.

[0147] The capillary breaks 178, 179 may also serve as reservoirs, holding a portion of the sample during the early stages of the mixing process. In some embodiments, reagents may be stored in a stable, dried form within the cartridge 150. For example, the reagents may be lyophilized prior to the analytical methods of the present disclosure. In such cases, the mixing of the plasma and lyophilized reagents that occurs within the mixing chamber 175 may result in the release of air trapped during the lyophilization process. Again, due to the centrifugal force caused by the rotation of the cartridge, this air will move radially inward and outward from the sample as the mixing process occurs. Thus, the overall volume occupied by the sample when it first reaches the mixing chamber is greater than later in the mixing process when the air is released. The capillary breaks 178, 179 may serve as reservoirs, holding a portion of the sample until the air is released and allowed to escape from the sample.

[0148] In some embodiments, controller 140 may be configured to capture an image of mixing chamber 175, or a portion thereof, using a process quality control camera after transfer from separation area 160. Controller 140 may further be configured to analyze the image and determine the fill level of mixing chamber 175. Knowledge of the precise volume of the sample being analyzed can be useful in determining the exact concentration of a target analyte. Thus, controller 140 may be configured to proceed with the analysis in response to determining that the volume in mixing chamber 175 exceeds a threshold value. Furthermore, controller 140 may be configured to use the volume of the sample being analyzed to normalize data resulting from the analysis.

[0149] 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 a portion of the sample remains in the pre-mixing chamber channel 173 and the post-mixing chamber channel 174. Thus, the controller 140 may 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.

[0150] 23 shows another embodiment of a fluidic circuit 351 according to the present disclosure. The fluidic circuit 351 includes a port 352 and a channel 355, which is coupled to a mixing chamber 375. The port 352 and the channel 355, in use, allow for the introduction of a liquid reagent 457 into the fluidic circuit 351 (and associated cartridge). This may occur, for example, when a sample is loaded into the sample chamber 358. Rotation of the cartridge in which the fluidic circuit 351 is included moves the liquid reagent 457 radially outward as a result of "centrifugal force" into the mixing chamber 375. This may occur at the same time that the sample is transferred to the separation chambers 361 and 362. The liquid reagent 457 may remain within the mixing chamber 375 throughout the centrifugation process, as described above. After centrifugation is completed and the supernatant is transferred into mixing chamber 375, the supernatant may be mixed with the binding partner contained in liquid reagent 457 in a manner similar to that described above, where the binding partner is contained in a lyophilized pellet.

[0151] 23 shows a liquid reagent port 352 and channel 355 to the right of the mixing chamber 375, where the reagent channel intersects with the siphon channel, but in other embodiments, the liquid reagent channel and port may be provided elsewhere. For example, in some embodiments, the liquid reagent channel may connect directly to the mixing chamber between the inlet and outlet channels of the mixing chamber, with the liquid reagent port located radially inward. Magnetic transfer of the sample

[0152] In some embodiments, the analyzer 100 may include one or more magnets 145 configured to move the paramagnetic capture beads, as described in more detail below. Each may be coupled to a movable stage 147, 148, as illustrated in the cross-sectional portion of the analyzer 100 shown in FIG. 24. The magnets 145, 146 may be positioned above or below the cartridge 150 to allow movement of the paramagnetic capture beads 177 from outside the cartridge 150. The linear movement of the magnets 145 in the radial and axial directions, combined with the rotation of the cartridge 150 by the manifold positioning motor 110, allows the magnets 145 to be positioned over any portion of the cartridge 150 without having to move the magnets 145 in the circumferential direction. Thus, in some embodiments, stage 148 may be enabled to move magnets 145, 146 radially forward and backward along cartridge 150 and axially toward and away from cartridge 150 using radial magnet stage 148, and to introduce or remove magnetic attraction of paramagnetic capture beads 177 using axial magnet stage 147. In other embodiments, the movable stage may be operable to move in three dimensions to move over any portion of cartridge 150 without cartridge 150 having to be rotated. In some embodiments, the magnets may be electromagnets, while in other embodiments, the magnets may be permanent magnets. Additionally, in some embodiments, the electromagnets may be activated using AC current to further facilitate manipulation of the paramagnetic beads.

[0153] Once the contents of the mixing chamber 175 have been thoroughly mixed and the target analytes have attached to the dispersed paramagnetic capture beads 177 (as shown in FIG. 17 ), the paramagnetic capture beads 177 may be immobilized within a portion of the mixing chamber 175 by a magnet or other means. Once the paramagnetic capture beads 177 are immobilized within the mixing chamber 175, a wash buffer 270 may be pumped through the mixing chamber 175 to remove blood plasma 201 therefrom, as shown in FIG. 19 . As the wash buffer 270 from the delivery line 115 is pumped into the cartridge, it travels along path 156 into the mixing chamber 175, pushing the blood plasma 201 and any other contaminants not bound to the paramagnetic beads 177 out of the mixing chamber 175 toward the waste chamber. As the wash buffer 270 continues to flow across the paramagnetic beads 177 and the walls of the cartridge, the undesired contaminants are washed away. With repeated washing, the concentration of such contaminants can be reduced to undetectable or manageable levels.

[0154] Magnets 145, 146 may be introduced to facilitate movement of the paramagnetic capture beads 177 through the cartridge 150. Once the magnet 145 is placed adjacent to the mixing chamber 175, the cartridge 150 may be rotated back and forth over the magnet 145 to collect the paramagnetic capture beads 177 into a bolus, as shown in FIG. 18 . In some embodiments, the controller 140 is configured to capture an image of the bead bolus after the paramagnetic capture beads are collected using the magnet 145. This may occur within the mixing chamber or at some other location in the circuit. Additionally, in some embodiments, the controller 140 is configured to measure the size of the paramagnetic bead bolus and proceed with the analysis if the bead bolus size is within a predetermined range. If not, the controller 140 may identify an error and abort the analysis or invalidate the results in final processing.

[0155] In some embodiments, the magnets 145, 146 may be introduced before the introduction of the wash buffer. In such embodiments, during purging of the blood plasma 201 from the mixing chamber 175, the bolus of paramagnetic capture beads 177 may be kept within a specific location in the mixing chamber 175 to avoid dispersion of the bolus. For example, the bolus may be positioned at a corner of the mixing chamber 175 during purging of the blood plasma.

[0156] In some embodiments, wash buffer 270 is introduced into cartridge 150 using pump 118 from fluid delivery line 115 via manifold 108, as shown in FIG. 8. For example, in some embodiments, pump 118 moves a portion of the contents of pre-filled priming line 116 into cartridge 150 via manifold 108. Because pathway 156 ( FIG. 11 ) in fluid circuit 151 is isolated between inlet port 154 and mixing chamber 175, simply injecting the contents of priming line 116 into cartridge 150 will force wash buffer 270 through circumferential channel 182, through radial channel 181, around elbow 180, and through post-mixing chamber channel 174 into mixing chamber 175. The term “isolated” as used herein means that there are no separate branches extending from the pathway or openings in the pathway for the fluid to escape. Thus, fluid pumped into the cartridge through inlet port 154 will eventually arrive at mixing chamber 175. In the illustrated embodiment, the isolated pathway continues from mixing chamber 175 to separation area 160. However, in other embodiments, the fluid circuit may include other branches connected to the mixing chamber. Additionally, aspects of the present disclosure may be utilized without such an isolated pathway.

[0157] In some embodiments, the controller 140 may be configured to capture an image of at least a portion of the mixing chamber 175 after it has been filled with the wash buffer 270. Additionally, the controller 140 may be configured to analyze the image of the mixing chamber 175 to confirm the absence of air in the mixing chamber 175 or to confirm that the volume of any air bubbles in the mixing chamber 175 is below a predetermined threshold. For example, the controller 140 may be configured to calculate the shape of any air bubbles in the mixing chamber 175 and calculate the overall volume of air in the mixing chamber 175. If the calculated volume of air is above a predetermined threshold, the controller may be configured to pump additional fluid in or abort the analysis. Similarly, the controller may be configured to continue the analysis if the calculated volume of air is below a predetermined threshold or is zero.

[0158] Once the paramagnetic capture beads 177 are collected in a bolus within the mixing chamber 175, as shown in FIG. 19 , the magnet 145 may be moved in conjunction with the rotation of the cartridge 150 by the movable stage 148 to transport the bolus of paramagnetic capture beads 177 around the elbow 180 and into the radial channel 181, which may act as an additional wash zone for the cartridge 150. In some embodiments, the controller 140 may be configured to capture an image of at least a portion of the radial channel 181 after the bead bolus of paramagnetic capture beads 171 is transferred into the radial channel 181 and verify that transfer has occurred. Furthermore, in some embodiments, the controller 140 is configured to measure the size of the paramagnetic bead bolus within the radial channel 181 and proceed with the analysis if the size of the bead bolus within the radial channel 181 is within a predetermined range. If not, the controller 140 may identify an error and abort the analysis or invalidate the results at the end of processing.

[0159] Once the paramagnetic capture beads 177 are positioned within the radial channels 181, the movable stage 148 may be moved back and forth, as shown diagrammatically in Figure 20, to effectively wash the paramagnetic capture beads 177 and remove appreciable levels of contaminants from the sample, except for the target analyte, detection label, and any controls used in the system. In some embodiments, the used wash buffer may be repeatedly swept out of the radial channels 181 and a new volume of wash buffer 270 may be added to the radial channels 181 before repeating the wash step. The wash step may be performed several times, for example, three or more times.

[0160] In some embodiments, a second magnet 146 may be introduced during the wash step to disperse and recondense the paramagnetic capture beads 177 during the sequence of steps in the wash operation. In particular, the magnet 145 and the second magnet 146 may be positioned on opposite sides of the cartridge 150 to disperse and recondense the paramagnetic capture beads 177 as they move along the radial channel 181. Dispersing the paramagnetic capture beads 177 allows them to be washed more efficiently by the wash buffer than if the beads were held together in a bolus. Thus, the time and number of cycles required for the wash step may be reduced compared to conventional wash methods.

[0161] 25 and 26 illustrate two exemplary embodiments of a washing operation according to the present invention. FIG. 25 illustrates a washing operation in which two magnets 145, 146 are moved in a sawtooth pattern relative to a radial channel 181. In particular, FIG. 25 illustrates five discrete locations P1-P5 occupied by a first magnet 145 and a second magnet 146 during the sawtooth washing operation. At position P1, the first magnet 145 is adjacent to the cartridge 150 while the second magnet 146 is remote from the cartridge 150, which causes the paramagnetic capture beads to form a bolus adjacent to the first magnet 145. The magnets 145, 146 are then moved axially such that the second magnet 146 approaches the vicinity of the cartridge 150 while the first magnet 145 moves away from the cartridge 150. In conjunction with this movement, the movable stage 148 may also be moved so that the magnets 145, 146 are also repositioned laterally along the radial channel 181. As the first magnet 145 moves away from the paramagnetic capture beads 177, the bolus is dispersed into the wash solution so that unwanted components of the blood plasma can be separated and washed from the paramagnetic capture beads 177. The dispersion of the paramagnetic capture beads 177 is illustrated between positions P1 and P2 in FIG. 25. As the second magnet 146 approaches the radial channel 181, the paramagnetic capture beads 177 are pulled out of suspension and again reassembled into a tight bolus. The dispersion and recondensation 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 be continued in a sawtooth pattern for several additional steps.

[0162] FIG. 26 illustrates another embodiment of a wash operation in which two magnets 145, 146 are moved in a square wave or trapezoidal pattern relative to a radial channel 181. In particular, FIG. 26 illustrates nine discrete locations P1-P9 occupied by a first magnet 145 and a second magnet 146 during a sawtooth wash operation. Again, at position P1, the second magnet 146 is remote from the cartridge 150, while the first magnet 145 is adjacent to the cartridge 150, which causes the paramagnetic capture beads to form a bolus adjacent to the second magnet 146. The movable stage 148 is then moved such that the magnets 145, 146 move relative to the radial channel 181. Advantageously, the stage 148 can be moved at a speed sufficient to diffuse 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 such that the second magnet 146 approaches the vicinity of the cartridge 150 while the first magnet 145 moves away from the cartridge 150. Again, as the first magnet 145 moves away from the paramagnetic capture beads 177, the bolus is dispersed into the wash solution so that unwanted components of the blood plasma can be separated and washed from the paramagnetic capture beads 177. Similarly, as the second magnet 146 approaches the cartridge 150, the paramagnetic capture beads 177 are drawn out of suspension against the wash chamber wall, as shown in position P3.

[0163] 25 and 26 involve recondensing the paramagnetic capture beads into a tight bolus, in other embodiments, the paramagnetic capture beads may be directed through the channel without strictly coalescing into a bolus during the operation. For example, during the operation steps, the beads may remain relatively dispersed in the wash fluid but be moved by a magnet back and forth along the length of the radial channel.

[0164] As described above, in some embodiments, the magnet 145 and the second magnet 146 are positioned on opposite sides of the cartridge 150, e.g., above and below the cartridge 150. In other embodiments, the magnets 145, 146 are positioned on the same side of the cartridge 150 but on opposite sides of the radial channel 181 relative to the circumference. Furthermore, in some embodiments, the magnets 145, 146 cause the paramagnetic capture beads 177 to diffuse 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 may facilitate the breakup of the bolus of paramagnetic capture beads 177, improving the wash operation.

[0165] After the wash operation, the paramagnetic capture beads 177 may again be collected using the first magnet 145 and moved to the circumferential channel 182. The pump 118 may then be activated to push fluid from the priming line 116 further along the path 156, which extends from the inlet port 154 to the mixing chamber 175. Specifically, the pump 118 operates until the first beads of air 273 are pushed past the bolus of paramagnetic capture beads 177 such that the paramagnetic capture beads 177 are immersed in the elution buffer 271, as shown in FIG. 21 . While retaining the paramagnetic capture beads 177 using one or more magnets 145, 146, the cartridge 150 may be rotated back and forth, passing the paramagnetic capture beads 177 through the circumferential channel 182 and the elution buffer 271, which removes binding between the paramagnetic capture beads 177 and the target analyte and between the label and the target analyte. This leaves a pure fluorescent dye complex suspension in elution buffer 271 within circumferential channel 182 as shown in FIG.

[0166] To enhance elution of target analytes and labels, a magnetic elution operation may be used similar to the wash operation described above. For example, the magnets 145, 146 may be moved in a particular pattern, such as that shown in Figures 25 and 26, relative to the circumferential channel 182, while the cartridge is rotated relative to the magnets. Controlling the paramagnetic beads in a controlled manner similar to that of the wash operation enhances the magnetic elution operation.

[0167] While the described embodiment uses radial channel 181 as a wash zone and circumferential channel 182 as an elution zone, in other embodiments, the areas 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 in an area of ​​the circumferential channel adjacent to the radial channel, while elution occurs in a portion of the circumferential channel closer to inlet port 154. However, with regard to methods for maintaining distinct washing and elution zones along path 156 from the inlet port to mixing chamber 175, it may be advantageous for the elution zone to be closer to inlet port 154 than the washing zone. When fluid flows unidirectionally through fluid circuit 151 along path 156, having the elution zone closer to the inlet port may help ensure that elution occurs in a location where used wash buffer is not traveling.

[0168] After the elution process is performed, the paramagnetic capture beads 177 may be moved to the outside of the circumferential channel 182 or to one end of the circumferential channel 182 to avoid interfering with the optical system 120, as shown in Figure 22. The optical system 120 of the analyzer 100 may then be activated and analyze the solution within the circumferential channel to determine the presence or concentration of the target analyte in the volume of fluid within the circumferential channel, as described above. Thus, at least a portion of the circumferential channel 182 may serve as both an elution zone, where an elution buffer removes the label from the paramagnetic beads, as well as a detection zone.

[0169] Figure 27 shows, from left to right, the signal from the elution buffer where no elution has occurred. Within that region, the signal is generally below 30 photons in each signal bin. Near the center of the image, a zone where the label has eluted is clearly visible as a series of bins, with a signal above 30 photons. To the right of the elution zone, another region is visible where the label has not eluted, and the signal is also generally below 30 photons per bin. To the right of that zone is the region where the bead bolus was retracted after the elution process. Because the bead substrate fluoresces and strongly scatters light, a very strong signal, well above 150 photons, is observed in this region. Finally, to the right of the bolus region is another region of clean elution buffer where no elution has occurred.

[0170] The overall purification process, briefly described herein, is designed to create 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 readout process. After the elution sequence, in which the target analyte bond is cleaved, the released paramagnetic capture beads may be attracted to a preferred location in the elution chamber, as just described. The readout process shown in Figure 27 may cover multiple regions in the elution channel, including a fresh elution buffer zone, an elution zone, and a bead bolus zone. These individual sections may be analyzed to identify and quantify single molecules from the fluorophore label that was transported to the elution zone via binding to the target analyte.

[0171] While the fluid circuits shown in Figures 10-22 include a variety of different structures for processing samples using the systems described herein, the methods described herein may be practiced using other fluid circuits. For example, in some embodiments, a fluid circuit may be formed without any separation chambers or transfer structures. For example, a fluid circuit may include a more direct route from a sample port to a mixing chamber. Furthermore, in some embodiments, a fluid circuit may be configured without a separate waste chamber. For example, the flow path of fluid delivered to the cartridge may be from a fluid inlet port to a sample port, with the sample chamber also acting as a waste chamber.

[0172] For example, FIG. 28 shows another embodiment of fluid circuit 551 according to the present disclosure, in which the initial centrifugation step may be omitted. In contrast to circuit 151, described above, separation chambers 161 and 162 have been eliminated. In an embodiment using circuit 551, a sample is injected into sample chamber 558 through sample port 553. When the cartridge is spun as described above, the sample moves into mixing chamber 575 due to centrifugal force, without the need for a priming step. In this embodiment, the sample volume input may be controlled such that the overflow channel and overflow chamber may be omitted. The same processes of mixing, washing, elution, and reading as described above for circuit 151 may be performed using circuit 551. Buffer is input into inlet port 554 in a manner similar to that described above for circuit 151. As the wash buffer is injected, the sample is forced out of mixing chamber 575 and into sample chamber 558, which also serves as a waste chamber. In a later step, an elution buffer is also input, forcing the wash buffer into chambers 575 and 558. In some embodiments, different portions of channel 582 may be used for washing, elution, and reading. As described above with respect to circuit 151, analytes generally migrate toward inlet port 554, and buffer and waste migrate toward sample port 553. In this manner, analytes are generally exposed to clean, uncontaminated portions of the cartridge and clean buffer. In embodiments of circuit 551, reading occurs radially along channel 582. However, in other embodiments, such a channel may have an elbow region where the channel changes direction, eventually bending circumferentially just below the mixing chamber and eventually returning toward the center of rotation.

[0173] Thus, circuit 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 this disclosure.

[0174] FIG. 29 illustrates another embodiment of a fluidic circuit 751 according to the present disclosure, in which the initial centrifugation step may be omitted. Similar to fluidic circuit 351 shown in FIG. 23, fluidic circuit 751 allows for liquid reagent 857 to be used. Fluidic circuit 751 includes port 752 and channel 755, allowing liquid reagent 857 to be added to the sample and input into circuit 751. Channel 755 keeps liquid reagent 857 from mixing with the sample until the disk containing fluidic circuit 751 begins to spin. In various embodiments, the chamber size may be adjusted to accommodate different liquid reagent and sample ratios. As described above, once the disk is spun, centrifugal force moves the sample and liquid reagent 857 radially outward into mixing chamber 775. The same processes of mixing, washing, elution, and reading may be performed using circuit 751, as described above with respect to circuit 151.

[0175] FIG. 30 illustrates another embodiment of a fluidic circuit 951 according to the present disclosure, in which centrifugal sample separation is omitted and liquid reagents are introduced into the fluidic circuit 951 through a reagent port 952. Both the reagent port 952 and the sample port 953 are provided within the sample chamber 938, but they are separated from each other by a divider 956. The divider 956 keeps the liquid reagents from mixing with the sample until the cartridge begins to rotate, forming the fluidic circuit 951. In various embodiments, the position of the divider 956 may be adjusted to accommodate different liquid reagent and sample ratios. Alternatively, in some embodiments, the fluidic circuit may include reagent and sample ports without a divider. Additionally, in some embodiments, the sample chamber may include a single port adapted to receive both the sample and the reagent.

[0176] As described above, once the cartridge is spun, centrifugal force moves the sample and liquid reagents radially outward, driving these components into mixing chamber 975. The same processes of mixing, washing, elution, and reading as described above with respect to circuit 151 may be performed using circuit 951.

[0177] Similar to fluid circuit 151, fluid circuit 951 includes a series of channels leading to an inlet port along an isolated path from mixing chamber 975. From mixing chamber 975, post-mixing chamber channel 974 extends radially inward such that fluid is driven into mixing chamber 975 as the cartridge is rotated. The path continues along post-mixing chamber channel 974 to elbow 980, where it reverses direction along radial channel 981 beyond 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 port 954. Each of these channels may be used for washing and / or elution as target analytes are transferred from mixing chamber 975 toward inlet port 954 through a new volume of wash buffer and / or elution buffer. The sample chamber 938 of circuit 951 also includes a vent 955 that allows the sample chamber 938 to also function as a waste chamber for used wash buffer as fluid continues to enter the fluid circuit 951 through the inlet port 954. The vent 955 allows fluid to fill the sample chamber 938 while preventing fluid from flowing into the manifold through the reagent port 952 or the sample port 953.

[0178] Each of the channels between the mixing chamber 975 and the inlet port 954 may also be used as a read chamber. The working channel 982 is particularly well suited to serve as a read chamber on a circular cartridge. The working channel 982 extends circumferentially around the cartridge at a constant radius from the center of rotation. As a result, directing electromagnetic radiation along a portion of the working channel 982 may be enabled through simple rotation of the cartridge while the source is activated. Alternatively, in some embodiments, the cartridge may be a linear cartridge configured to be moved along the cartridge axis during processing and reading. In such embodiments, the working channel may be straight rather than curved circumferentially.

[0179] For example, Figure 31 shows another embodiment of a fluidic circuit 1151 adapted for a linear cartridge. Similar to fluidic circuit 951, fluidic circuit 1151 includes a sample chamber 1138, along with a reagent port 1152 and a sample port 1153, which are separated by a divider 1156. From the sample chamber 1138, fluidic circuit 1151 extends along an isolated path through a mixing chamber 1175 toward an inlet port 1154. Sample chamber 1138 also includes a vent 1155, which allows fluid pumped into fluidic circuit 1151 through inlet port 1154 to be collected in sample chamber 1138 after flowing through the circuit.

[0180] From mixing chamber 1175, fluid circuit 1151 extends around elbow 1180 to working channel 1182, which may again function as a wash, elution, and / or read chamber. In contrast to working channel 982 of fluid circuit 951, working channel 1182 has a linear configuration. Thus, sliding lateral movement of the cartridge, which forms fluid circuit 1151, can be used to introduce electromagnetic radiation along the entire length of working channel 1182.

[0181] In another aspect, the present disclosure provides a cartridge including multiple fluid circuits according to the present disclosure. For example, FIG. 10 shows a cartridge 150 including three fluid circuits 151 having the configuration shown and discussed with respect to FIGS. 11-22. The cartridge 150 allows three samples to be processed on a single cartridge, such that three samples can be loaded in a single operation. FIG. 32 shows another embodiment of a cartridge 1150 including multiple fluid circuits. Specifically, the cartridge 1150 shown in FIG. 32 is formed as a linear cartridge and includes six fluid circuits having the configuration of the fluid circuit 1151 shown in FIG.

[0182] Each of the fluidic circuits 1151 of the cartridge 1150 has an identical configuration and is arranged in uniformly spaced rows. To move the sample from the sample chamber to the mixing chamber, the cartridge is rotated, similar to the other cartridges described above, so that centrifugal force drives the fluid toward the mixing chamber. However, rather than rotating the cartridge in the plane of the fluidic circuits, the cartridge is rotated about an axis 1160 that extends parallel to the plane of the circuits. Because each of the fluidic circuits 1151 is spaced the same distance from the axis 1160, the centrifugal force on each sample is the same. Furthermore, because the fluidic circuits 1151 are aligned along rows, the samples may be processed simultaneously. To mix the sample and reagents within the fluidic circuits 1151 of the cartridge 1150, the cartridge 1150 is translated back and forth along the axis 1160. 33, a carrier 1144 supporting six uniformly spaced magnets 1145 may be moved relative to the cartridge 1150 to simultaneously transport all six magnets 1145 in the same manner along the fluid circuit path. In this manner, the paramagnetic beads in each fluid circuit may be simultaneously drawn along the fluid circuit path. A second carrier may be used on the opposite side of the cartridge for washing or elution, as described above.

[0183] Embodiments of a linear cartridge may include more or fewer rows of fluid circuits than the six circuits shown in cartridge 1150. Additionally, in some embodiments, a linear cartridge may include multiple rows. For example, diagram 1350 includes a first row 1352 of fluid circuits on one side of the cartridge and a second row 1353 of fluid circuits on the opposite side of the cartridge. All of the fluid circuits have the same configuration and are formed in the same plane. To equalize the centrifugal forces on the samples in each fluid circuit, the cartridge may be rotated about central axis 1360 during an initial phase that moves fluid into the mixing chamber.

[0184] While the preceding examples of cartridges with multiple fluid circuits show complex circuits in a rotary cartridge and simple fluid circuits in a linear cartridge, both cartridge types may be used with various configurations of fluid circuits. For example, fluid circuit 1151 in FIG. 30 has a wedge shape so that several instances of fluid circuit 1151 can fit on a single rotary cartridge. Similarly, FIG. 35 shows a linear cartridge 1550 including four similar fluid circuits 1551 configured to separate components of a sample. Fluid circuit 1551 includes a separation area and an overflow channel, similar to fluid circuit 151 in FIGS. 11-22. A siphon, activated by capillary action, draws fluid from the separation area into a mixing chamber, where the sample and reagents are mixed before washing, elution, and reading, as described above.

[0185] In some embodiments, a fluid processing cartridge with multiple fluid circuits includes a group of fluid circuits, each having a working channel that extends along a common linear or circular path. Such a configuration allows a single read operation to be used to identify target analytes in the working channels of each fluid circuit. For example, the working channels of multiple fluid circuits may extend along a single circular path in a rotating cartridge, such as cartridge 150, and the cartridge may simply be rotated to take a read along each working channel. Similarly, the working channels of multiple fluid circuits may extend along a straight line across a linear cartridge, such as cartridge 1150, and the cartridge may be moved laterally to take a read along each working channel.

[0186] Each of the illustrated fluidic circuits is configured such that binding of the target analyte, label, and paramagnetic beads is facilitated together in a mixing chamber in a single step, although in other embodiments, this binding may occur in more than one step. For example, in some embodiments, the fluidic circuit may include a pre-mixing chamber, where the target analyte is bound to the paramagnetic beads. The paramagnetic beads may then be moved to the mixing chamber, where the label is bound to the target analyte.

[0187] 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 operations. In some embodiments, the analyzer's second electromagnetic radiation source 128 and second detector 129 may be used to determine the presence of a second target analyte in a sample. In other embodiments, the second electromagnetic radiation source 128 and second detector 129 may be used to measure the concentration of a control analyte in the cartridge 150. For example, the cartridge 150 may contain a precise, known amount of the control analyte. Thus, the measured concentration of the control analyte may be used as a comparison standard for the target analyte. This measured concentration can then be used to adjust the detected concentration of the target analyte.

[0188] 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 adjust the detected concentration of the target analyte. For example, the controller 140 may determine that the analyzer 100 is detecting only 95% of the target analyte in the sample and adjust the calculated concentration accordingly.

[0189] In some embodiments, the electromagnetic radiation from first electromagnetic radiation source 121 and second electromagnetic radiation source 128 is directed at the cartridge using the same objective lens. Indeed, in some embodiments, the electromagnetic radiation from the two sources is directed into the same interrogation 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.

[0190] The present disclosure provides systems and methods for highly sensitive detection and quantification of one or more target analytes, such as markers for a biological state. Singleplex and multiplex assays

[0191] In one aspect, the present disclosure provides systems and methods that can perform a "singleplex" assay of a sample to detect and analyze a single type of target analyte within the sample. In another aspect, the present disclosure provides systems and methods that can perform a "multiplex" assay of a sample to detect and analyze multiple (e.g., two, three, or more) different types of target analytes within the sample. Using the multiplexing systems and methods described herein can provide more rapid detection and analysis of multiple target analytes using reduced sample and reagent volumes than would be required to perform a similar analysis of those target analytes via a singleplex assay. Furthermore, the multiplexing systems and methods described herein can allow analysis of a sample containing a target analyte to be compared to a control assay of known concentration.

[0192] To detect and analyze multiple different types of target analytes in a sample, a multiplexed analyzer system can distinguish one type of target analyte from another. This can be achieved, in part, by labeling different target analytes with different labels that have different excitation and / or emission wavelength bands. In some implementations, the different labels have excitation and / or emission wavelength bands with relatively little or no overlap. In other implementations, there may be some overlap between the excitation and / or emission wavelength bands of the labels. Multiplexing can also be achieved by implementing more than one fluidic circuit on the same cartridge, each fluidic circuit being spatially distinct, and carrying reagents for different target analytes. With different fluidic circuits, it is not necessary for different target labels to have different excitation and emission wavelengths. Additional circuits may collect samples from the same sample chamber or from different sample chambers. Electromagnetic radiation power and bin size

[0193] In an optical system, the electromagnetic radiation source 121 may be configured so that the wavelength of the electromagnetic radiation is sufficient to excite the fluorescent label attached to the target analyte. In some embodiments, the electromagnetic radiation source 121 is a laser emitting 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 fluorescent moieties such as those used in the methods and compositions of the present disclosure can be used without departing from the scope of the present disclosure. Power settings for the laser are generally between 1 mW and 100 mW. However, those skilled in the art will understand that the laser power can be set at any setting to achieve the optimal signal-to-noise ratio of the measurement. To do so, the laser power should be set to achieve as many excitation-emission cycles as possible during the label's residence time in the interrogation space. The detector bin time should also be set accordingly. A bin time longer than the time required to photobleach the label and / or longer than the residence time of the label within the interrogation space will simply allow for the collection of excessive noise. Laser power settings that are too low or too high, or bin time settings that are too long, will not result in the highest possible signal-to-noise ratio.

[0194] As the interrogation space within the analyzer 100 passes over the labeled target analytes, photons emitted by the fluorescent particles are registered by the detector 122 with a time delay indicating the time for the interrogation space to pass over the labeled particles. Photon intensity is recorded by the detector 122, and the sampling time is divided into bins, which are uniform, arbitrary time segments with freely selectable time channel widths. The number of signals contained in each bin is evaluated. One or more of several statistical analysis methods are used to determine when a label or particle is present, or when a fraction of a bin contains an artifact. Single bins or fractions of bins containing labels are counted, while fractions of bins containing artifacts are discarded. The number of labels counted indicates the number of target analytes present in the sample. Interrogation Volume

[0195] The interrogation volume can be thought of as the effective volume of a sample where the target analyte, when present, can be detected. While various methods exist for calculating the interrogation volume of a sample, the simplest way to determine the effective volume (V) of the interrogation volume is to calculate the effective cross-section of the detection volume. Because the detection volume is typically swept through the sample by translating the detection volume through a stationary sample, the volume is typically a result of the cross-sectional area of ​​the detection volume being swept through a distance during the measurement time. As discussed above, the lateral extent of the cross-sectional area of ​​the interrogation volume (perpendicular to the direction of laser motion relative to the sample and perpendicular to the direction of laser light propagation) is limited by the numerical aperture at which the laser source is imaged in sample space. The longitudinal size of the interrogation volume (along the direction of laser propagation) is determined by the size of the confocal aperture selected. If the sample concentration (C) is known and the number of molecules detected over a period of time (N) is known, the sample volume consists of the number of molecules detected divided by the concentration of the sample, i.e., V=N / C (sample concentration has units of molecules per unit volume).

[0196] For example, in some embodiments of the systems described herein, all detected photons are counted and summed in 100-microsecond intervals (photon counting bins). If a molecule of interest is present in a 100-microsecond interval, the detected photon counts are typically significantly higher than the background. Therefore, the distance the detection volume travels relative to the sample is the appropriate distance to use to calculate the volume sampled within a single compartment, i.e., the interrogation volume. In this example, if a sample is analyzed over 60 seconds, effectively 600,000 compartments are scanned. When the effective volume is divided by the number of compartments, the resulting volume is essentially the volume of a single compartment, i.e., the interrogation volume. Mathematically, the volume of a single compartment, i.e., the interrogation volume (Vs), is equal to the number of molecules detected (N) divided by the concentration of the sample multiplied by the number of compartment bins (C·n, where n represents the number of compartment bins during which the number of molecules N was counted). For illustrative purposes only, consider that a known standard of 1 femtomolar concentration is run through 600,000 compartments and 20 molecules of the standard are detected. Therefore, the interrogation volume V is N / (C n), or 20 / (602.214 6E5), or 55.351 μm 3 Therefore, in this example, the interrogation space volume, which is the effective volume of one sample corresponding to one photon counting bin, is 55.351 μm 3 is. detector

[0197] In some embodiments, light emitted by the fluorescent label after exposure to electromagnetic radiation is detected. The emitted light can be, for example, ultraviolet, visible, or infrared. For example, the first detector 122 may capture the amplitude and duration of photon bursts from the fluorescent moiety and convert the amplitude and duration of the photon bursts into an electrical signal. Detection devices such as CCD cameras, video input module cameras, and streak cameras can be used to generate images with continuous signals. Other embodiments use devices such as bolometers, photodiodes, photodiode arrays, avalanche photodiodes, and photomultiplier tubes that generate sequential signals. Any combination of the aforementioned detectors can be used. Molecules for concentration analysis

[0198] The devices, kits, and methods of the present disclosure can be used for sensitive detection and determination of the concentration of several different types of target analytes, such as markers of biological states.

[0199] 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 are useful in detecting protein and small molecule target analytes in biological samples and determining the concentrations of such molecules in the samples.

[0200] The molecules detected by the present systems and methods may be free of or 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.

[0201] In some embodiments, the present disclosure provides compositions and methods for the sensitive detection of biological markers and for the use of such markers in determining diagnostic, prognostic, and / or therapeutic methods.

[0202] A marker can be, for example, any composition and / or molecule, or complex of compositions and / or molecules, associated with the biological state of an organism (e.g., a symptom, such as a disease or non-disease state). Markers can be, for example, small molecules, polypeptides, nucleic acids such as DNA and RNA, lipids such as phospholipids or micelles, cellular components such as mitochondria or chloroplasts, etc. Markers contemplated by the present disclosure can 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 symptom of interest, while in other embodiments, known polypeptides are identified as markers for a biological state or symptom of interest. It is conceivable that the systems of the present disclosure can be used to observe markers, e.g., polypeptides, with high potential use in determining the biological state of an organism but that are present only in low concentrations, such as those "leached" from diseased tissue. Other potentially useful markers or polypeptides can be those associated with disease, e.g., those produced in the tumor host environment. Any suitable marker that provides information about a biological state can be used in the methods and compositions of the present disclosure. "Marker," as that term is used herein, encompasses any molecule that can be detected in a sample from an organism, the detection or quantification of which provides information about the biological state of the organism.

[0203] Biological states include, but are not limited to, phenotypic states, conditions affecting an organism, developmental states, age, health, pathology, disease detection, process, or staging, infectious diseases, toxicity, or response to chemical, environmental, or drug agents (such as drug response phenotyping, drug toxicity phenotyping, or drug efficacy phenotyping).

[0204] The term "organism" as used herein refers to any living thing consisting of at least one cell. An organism can be as simple as a single-celled organism or as complex as a mammal. The organism of the present disclosure is preferably a mammal. Such a mammal can be, for example, a human or an animal such as a primate (e.g., monkey, chimpanzee, etc.), a domesticated animal (e.g., dog, cat, horse, etc.), a livestock animal (e.g., goat, sheep, pig, cow, etc.), or a laboratory animal (e.g., mouse, rat, etc.). Preferably, the organism is a human. sign

[0205] In some embodiments, the present disclosure provides methods and compositions that include labels for highly sensitive detection and quantitation of molecules, eg, markers.

[0206] Many strategies can be used to label target analytes and enable their detection or discrimination in a mixture of particles. Labels can be attached by any known means, including methods that utilize non-specific or specific interactions between the label and the target analyte. Labels can provide a detectable signal or affect the mobility of particles in an electric field. Labeling can be accomplished directly or through a binding partner.

[0207] Labels can include, but are not limited to, one or more of the following: fluorophores, chromatophores, 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.

[0208] In some embodiments, the label consists of a binding partner to the molecule of interest, where the binding partner is attached to a fluorescent moiety. The compositions and methods of the present disclosure can use highly fluorescent moieties. Suitable moieties for the compositions and methods of the present disclosure are described in more detail below. The fluorescent molecule may be attached to the binding partner by any known means, such as direct conjugation, or indirectly (e.g., biotin / streptavidin).

[0209] The fluorescent moiety 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 Brilliant Violet 421. TM , Brilliant Violet 510 TM , Brilliant Violet 570 TM , Brilliant Violet 605, etc. ALEXA FLUOR® 700 Brilliant Violet TM Molecules (BD Biosciences), and ATTO TM ATTO such as 532 TM dye (ATTO TECH GmbH). In some embodiments, the dye molecule is Alexa Fluor® 647 dye molecule. Bonding Partners

[0210] In some embodiments, the binding partner comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0211] The antibody may be specific for any suitable marker, hi some embodiments, the antibody is specific for a marker selected from the group consisting of a cytokine, a growth factor, a tumor marker, a marker of inflammation, an endocrine marker, an autoimmune marker, a thyroid marker, a cardiovascular marker, a marker for diabetes, a marker for infectious diseases, a neurological marker, a respiratory marker, a gastrointestinal marker, a musculoskeletal marker, a skin disease, and a metabolic marker.

[0212] Any suitable binding partner with the required specificity for the form of the molecule to be detected, e.g., marker, can be used. If the molecule, e.g., marker, has several different forms, various specificities of the binding partner are possible. Suitable binding partners are known in the art and include antibodies, aptamers, lectins, and receptors. A useful and versatile type of binding partner is an antibody.

[0213] A capture binding partner and a detection binding partner pair, for example, a capture and detection antibody pair, can be used in the embodiments of the present disclosure. Thus, in some embodiments, a heterogeneous assay protocol is typically used, in which two binding partners, for example, two antibodies, are used. One binding partner is usually a capture partner immobilized on a solid support, and the other binding partner is typically a detection binding partner to which a detectable label is attached. Antibody pairs can be designed and prepared by methods well known in the art. The compositions of the present disclosure include antibody pairs, in which one element of the antibody pair is a label as described herein, and the other element is a capture antibody.

[0214] In some embodiments, it is useful to use antibodies that cross-react with various species, either as capture antibodies, detection antibodies, or both. Such embodiments include, for example, measuring drug toxicity by determining the release of cardiac troponin into the blood as a marker of cardiac damage. Cross-reacting antibodies allow toxicity studies to be conducted in one species, e.g., a non-human species, and the results can be directly transferred to studies or clinical observations in another species, e.g., humans, using the same antibody or antibody pair in the assay reagents, thus reducing variability between assays. Thus, in some embodiments, one or more of the antibodies used as binding partners for a marker of a molecule of interest, e.g., a cardiac troponin such as cardiac troponin I, can be cross-reacting. In some embodiments, the antibody cross-reacts with markers, e.g., cardiac troponins, from at least two species selected from the group consisting of human, monkey, dog, and mouse. In some embodiments, the antibody cross-reacts with markers, e.g., cardiac troponins, from the entire group consisting of human, monkey, dog, and mouse.

[0215] The above detailed description describes various features and functions of the disclosed systems, devices, and methods with reference to the accompanying figures. In the figures, like symbols typically identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, figures, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the subject matter presented herein. In general, it should be readily understood that aspects of the present disclosure, as described herein and illustrated in the figures, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0216] While various aspects and embodiments are 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 are not intended to be limiting, with the true scope being indicated by the following claims. Embodiment

[0217] Embodiment 1. An analyzer system for detecting the presence of a target analyte in a sample, the analyzer system comprising: A motor; a dock coupled to the motor so as to be rotated by actuation of the motor; a cartridge held within the dock, the cartridge including a fluidic circuit configured to receive a sample, isolate a target analyte of the sample, and collect an amount of a first label proportional to the amount of the target analyte in the sample, the fluidic circuit comprising: a sample port configured to receive a sample; a mixing chamber in fluid communication with the sample port, the mixing chamber configured to mix at least a portion of the sample to combine the target analytes and the first label; a fluid inlet port in fluid communication with the mixing chamber and configured to receive a wash buffer and an elution buffer; the fluid circuit including an isolated pathway extending from the fluid inlet port to the mixing chamber; a fluid delivery line configured to be coupled to the fluid inlet port to deliver fluid to the cartridge through the fluid inlet port and force the fluid along an isolated path toward the mixing chamber; a first magnet, the first magnet being movable relative to the cartridge and secured to a stage configured to move the paramagnetic beads within the cartridge; a first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within the detection chamber of the cartridge; a first detector configured to detect electromagnetic radiation emitted within the interrogation space by the label when the label is present within the interrogation space; a controller configured to identify the presence of a target analyte in the sample based on the electromagnetic radiation detected by the first detector; An analyzer system comprising:

[0218] Embodiment 2. The analyzer system of embodiment 1, further comprising a second magnet arranged to be positioned on an opposite side of the cartridge from the first magnet.

[0219] Embodiment 3. The analyzer system of embodiment 1 or embodiment 2, further comprising a pump configured to force wash buffer and elution buffer through the fluid delivery line and into the cartridge via the inlet port.

[0220] Embodiment 4. The analyzer system of any of embodiments 1-3, further comprising a distribution valve coupled to the fluid delivery line, the wash buffer port, the elution buffer port, and the pump.

[0221] Embodiment 5. The analyzer system of embodiment 4, further comprising a priming line disposed between the distribution valve and the pump, the distribution valve configured to sequentially connect the elution buffer port and the wash buffer port to the priming line, load the priming line, and then connect the priming line to the fluid delivery line.

[0222] Embodiment 6. An analyzer system according to any of embodiments 1-5, wherein the cartridge contains a lyophilized reagent in the mixing chamber.

[0223] Embodiment 7. An analyzer system according to any of embodiments 1-5, wherein the cartridge includes a liquid reagent port.

[0224] Embodiment 8. An analyzer system according to any one of embodiments 1-7, wherein the fluid circuit is one of multiple fluid circuits within a cartridge.

[0225] Embodiment 9. An analyzer system as described in embodiment 8, wherein each of the fluid circuits has an identical configuration and includes a working channel along an isolated path between the mixing chamber and the inlet port.

[0226] Embodiment 10. An analyzer system as described in embodiment 9, wherein the working channel extends along a common circular line.

[0227] Embodiment 11. An analyzer system as described in embodiment 10, wherein the motor and dock are arranged to rotate the cartridge about an axis that is perpendicular to the plane of the fluid circuit.

[0228] Embodiment 12. An analyzer system as described in embodiment 9, wherein the working channels extend along a common straight line.

[0229] Embodiment 13. An analyzer system as described in embodiment 12, wherein the motor and dock are arranged to rotate the cartridge about an axis that is parallel to the plane of the fluid circuit.

[0230] Embodiment 14. A method comprising: receiving the cartridge within the analyzer system such that the cartridge is coupled to a motor of the analyzer system; rotating the cartridge using a motor to move a volume of sample toward a mixing chamber within the cartridge; mixing said 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 the primed fluid delivery line into the cartridge through the fluid inlet ports, the series of fluids including a wash buffer and an elution buffer; forcing a series of fluids along isolated paths in a first direction from the fluid inlet port to a mixing chamber; using a magnet to move the paramagnetic capture beads out of the mixing chamber along an isolated path in a second direction toward the fluid inlet port; A method comprising:

[0231] Embodiment 15. directing electromagnetic radiation from an electromagnetic radiation source to form an interrogation space within the cartridge; receiving, at a detector, electromagnetic radiation emitted within the interrogation space if the fluorophore is present within the interrogation space; using the controller to identify the presence of a target analyte in the sample based on the electromagnetic radiation detected by the detector; 15. The method of embodiment 14, further comprising:

[0232] Embodiment 16. The method of embodiment 15, wherein the label is a fluorophore label.

[0233] Embodiment 17. The method of any of embodiments 14-16, further comprising the step of sequentially loading the primed fluid delivery line with fluids by coupling the fluid delivery line to the elution buffer port and the wash buffer port using a distribution valve.

[0234] Embodiment 18. The method of any of embodiments 14-17, wherein the cartridge is rotated about an axis that is perpendicular to the plane of the fluid circuit.

[0235] Embodiment 19. The method of embodiment 18, wherein the step of moving the cartridge and mixing said volume of said sample further comprises the step of rotating the cartridge.

[0236] Embodiment 20. The method of embodiment 18 or 19, wherein the fluid circuit is one of multiple fluid circuits arranged around the center of the cartridge.

[0237] Embodiment 21. The method of embodiment 20, wherein each of the fluid circuits has an identical configuration and includes a working channel along an isolated path between the individual mixing chambers and the inlet port.

[0238] Embodiment 22. The method of embodiment 21, wherein the working channel extends along a common circular line disposed at a fixed radius from the center of the cartridge.

[0239] Embodiment 23. The method of any of embodiments 14-17, wherein the cartridge is rotated about an axis that is parallel to the plane of the fluid circuit.

[0240] Embodiment 24. The method of embodiment 23, wherein the step of moving the cartridge and mixing the amount of said sample comprises moving the cartridge in a direction parallel to the plane of the fluid circuit.

[0241] 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.

[0242] Embodiment 26. The method of embodiment 25, wherein each of the fluid circuits has an identical configuration and includes a working channel along an isolated path between the individual mixing chambers and the inlet port.

[0243] Embodiment 27. The method of embodiment 26, wherein the working channels extend along a common straight line.

Claims

1. 1. An analyzer system for detecting the presence of a target analyte in a sample, the analyzer system comprising: A motor; a dock coupled to the motor so as to be rotated by actuation of the motor; a cartridge held within the dock, the cartridge including a fluidic circuit configured to receive a sample, isolate a target analyte of the sample, and collect an amount of a first label proportional to the amount of the target analyte in the sample, the fluidic circuit comprising: a sample port configured to receive a sample; a mixing chamber in fluid communication with the sample port, the mixing chamber configured to mix at least a portion of the sample to combine the target analyte and the first label; a fluid inlet port 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 port to the mixing chamber; a fluid delivery line configured to be coupled to the fluid inlet port to deliver fluid to the cartridge through the fluid inlet port and force the fluid along the isolated path toward the mixing chamber; and a first magnet, the first magnet being movable relative to the cartridge and secured to a stage configured to move paramagnetic beads within the cartridge; a first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within a detection chamber of the cartridge; and a first detector configured to detect electromagnetic radiation emitted within the interrogation space by a label when the label is present within the interrogation space; a controller configured to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector; An analyzer system comprising:

2. 10. The analyzer system of claim 1, further comprising a distribution valve coupled to the fluid delivery line, a wash buffer port, an elution buffer port, and the pump.

3. 3. The analyzer system of claim 2, further comprising a priming line disposed between the distribution valve and the pump, the distribution valve configured to sequentially connect the elution buffer port and the wash buffer port to the priming line, load the priming line, and then connect the priming line to the fluid delivery line.

4. The analyzer system of claim 1 , wherein the cartridge contains a lyophilized reagent in the mixing chamber.

5. The analyzer system of claim 1 , wherein the cartridge includes a liquid reagent port.

6. the fluid circuit is one of a plurality of fluid circuits within the cartridge; The analyzer system of claim 1 , wherein each of the fluid circuits has an identical configuration and includes a working channel along the isolated path between the mixing chamber and the inlet port.

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

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

9. 1. A method comprising: receiving the cartridge within the 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 within the cartridge; mixing the volume of the 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 fluid inlet ports, the series of fluids including a wash buffer and an elution buffer; forcing the series of fluids along isolated paths in a first direction from the fluid inlet port to the mixing chamber; using a magnet to move the paramagnetic capture beads out of the mixing chamber along the isolated path in a second direction toward the fluid inlet port; A method comprising:

10. directing electromagnetic radiation from an electromagnetic radiation source to form an interrogation space within said cartridge; receiving, at a detector, electromagnetic radiation emitted within the interrogation volume when a fluorophore is present within the interrogation volume; using a controller to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the detector; 10. The method of claim 9, further comprising:

11. The method of claim 10, wherein the label is a fluorophore label.

12. 10. 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 distribution valve.

13. 10. The method of claim 9, wherein the cartridge is rotated about an axis that is perpendicular to the plane of the fluid circuit, and moving the cartridge and mixing the volume of the sample comprises further rotating the cartridge.

14. 14. The method of claim 13, wherein the fluid circuit is one of a plurality of fluid circuits arranged around a center of the cartridge, each of the fluid circuits having an identical configuration and including a working channel along the isolated path between the individual mixing chambers and inlet ports, the working channel extending along a common circular line arranged at a fixed radius from the center of the cartridge.

15. 10. The method of claim 9, wherein the cartridge is rotated about an axis that is parallel to the plane of the fluid circuit, and moving the cartridge and mixing the volume of the sample comprises moving the cartridge in a direction parallel to the plane of the fluid circuit.

16. 16. The method of claim 15, wherein the fluid circuit is one of a plurality of fluid circuits arranged in a row across the cartridge, each of the fluid circuits having an identical configuration and including a working channel along the isolated path between the individual mixing chambers and inlet ports, the working channels extending along a common straight line.

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