Point of care concentration analyzer

Abstract

To provide automatic sample processing, measurement and analysis of a sample.SOLUTION: An analyzer system includes a cartridge configured to receive a sample. The cartridge has a plurality of chambers for isolating a target analyte of the sample and collecting a quantity of a first label that is proportional to a quantity of the target analyte in the sample. The system includes an analyzer with a first electromagnetic radiation source, a first detector and a controller. The first electromagnetic radiation source is configured to provide electromagnetic radiation to form an interrogation space within a detection chamber of the cartridge. The first detector is configured to detect electromagnetic radiation emitted in the interrogation space by the first label if the first label is present in the interrogation space. The controller is configured to identify the presence of the target analyte in the sample on the basis of electromagnetic radiation detected by the first detector.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 817,433, filed Mar. 12, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention generally relates to the automated sample processing, measurement, and analysis of samples for isolating, labeling, detecting, and determining the amount of a specific target analyte that may be present at very low concentrations.

Background Art

[0003] Advances in the study and understanding of the underlying causes and progression of diseases have shown that the detection of early disorders associated with the detection of infectious agents or appropriate treatment substantially improves clinical outcomes. Many symptoms that formerly required the use of expensive diagnostic imaging, which requires specialists trained 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 proteins, nucleic acids, or other molecules that are up - or down - regulated and are highly specific for a medical condition or infection.

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

[0005] In some medical conditions, the concentration of a biomarker or analyte of interest is relatively high, and simple, low-cost lateral flow devices can 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 rapidly with relatively little or no training at the point of care. However, lateral flow assays also tend to suffer from poor accuracy in performing minimal-quality quantitative measurements, even when an objective reader system is used to measure the strip. Also, depending on the stage of the disease or infection, the concentration of the target analyte is often too low to be detected using lateral flow within blood, urine, saliva, or other sample types.

[0006] In these cases, sample processing and readout are more complex. This often requires precise measurements to assess concentration, high efficiency to avoid loss of the target analyte, and centrifugation as a primary step in the purification process. In addition to 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 conjugated molecules on their surfaces, complementary molecules, or both. Once binding occurs, additional process steps must then be taken to wash, further isolate, and suspend the target analyte in a fresh buffer solution or place it on a clean surface prior to measurement. To perform these processing steps, multiple devices including centrifuges, mixers, incubators, precision pipettes, and thermal cyclers are used, and the sample is often transferred and measured between processing steps using multiple disposable tips, tubes, plates, and other sample containers. Once processing is complete, extremely sensitive and precise instruments are used to measure the processed sample and determine the presence and / or abundance of the target analyte.

[0007] The analysis of low-concentration biomarkers can take several forms, but generally, the processing and measurement of samples 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 the target analyte and bind it to the substrate 4. Introduction of buffers and steps to wash away unbound labels and other contaminants 5. Sterile containers for precise measurement during processing and sample storage 6. An efficient and precise means of sample transfer 7. A means of measurement that provides high sensitivity and accuracy for measuring the presence and abundance of the target analyte

[0008] Currently, the processing and measurement of low-concentration biomarkers must be performed by trained staff or by using highly specialized equipment in a centralized location. As a result, the time required from sample acquisition to result is long, the instrumentation cost is high, and the measurement cannot be performed at the point of care.

[0009] Therefore, the inventors recognize that an improved technique capable of addressing the key characteristics listed above for low-concentration biomarker processing and measurement is desirable. This technique should be suitable for a point-of-care environment with minimal consumables, precise measurement, fast turnaround time, and sensitivity to overcome the limitations of the prior art.

[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 highly sensitive detection. This disclosure provides further development within the art. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0011] Disclosed herein are an analyzer system, a cartridge, and a method for detecting a target analyte in a sample. Advantageously, embodiments of the analyzer system use a compact cartridge for processing and analyzing a sample that enables the analyzer to be of a reduced size so as to be provided at the point of care.

[0012] Accordingly, in a first aspect, the present disclosure is a cartridge configured to receive a sample, the cartridge including a plurality of chambers for isolating a target analyte of the sample and collecting an amount of a first label proportional to the amount of the target analyte in the sample, a first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within a detection chamber of the cartridge, a first detector configured to detect electromagnetic radiation emitted within the interrogation space by the first label when the first 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, and providing an analyzer system comprising the same.

[0013] This aspect as well as other aspects, advantages, and alternatives will become apparent to those of ordinary skill in the art by reading the following detailed description of the invention. The present invention provides, for example, the following. (Item 1) An analyzer system for measuring the concentration of a target analyte in a sample, the analyzer system comprising a motor, a dock coupled to the motor and configured to be rotated by the operation of the motor, A cartridge, the cartridge being held within the dock, including a fluid system, the fluid system 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 fluid system an inlet chamber, and a mixing chamber, the mixing chamber being downstream of the inlet chamber and configured to mix at least a portion of the sample to bind the target analyte with the first label, a washing chamber, the washing chamber being downstream of the mixing chamber and connected to the mixing chamber by a channel, the washing chamber being radially offset from the mixing chamber so as to prevent flow of the sample into the washing chamber during the mixing process performed in the mixing chamber, a cartridge including a first electromagnetic radiation source, the first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within a detection chamber of the cartridge, a first detector, the first detector configured to detect electromagnetic radiation emitted within the interrogation space by the first label when the first label is present within the interrogation space, a controller, the 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. (Item 2) The channel extending from the mixing chamber to the washing chamber includes a capillary break, the capillary break having a cross-sectional area that expands in a direction leading away from the mixing chamber, the analyzer system according to item 1. (Item 3) The fluid system of the cartridge further includes a separation area disposed between the inlet chamber and the mixing chamber, the separation area including a radially inner separation chamber and a radially outer separation chamber connected by a reduced diameter portion, the analyzer system according to item 1. (Item 4) The analyzer system according to item 3, wherein a siphon extends from the radially inner separation chamber to the mixing chamber. (Item 5) A method of mixing liquids within a cartridge in the form of a flat disk, the method comprising: introducing a liquid into the mixing chamber of the cartridge through a channel extending radially inward from the mixing chamber, the mixing chamber containing a mixing ball therein; rotating the cartridge in a first circumferential direction so as to radially outwardly press the liquid and retain the liquid within the mixing chamber; intermittently accelerating and decelerating the rotation of the cartridge so as to recursively move the mixing ball back and forth through the mixing chamber comprising a method. (Item 6) The mixing chamber is provided with a lyophilized reagent prior to introduction of the liquid, mixing due to movement of the mixing ball through the mixing chamber releases gas from the lyophilized reagent into the liquid, The method according to item 5. (Item 7) The method according to item 6, wherein the released gas moves radially inwardly out of the mixing chamber. (Item 8) The method according to item 5, wherein the mixing ball is non-magnetic. (Item 9) A method of detecting the presence of a target analyte in a sample, the method comprising: Introducing the sample into a cartridge, the cartridge including a fluid system for isolating a target analyte of the sample and collecting an amount of a first label proportional to the amount of the target analyte in the sample, the fluid system comprising: A fluid line including a fluid inlet port configured to receive fluid, a first chamber, and a fluid outlet port; A detection chamber; A connection passage between the first chamber and the detection chamber; And; Transferring the target analyte to the first chamber; Pressurizing fluid into the first chamber along the fluid line while maintaining a mass of air in the connection passage; Transferring the target analyte to the detection chamber; Directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the detection chamber of the cartridge; Receiving, in a first detector, electromagnetic radiation emitted within the interrogation space by the first label if the first label is present within the interrogation space; Using a controller to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector; A method comprising. (Item 10) The method according to item 9, further comprising transferring the target analyte to the detection chamber, the target analyte being conveyed into the detection chamber by paramagnetic beads transported using a magnet. (Item 11) The detection chamber is disposed within an elution line including an elution inlet port and an elution outlet port; The method further comprises pressurizing an elution fluid into the elution line to release the target analyte from the paramagnetic beads; The method according to item 10. (Item 12) The method according to item 9, wherein the fluid is pumped along the fluid line by feeding the fluid into the fluid line at the fluid inlet port and withdrawing the fluid from the fluid line at the fluid outlet port. (Item 13) A method for detecting the presence of a target analyte in a sample, the method comprising: introducing the sample into a cartridge, the cartridge including a fluid system for isolating the target analyte of the sample and collecting an amount of a first label proportional to the amount of the target analyte in the sample, the fluid system comprising: a first chamber; a second chamber; a channel extending from the first chamber to the second chamber; and; binding the target analyte to a substrate consisting of paramagnetic beads; positioning a first magnet near a first surface of the cartridge and adjacent to the first chamber; facilitating relative movement of the first magnet and the cartridge so as to draw the paramagnetic beads out of the suspension and into a bolus, the relative movement being facilitated by at least one of moving the first magnet across the first surface or rotating the cartridge; directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the cartridge; receiving, in a first detector, electromagnetic radiation emitted within the interrogation space by the first label if the first label is present within the interrogation space; using a controller to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector; and. (Item 14) The method according to item 13, further comprising promoting relative movement of the first magnet and the cartridge so as to transfer the paramagnetic beads and the target analyte from the first chamber to the second chamber. (Item 15) The method according to item 14, further comprising performing a washing operation in the second chamber so as to isolate the target analyte from other components of the sample. (Item 16) The washing operation moving the first magnet away from the first surface of the cartridge so that the paramagnetic beads are dispersed in the second chamber; moving the second magnet towards the second surface of the cartridge so that the paramagnetic beads gather near the second magnet; moving the second magnet away from the second surface of the cartridge so that the paramagnetic beads are dispersed in the second chamber; moving the first magnet towards the first surface of the cartridge so that the paramagnetic beads gather near the first magnet and including, the method according to item 15. (Item 17) A method for detecting the presence of a target analyte in a sample, the method comprising: introducing the sample into a cartridge, the cartridge including a fluid system for isolating the target analyte of the sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluid system comprising: an inlet chamber; a separation area connected to the inlet chamber, the separation area including an inner separation chamber and an outer separation chamber; a detection chamber downstream of the separation area; and comprising; transferring the blood sample from the inlet chamber to the separation area; Rotating the cartridge using a centrifuge to move the red blood cells of the blood sample towards the outer separation chamber and to move the plasma towards the inner separation chamber; Capturing an image of the blood sample within the separation area using a camera; Analyzing the image of the blood sample within the separation area using a controller and determining the position of the red blood cells within the separation area; Transferring plasma from the inner separation chamber to the mixing chamber; Isolating the target analyte from the plasma; Transferring the target analyte to the detection chamber; Directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the detection chamber of the cartridge; Receiving, within a first detector, electromagnetic radiation emitted within the interrogation space by the first label if the first label is present within the interrogation space; Identifying the presence of the target analyte within the sample based on the electromagnetic radiation detected by the first detector using a controller A method comprising. (Item 18) The method of item 17, further comprising rotating the cartridge further using the centrifuge to move the red blood cells further towards the outer separation chamber in response to the determined position of the red blood cells. (Item 19) The method of item 17, further comprising analyzing an image of the blood sample using the controller and determining the transparency of the plasma within the inner separation chamber after rotating the cartridge using the centrifuge, wherein transferring a portion of the sample from the separation area to the mixing chamber is performed in response to the plasma transparency exceeding a predetermined value. (Item 20) Capturing an image of the plasma within the mixing chamber; Using the controller, analyzing an image of the plasma in the mixing chamber and calculating a volume of the plasma in the mixing chamber The method according to item 17, further comprising

Brief Description of the Drawings

[0014] The accompanying drawings are included to provide a further understanding of the methods and devices of the present disclosure, are incorporated herein, and constitute a part hereof. The drawings are not necessarily to scale, and the 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 operations of the present disclosure.

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[0037] The following embodiments for carrying out the invention present an overview of exemplary embodiments of the method and system according to the present invention. After this overview, further descriptions of various exemplary embodiments of the method, system, and apparatus follow in connection with the present invention. (Overview of Exemplary Embodiments) Separation and Measurement

[0038] The present invention is directed to a sample processing and analysis system for isolating target analytes and determining their concentrations. A cartridge in the form of a single disposable disc 150, as shown in FIGS. 9 - 19, may be used for all sample processing, measurement, and storage of the resulting processed samples during measurement. The processing involves high - speed rotation of the disc to sediment the concentrated elements contained in the original sample. During an initial centrifugation step, as shown in FIG. 11, the sample 152 is transferred from the sample chamber 158 to the separation chambers 161, 162 shown in FIG. 12. The cartridge 150 is then rotated at a higher speed, for example, 7,000 rpm, to separate the concentrated elements into the separation area 162. The resulting supernatant within the separation area 161 is then transferred to a mixing chamber 175 containing a reagent consisting of a binding partner. The volumes of the separation chamber 161 and the mixing chamber 175, as well as the method of transferring the supernatant, act to measure the amount of sample used in the process in order to maintain accuracy. The sedimentation process and transfer may be imaged by a process quality control camera and analyzed during the process to ensure proper separation and measurement. Reagents and binding partners

[0039] The binding partners can include several species, which can be in the form of the dried reagent or lyophilized pellets 180 shown in FIG. 13 and may be stored in the mixing chamber. The first species is a paramagnetic bead substrate functionalized with a molecule having a binding site specific for the target analyte. The second species of binding partner includes a fluorescent label conjugated to a molecule with a binding site specific for a distinct and clearly different moiety of the target analyte. Another component within the reagent may include a control analyte consisting of an engineered protein or other molecule that would not be found in a patient sample containing the target analyte. Related to the control analyte are another set of paramagnetic capture beads and labels that are specific for binding sites on the control analyte in the same manner that the first set of paramagnetic capture beads and labels are specific for the target analyte. The control will undergo the same processes and measurements as the target analyte. However, the amount of the control, unlike the target analyte, is precisely known prior to the process. Thus, when the control is measured, since the concentration is known a priori, it can serve as a monitor of the effectiveness of the means for normalizing the imprecision of the sample processing and the process. Mixing and Incubation

[0040] Once the supernatant and reagents enter the mixing chamber, a mixing process will occur where the disk 150 will rotate at a variable rpm, albeit slowly. Specifically, the disk will rotate, accelerate, and decelerate at a controlled rate and execute a preferred motion profile during rotation. As shown in FIG. 13, mixing balls 176 with a higher density than samples 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, facilitate the dissolution and distribution of the dry reagents, and ensure 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. Further, the chamber may contain various features to further facilitate mixing and incubation and to 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 the binding of the target analyte to the paramagnetic capture beads and labeled molecules. To ensure accuracy, the mixing process can be carried out over a controlled amount of time.

[0041] Cartridge 150 also includes a wash chamber 181 that is coupled to the mixing chamber through channel 173. The supernatant can freely flow into the wash chamber through channel 173 in another manner, but the wash chamber may advantageously be positioned at a smaller radius from the center of rotation so that centrifugal force keeps the supernatant and reagents within the mixing chamber. Capillary breaks in the form of wide channel widths 178, 179 as shown in FIG. 14 may be present to further prevent capillary action from drawing the supernatant and mixed suspension from the mixing chamber into the wash chamber. Capillary breaks 178, 179 can also serve to store the supernatant, which will displace the air trapped within the lyophilized pellet during pellet manufacture.

[0042] Once the mixing and incubation process is complete, the cartridge 150 may be rotated to align with a manifold 108 (FIG. 5) having four ports 111, 112, 113, 114 and associated seals that will move into interfacial contact with the cartridge. The manifold may be coupled to a motor 110 for precise rotational movement, and the manifold may also be held on a suspension system to ensure contact and sealing of the ports 111, 112, 113, 114 on the cartridge. The manifold may also be on a movable arm 109 as shown in FIG. 1 to lower the manifold to the cartridge and then raise it therefrom. The manifold may also contain features to precisely align it with the cartridge and ensure various ports are aligned with sufficient positional accuracy. Once the manifold is aligned and in contact with the cartridge, the manifold motor ensures precise 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 rotation stage for the cartridge. The manifold is connected to a series of pumps and valves to allow introduction of buffer agents from an external reservoir through a precision syringe pump into the cartridge. The buffer agents preferably include a wash buffer, an elution buffer, and deionized water for cleaning and storage. Similarly, the ports on this cartridge may include a wash buffer inlet port 154, a wash buffer draw port 156, an elution buffer inlet port 155, and an elution buffer draw port 157 as shown in FIG. 9.

[0043] After the manifold 108 is aligned on the cartridge 150, one or more magnets are moved to a fixed position across the mixing chamber. The magnets are positioned above and below the cartridge on the Z stage, with an axis of movement perpendicular to the flat surface of the cartridge, as shown in FIG. 20. This allows the magnet 130 to move closer to the cartridge 150, increasing its effective attraction on the paramagnetic capture beads, while the other magnet 131 moves away from the cartridge 150, reducing its influence. The magnet Z stage 132 is also coupled to the radial stage 133. The radial stage allows the magnet to move closer to or away from the axis of rotation of the cartridge. As will be discussed later, the various channels and chambers on the cartridge are arranged radially or circumferentially. The various Z and radial stages, in combination with the manifold motor, allow the magnets to be placed at any desired position relative to the chambers and channels contained within the cartridge 150.

[0044] After the magnets are introduced, the magnet Z and radial stages perform a predetermined series of movements and are controlled with partial cartridge rotation to draw out of suspension all of the paramagnetic capture beads that are currently bound to the target analyte and the analyte of interest, as shown in FIG. 15. The magnet 130 on the bottom side surface of the cartridge is positioned close to the surface of the cartridge at a preferred location to draw the beads into a tight bolus. The bead bolus may be imaged by a process quality control camera and analyzed to ensure that the beads are being properly drawn out of suspension. Introduction and washing of the wash buffer

[0045] At this point, the wash valve connects the wash pump to the wash inlet port 154 on the cartridge, and the draw pump is connected to the wash draw port 156. The other ports remain closed. The wash pump and the draw pump are controlled to empty and draw, respectively, at a defined volumetric flow rate. This fills the wash buffer into the wash chamber and the channels leading to the mixing chamber as shown in FIG. 16. The sequence continues until the supernatant is pushed out of the mixing chamber and pushed back into the separation chamber. This serves to wash away most of the unbound label and contaminants from the bead borosil held in place by the magnet. Once the wash buffer filling sequence is complete, an image of the wash chamber may be collected from the process quality control camera and processed to ensure complete and proper filling of the wash chamber, channels, and mixing chamber with the wash buffer. Magnetic transfer for washing

[0046] At this point, the magnet stage and the manifold motor perform a series of movements to radially pull out the paramagnetic bead borosil from outside the mixing incubation chamber, along the channel between the wash chamber and the mixing chamber, and into the wash chamber. At various points in the process, images are captured and the process is processed in a manner that ensures that the bead borosil is of the appropriate size and that all of the bead borosil has been drawn into the wash chamber. Wash sequence

[0047] When the bead bolus is transferred to the wash chamber, the manifold motor, magnetic Z stage, and radial stage perform a series of movements to disperse the beads and are controlled to re-aggregate the bead bolus using alternating magnets on different sides of the wash chamber along the length of the wash chamber. This process is performed to remove any remaining unbound label or other contaminants that may be trapped in the bead bolus. When the wash sequence is complete, the paramagnetic capture beads are drawn back into a tight bolus. A clean wash buffer is then pumped into the wash chamber while the contaminated buffer is pushed through the mixing chamber and into the wash waste chamber 166 located on this cartridge. The wash waste chamber 166 is sized such that contaminated wash, original sample, and supernatant never completely fill the wash waste chamber and exit through a wash draw port located at the distal end of the wash waste chamber. This ensures that the manifold never comes into contact with any sample or any buffer that may have been mixed with a sample. Air Dam Removal and Elution Buffer Filling

[0048] The washing chamber 181 is connected to the elution chamber 184 at the opposite end of the mixing chamber via a connection passage 187. The elution chamber 184 is also connected to an elution port 155 and an elution draw port 157. When the washing chamber is filled as discussed above, the sequence is executed in a manner that prevents the wash buffer from entering the channels and the elution chamber 184. This is important because the contaminated wash buffer may contain unbound labels and other elements that can be sources of noise during subsequent measurements. A sufficient gap between the washing chamber 181 and the elution chamber 184 may be confirmed via analysis of images taken by a process quality control camera between each wash filling sequence. After the final magnetic washing sequence and after the washing chamber is refilled with clean wash buffer, the sequence is performed to fill the gap (if desired) in the connection passage 187 between the washing chamber and the elution chamber 184 with wash buffer. The wash draw port valve is closed, the elution draw port is opened, and the draw pump is connected to the elution draw port 114. A pump sequence is executed using the wash and draw pumps at a predetermined volumetric flow rate to fill the channel between the washing chamber and the elution chamber. This process may be confirmed via image collection and analysis.

[0049] When complete, another sequence is executed where the elution pump fills the elution chamber with elution buffer. Image processing may be performed to ensure that the channels and the elution chamber are filled correctly. At this point, a pre-read of the pure elution buffer, prior to introduction of the sample into the elution chamber, may be performed in the same manner that the processed sample will be read so that it serves as a negative control for the assay. The elution buffer is designed to cleave the bond between the target analyte and its associated label as discussed below. It is important that this process is avoided when the sample bead bolus is outside the elution chamber. Magnetic transfer to the detection chamber

[0050] Once the elution chamber is filled with elution buffer, the magnet stage and the manifold motor are controlled in a predetermined sequence to draw the bead bolus from the wash chamber 181 through the connection passage 187 into the elution chamber 184. Preparation for measurement

[0051] Once the bead bolus enters the elution chamber, a process similar to the washing process is performed within the elution chamber. The washing step left the contaminants in a suspended and wash - away state, while the elution process is designed to leave the labels that were once bound to the target analyte (and control analyte) in a dissociated state suspended in the solution. The combined movement of the upper and lower magnets, combined with the precise rotational movement of the cartridge, repeatedly disperses and re - aggregates the bead bolus across the length of the channel. The elution buffer cleaves the non - covalent bond between the target analyte and the paramagnetic capture beads, as well as the bond between the target analyte and its label, resulting in a homogeneous solution of the eluted label within the elution chamber. The same cleavage occurs for the control analyte. The entire purification process briefly described herein is designed to produce a suspension containing only the target analyte isolated in a 1 - to - 1 relationship and the label that was once bound to that target analyte. The paramagnetic capture beads used to capture the target analyte are a source of noise for the reading process. After the elution sequence in which the target analyte binding is cleaved, the dissociated paramagnetic capture beads may be drawn via the magnet to a preferred location within the elution chamber and away from the location where the processed sample will be read. Measurement

[0052] In the measurement step, a confocal laser-based optical system is focused within the elution chamber, for example, at a point within the elution chamber remote from the walls, top surface, and bottom surface of the chamber. The measurement and detection of the analyte occur within the elution chamber 184, and thus, chamber 184 serves both as the elution chamber and the detection chamber, and both terms are used throughout this disclosure to refer to chamber 184. The cartridge itself may be made of a material with ultra-low autofluorescence, and the elution buffer, pump materials, valves, fluid lines, etc. may be selected so as not to eject or leach materials that can emit autofluorescence when transported into the elution chamber. A small interrogation space is scanned through the liquid within the elution chamber by rotating the cartridge at a predetermined rpm back and forth via a manifold motor. The interrogation space is defined by the lateral extent of the laser spot and the lateral extent of the cone angle of the light forming the laser spot. The interrogation space is further defined along the optical axis by the size of a confocal stop positioned conjugate to a field within the optical system. As will be understood by those skilled in the art, the confocal architecture is used to remove light from positions remote from the focal plane. The farther away from the focal plane and the smaller the confocal stop, the more light originating from remote positions is attenuated. In imaging applications, this reduction of out-of-focus light reduces noise and provides sharp image slices. Light originating from locations remote from the focal plane (or image slice) does not represent structures within the image slice and is thus noise. The same process of noise reduction may be employed in the present invention, however, in this case, the confocal system is not used for imaging. As the laser spot scans through the fluid, it may encounter fluorescent labels from the target analyte. When it does, the laser excites fluorescence from the label, and individual photons are emitted from the label and directed by the optical system onto a detector where they are counted. On the way towards and beyond the focal plane, the laser light may encounter elements that emit autofluorescence, including glass and bonding materials comprising the optical system, the window on the cartridge, the backside of the elution chamber, the elution buffer, and any other material through which it can progress within the elution chamber.Any fluorescence from those components is noise because it does not originate from the target analyte label. The confocal architecture attenuates those signals by preferentially enabling signals from the target analyte label in or near the focal plane. As a result, when the laser passes over the target label, the flow of photons received and counted by the detector increases compared to the background photon level. The processing algorithm detects and classifies the elevated photon counts as the molecules of interest. In this way, individual molecules from the target analyte can be counted to determine the concentration of the target analyte in the original sample.

[0053] The invention described herein enables substantial advantages over the prior art for precisely detecting and quantifying the number of target analytes in a sample where the concentration of the target analyte in the sample is low. Further, the methods and apparatus of the invention have characteristics suitable for deployment in a point-of-care setting. Other aspects of the invention disclosed herein are directed to methods of sample processing and analysis to isolate target analytes and determine their concentrations. These methods generally implement steps that are consistent with the sample processing and measurements described above. (Exemplary Embodiment)

[0054] Examples and systems are described herein. It is to 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 "exemplary" or "example" 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.

[0055] The exemplary embodiments described in this specification are not intended to be limiting. Generally, aspects of the disclosure as 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 not explicitly considered herein.

[0056] Unless otherwise indicated, the terms "first", "second", etc. are used herein merely as labels and are not intended to impose an ordinal, positional, or hierarchical requirement on the items they refer to. Further, for example, a reference to a "second" item does not require or exclude the presence of, for example, a "first" or lower numbered item and / or, for example, a "third" or higher numbered item.

[0057] References to "an embodiment", "embodiment", "an example", or "example" in this specification mean that one or more features, structures, or characteristics described in connection with the embodiment are included in at least one implementation. The phrase "an embodiment" or "an example" in various places in this specification may or may not refer to the same embodiment.

[0058] As used herein, a system, apparatus, device, structure, article, element, component, or hardware that is "configured to" perform a specified function is not merely one that has the potential to perform the specified function after further modification, but is one that can actually perform the specified function without any modification. 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" represents the existing characteristics of a system, apparatus, structure, article, element, component, or hardware that enable it to perform a specified function without further modification. For purposes of the present disclosure, a system, apparatus, structure, article, element, component, or hardware that is described as being "configured to" perform a particular function may additionally or alternatively be described as being "adapted to" and / or "operable to" perform that function.

[0059] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the concepts disclosed herein, which may be practiced without some or all of these details. In other instances, details of known devices and / or processes are omitted so as not to unnecessarily obscure the present disclosure. Although some concepts will be described in conjunction with specific embodiments, it should be understood that these embodiments are not intended to be limiting. Exemplary Analyzer System

[0060] In one aspect, the present disclosure provides an analyzer system shown in FIG. 1, including an analyzer 100 and a cartridge 150. The cartridge 150 is configured to receive a sample, isolate a target analyte of the sample, and includes a plurality of chambers for collecting a quantity of a first label proportional to the quantity of the target analyte in the sample. The analyzer 100 includes an optical system 120, which is more clearly seen in the rear view of the analyzer 100 in FIG. 2. Further, the components of the optical system 120 are shown separately from the other parts of the analyzer in FIG. 3 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 will also be described in more detail below.

[0061] The analyzer 100 also includes a controller 140, schematically represented in FIG. 1. The controller 140 includes a non-transitory computer-readable medium with program instructions stored thereon for performing the steps performed by the analyzer 100, and identifies the presence of a target analyte in the 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.

[0062] The processor 141 of the controller 140 includes computer processing elements 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 that temporarily stores the executed instructions and corresponding data, as well as cache memory that temporarily stores the 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, solid state drive, or hard disk drive. In some embodiments, the memory 142 stores program instructions that are executable by the controller 140 to execute 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 (Wifi) or BLUETOOTH®. Other communication conventions are also possible.

[0063] In some embodiments, the analyzer 100 includes at least one motor configured to rotate the cartridge in order to manipulate any sample placed within the cartridge and to align the cartridge with the analyzer. In some embodiments, the motor is a centrifuge drive motor, and in other embodiments, the motor is a positioning motor. Further, 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.

[0064] In analyzer 100, centrifuge 101 is coupled to cartridge 150 to rotate the cartridge at a speed of at least 100 rpm. The details of centrifuge 101 are shown more clearly in FIG. 4. As shown, centrifuge drive motor 103 is configured to couple to the cartridge using dock 102. Dock 102 may include alignment mechanisms such as a plurality of alignment pins 105 and cantilever clips 104 that engage the mounting opening 153 of cartridge 150 (shown in FIG. 9). In some embodiments, cantilever clip 104 is at the end of a leaf spring that clamps across the cartridge when the cartridge 150 is received within dock 102. Cantilever clip 104 may be configured to engage cartridge 150 in an outward direction. As centrifuge 101 rotates, cantilever clip 104 is pushed outward by centrifugal force that increases the holding force on cartridge 150. Thus, cartridge 150 may be firmly held within dock 102 when inserted into analyzer 100.

[0065] Dock 102 is connected to centrifuge drive motor 103 to rotate dock 102 and the cartridge 150 attached thereto. Further, centrifuge drive motor 103 may include an electronic drive phase sensor 106 and a flag wheel 107 for high-speed precision control of centrifuge 101 during operation. In some embodiments, dock 102 is directly driven by centrifuge drive motor 103, while in other embodiments, a power transfer system such as a gearbox or belt drive may be used to couple dock 102 to centrifuge drive motor 103. As described below, dock 102 may also be driven by manifold 108 (see FIG. 1). Specific embodiments of the operation of centrifuge 101 are described in more detail below.

[0066] In some embodiments, the analyzer includes a manifold 108 with a plurality of ports, each configured to couple to an individual port of the cartridge. A depiction of the manifold 108 coupled to the cartridge 150 is shown in FIG. 5. Further, a bottom view of the manifold 108 is shown in FIG. 6, illustrating ports 111-114. In some embodiments, the manifold includes supply ports 111, 112 for supplying fluid to the cartridge 150 and draw ports 113, 114 for drawing fluid from the cartridge 150. In some embodiments, supply ports 111, 112 are used to provide one or more liquids to the cartridge 150, while draw ports 113, 114 draw liquid into the cartridge 150 by extracting gas from the cartridge 150. In other embodiments, the draw ports extract liquid or both liquid and gas from the cartridge 150. To transfer fluid to and from the cartridge, the manifold 108 includes fluid lines 115 connected to ports 111-114.

[0067] The ports of the manifold 108 may each include a seal that covers the individual corresponding port of the cartridge 150 to isolate fluid transfer between the manifold 108 and the cartridge 150. For example, ports 111-114 may each include an O-ring or other feature for creating a seal between the manifold and the cartridge port that surrounds the individual port of the cartridge 150. In some embodiments, the ports of the cartridge 150 are already open when the cartridge 150 is installed within the analyzer. In other embodiments, the manifold is configured to pierce the cartridge 150 to open each of the ports of the cartridge 150.

[0068] In some embodiments, the manifold 108 is disposed on a movable arm 109 (shown in FIG. 1) that allows the manifold 108 to be separated from the cartridge 150 when the cartridge 150 is rotated at high speed by the centrifuge 101. The coupling of the manifold 108 to the cartridge 150 may be enabled by a mating structure. For example, the manifold 108 may include pins received within a mounting aperture 153 of the cartridge (see FIG. 9). In other embodiments, the pins 104 of the dock 102 may pass through the mounting aperture 153 of the cartridge 150 and into receiving holes within 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 contemplated as possible, as would be understood by one of ordinary skill in the art.

[0069] 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 that couples to the cartridge 150. The positioning motor 110 may be configured to pivot the cartridge 150 so as to align the detection chamber 184 of the cartridge 150 (see FIG. 9) with electromagnetic radiation from the first electromagnetic radiation source 121. Further, the positioning motor 110 may be used in cooperation with one or more magnets or by the hydrodynamic of the sample to circulate a target analyte through the chambers of the cartridge 150, 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 less than 1° increments. Specific examples of embodiments using the positioning motor 110 are described in more detail below.

[0070] 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 via 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.

[0071] In some embodiments, the analyzer 100 includes an optical system 120 (FIGS. 2-3) that directs electromagnetic radiation from the first electromagnetic radiation source 121 to the interrogation chamber 184 of the cartridge 150 and directs electromagnetic radiation emitted by the label to the first detector 122. The optical system 120 may include one or more mirrors and lenses for manipulating and directing electromagnetic radiation to and from the interrogation space. Additionally, the optical system may include an objective lens 123 as shown in FIG. 7 for focusing electromagnetic radiation from the first electromagnetic radiation source into the interrogation space within the cartridge 150. In some embodiments, the objective lens 123 is coupled to a movable stage 124 that enables movement of the objective lens relative to the cartridge 150.

[0072] In some embodiments, the optical system 120 is a confocal system. For example, the electromagnetic radiation source 121 is imaged as a spot within the focal plane of the objective lens 123 within the detection chamber 184. Light emitted from the label within the detection chamber 184 that is excited by the electromagnetic radiation source 121 is collected by the objective lens 123 and directed by the optical system 120 onto the confocal stop 125 within the optical system 120, as shown in FIG. 3. The confocal stop 125 is then imaged onto the detector 122. The confocal arrangement preferentially passes light from the label within the focal plane of the objective lens 123 while excluding light beyond the focal plane. In this way, the arrangement increases the signal-to-noise ratio by passing the signal from the label while excluding light from the liquid suspension, cartridge, and elements within the optical system that does not originate from the label. As is known to those skilled in the art, this arrangement may also use a dichroic filter 126 to reflect the laser light, pass the light emitted by the label, and allow only the light from the label to reach the detector while prohibiting the laser light from reaching the detector. Further, if more than one radiation source is used for the detection of additional labels, one or more additional dichroic filters 126 may be used to reflect the laser and label electromagnetic radiation from the first electromagnetic radiation source and label while passing the electromagnetic radiation from the second electromagnetic radiation source and the second label, as shown in FIGS. 2 and 3.

[0073] In some embodiments, all of the components of the analyzer 100 are disposed within a common housing. The common housing may be sized to fit on a countertop. For example, in some embodiments, the dimensions of the common housing are on the order of 1 meter in any direction. Further, in some embodiments, the common housing fits within a cube that is 30 inches × 30 inches × 30 inches.

[0074] In some embodiments, the controller 140 includes a network interface 143 for receiving control information from a user and outputting analysis data to the user. For example, in some embodiments, the analyzer communicates with the user via software on an external device such as a smartphone, tablet, notebook, computer, or desktop computer. The analyzer receives information from the user of the external device and outputs information to the user by communicating with the external device via the network interface. Such communication may be through a wireless or wired connection such as USB or other bus. In some embodiments, the analyzer 100 may include an input and / or output device for communicating directly with the user, such as a keyboard for receiving inputs, and a display for outputting information. Further, in some embodiments, the display may include a touch screen for both outputting information and receiving information from the user. In some embodiments, the analyzer includes a network interface, an input, and a display.

[0075] In some embodiments, the method of the present disclosure includes directing a portion of the sample through a chamber of the cartridge 150 without involving any valve-containing cartridge 150. Further, in some embodiments, the cartridge 150 does not include any valves.

[0076] In some embodiments, the liquid within the cartridge 150 is at least partially moved through the cartridge using pumps 116 - 118 and valves coupled to the supply and draw ports of the cartridge, as described in more detail below. A schematic of the fluid transfer components of the analyzer 100 is shown in FIG. 8. Manipulation of a portion of the sample within the cartridge 150 may also be facilitated using an external motive force such as a magnet, or by movement of the cartridge 150 and utilizing inertia and hydrodynamics to move a portion of the sample around the cartridge.

[0077] As shown in FIG. 8, in some embodiments, the cartridge 150 includes a plurality of ports 154-157 for introducing and extracting fluid from the cartridge 150. For example, in some embodiments, the cartridge 150 includes inlet ports 154, 155 and outlet ports 156, 157. The inlet ports 154, 155 may be configured to align with the supply ports 111, 112 of the manifold 108. Similarly, the outlet ports 156, 157 of the cartridge 150 may be configured to align with the draw ports 113, 114 of the manifold 108. The use of the inlet and outlet ports of the cartridge 150 is described in further detail below.

[0078] In another aspect, the present disclosure provides a plurality of chambers for isolating a target analyte of a sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample.

[0079] In some embodiments, the cartridge 150 is planar and the chambers of the cartridge are located in a single plane. For example, in some embodiments, the cartridge 150 is a flat cartridge and the chambers of the cartridge are circumferentially positioned 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" is not intended to mean extending around the entire circumference of the cartridge, but rather is intended to represent the circumferential direction within the plane of rotation. In some embodiments, at least a group of chambers may be circumferentially and continuously connected around a portion of the cartridge.

[0080] In some embodiments, the cartridge may include a base, a body disposed across the base, and a cover disposed across the body. The body includes an open path extending therethrough that defines a plurality of chambers of the cartridge 150. In some embodiments, the body may be a single integral part. Thus, for example, in some embodiments, all sidewalls of the chambers and interconnecting channels of the cartridge may be formed by a single integral part forming the body. Further, in some embodiments, both the body and the base together form a single integral part, and the cover is attached thereto. Similarly, in other embodiments, the body and the cover form a single integral part, and the base is attached thereto. For example, in the present embodiment, the body and the base may be a single molded part of a cyclic olefin polymer having a thickness of 5 mm, and the cover may be a laminate of a cyclic olefin polymer having a thickness of 188 microns. The laminate may be joined to the body using laser welding or ultrasonic welding so as to provide a joint that is as robust as the materials joined together. In some embodiments, the base, the body, and the cover may be layers of a laminated structure. For example, in some embodiments, both the base and the cover are laminated across opposite sides of the body. In some embodiments, the cover and the base of the cartridge 150 extend across and enclose the chambers and microfluidic channels of the cartridge, but they may include ports as described above for supplying or extracting fluid from the cartridge.

[0081] 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 measurement chamber for receiving the sample.

[0082] In some embodiments, the cartridge contains reagents stored within at least one of the plurality of chambers. For example, in some embodiments, the cartridge contains reagents that are stable and dried before the cartridge is inserted into the analyzer. For example, the reagents may be lyophilized or dried on the surface of one or more chambers of the cartridge. Or they may be in the form of lyophilized pellets that are placed within one or more of the chambers or the cartridge.

[0083] Although the cartridge is shown and described herein in the form of a disk that rotates within the analyzer, in other embodiments, the cartridge is not a disk. Further, some aspects of the present disclosure are implemented without using the cartridge at all. For example, in some embodiments, aspects of the present disclosure are implemented within discrete separate elements that form different chambers. Process quality control camera

[0084] In some embodiments, analyzer 100 includes a process quality control camera for monitoring the movement of substances through cartridge 150. For example, the process quality control camera may be mounted across cartridge 150 to view substances inside cartridge 150. In some embodiments, the process quality control camera is configured to output only representations of light detected within 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 situations. For example, controller 140 may be configured to detect the presence of unwanted air bubbles within the cartridge. Other exemplary embodiments of using the process quality control camera are described below.

[0085] 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 specific area of the cartridge 150 as it rotates. In particular, in some embodiments, the strobe may be used when the centrifuge 101 is rotating the cartridge 150. Optical quality control camera

[0086] 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 a mirror on the slide to obstruct the optical path before and after the confocal stop images the laser at the confocal stop to visualize that the electromagnetic radiation has the appropriate intensity, is focused in the correct place, and / or has the correct intensity profile. To image the laser at the confocal stop, the objective lens may be positioned such that the electromagnetic radiation source is imaged onto the surface of the window on the cartridge 150. When this is done, a portion of the radiation will reflect from the window back towards the objective lens due to the difference in refractive index between the window and the medium on the opposite side of the window. This radiation will be imaged onto the confocal stop by the optical system. The window on the cartridge may be dimensioned with the correct thickness to simulate the window thickness of 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 stop can be analyzed by the controller 140. The controller 140 analyzes the image from the optical quality control camera, verifies that the electromagnetic spot is the correct size, shape, intensity, and position relative to the confocal stop, and may be used to ensure that there are no abnormalities in the optical system. The measured size, shape, intensity, and position can be compared to known tolerance values for these parameters. If the measured values are outside the tolerance values or are approaching the limits of the tolerance values, the controller can notify the user of the analyzer or prevent the use of the analyzer. Exemplary method

[0087] Figures 10-18 illustrate exemplary cartridges and methods that utilize various embodiments of the present disclosure where the sample is blood. In other embodiments, the chambers of the cartridges and methods used may also be suitable for other sample types. For example, the analyzers, methods, and cartridges of the present disclosure may be suitable for use in conjunction with other biological fluids such as urine, diluted feces, or oral fluid. Other types of samples are also considered possible. Further, the sample may be neat or diluted. Loading and Sample Separation

[0088] As shown in FIG. 10, the cartridge 150 is first loaded with a sample 152 in an inlet chamber 158. The inlet chamber 158 includes an input port 151 that receives the sample 152 prior to analysis. In some embodiments, the sample 152 is received within 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 152 after the cartridge 150 has been received within the analyzer 100. As described above, in some embodiments, the input port 151 can be sealed prior to insertion of the sample 152, and the seal can be either punctured or removed to allow insertion of the sample 152. In other embodiments, the input port 151 can be a simple opening that is available for receiving the sample 152 without being “opened.” In some embodiments, the input port 151 can be sealed after the sample has been input. In other embodiments, the manifold 108 contains a seal for covering the ports when the manifold is in contact with the cartridge. In some embodiments, the inlet chamber 158 is a measurement chamber configured to receive a specific amount of sample, while in other embodiments, the inlet chamber 158 is sized large enough to accommodate more sample than will be used in the analysis. The inlet chamber 158 in the illustrated embodiment of FIGS. 9-18 is configured to receive approximately 200 μl of liquid.

[0089] Once sample 152 is loaded into inlet chamber 158 as shown in FIG. 10 and cartridge 150 is inserted into analyzer 100, cartridge 150 is coupled to centrifuge 101 such that centrifuge 101 can rotate within cartridge 150. As will be described in more detail below, the geometry of the chambers and channels within cartridge 150 is designed to affect the transfer of fluid through cartridge 150. To facilitate understanding of these geometries, the following description refers to the cylindrical / polarity direction. In particular, the use of the terms “inner,” “inward,” “outer,” “outward,” and similar descriptors typically refers to the radially inner and radially outer directions relative to the center of rotation of the cartridge, which is located near the geometric center of the cartridge. The description also refers to a first circumferential direction and a second circumferential direction that are related to the direction in which the cartridge is configured to be rotated in a first circumferential direction by the centrifuge. For example, the area at the first circumferential end of a chamber will pass a stationary reference position before the area at the second circumferential end of the same chamber. In the embodiments shown in FIGS. 9-18, the first circumferential direction is clockwise, however, other embodiments of the cartridge may be configured to rotate in the opposite direction such that in these embodiments the first circumferential direction is counterclockwise.

[0090] When the cartridge 150 is loaded into the analyzer 100, the centrifuge 101 is activated to rotate the cartridge 150 to move the sample 152 from the inlet chamber 158 through the inlet channel 159 into the separation area 160, as shown in FIG. 11. The rotation of the cartridge 150 moves the sample 152 radially outward as a result of "centrifugal force", i.e., the inertial phenomenon that moves an object 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 such that once the sample is loaded, the rotation of the disk-shaped cartridge 150 will keep the sample and any other liquid received within the cartridge 150 away from the input port 151. Further, in some embodiments, the input port 151 may be aligned on the cartridge 150. In some embodiments, to move the sample from the inlet chamber 158 to the separation area 160, the cartridge may be rotated, for example, from 0 rpm to 1,000 rpm at a rate of 2,000 rpm / second and held at that speed for a few seconds, e.g., 2 - 10 seconds. Thus, sample transfer can occur very quickly. The rotation rates and accelerations provided are exemplary, and the actual rates selected will depend on the sample being processed and the rotation can vary from 100 to 10,000 rpm with accelerations varying between 100 rpm / second and 8,000 rpm / second.

[0091] In some embodiments, the separation area may include an inner separation chamber 161 and an outer separation chamber 162 configured to hold 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 rotates the cartridge 150, the more concentrated components of the sample are pushed radially outward into the outer separation chamber 162, while the less concentrated 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, for example, by a reduced diameter portion 163 located 22 mm from the center of rotation. The reduced diameter portion 163 has a cross-sectional area smaller than either of the chambers. For example, in some embodiments, the reduced diameter portion 163 may have a cross-sectional area of 3 mm 2 while the inner separation area 162 has an average 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, the diameter portion 163 is sized to facilitate the downward movement of the more concentrated components, while the less concentrated components move rapidly upward through the diameter portion 163. However, as discussed below, the reduced diameter portion 163 limits the movement of the more concentrated components into the inner separation area 161 when the cartridge is rapidly decelerated.

[0092] For further processing, an accurate volume of the sample should be ascertained in order to generate an accurate concentration value of the sample. If the sample cannot fill the separation area 160 and is unintentionally wasted, or if the separation area is sized to accept more than the sample volume, an accurate concentration value may be difficult to obtain. Thus, in some embodiments, the cartridge 150 may include various features for measuring a precise amount of fluid into the separation area 160.

[0093] For example, some embodiments of the cartridge 150 may include one or more features for avoiding entrapment of air within the cartridge, particularly during transfer of the sample from the inlet chamber 158 to subsequent chambers. As the sample is loaded therein, if air is trapped within the separation area 160, a portion of the sample may flow prematurely through the overflow channel 165, and accurate measurement of the sample into the separation area 160 may fail. Thus, it is beneficial to avoid formation of trapped air within the cartridge during loading.

[0094] In some embodiments, the inlet channel 159 is coupled to the first circumferential end of the inner separation chamber 161. As the sample moves outwards from the inlet chamber 158 and through the inlet channel 159 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 inlet channel 159 is coupled to the center of the inner separation chamber 161, additional precautions may be necessary to avoid formation of trapped air in the corner at the first circumferential end directed towards the inside of the inner separation chamber 161. However, if the inlet channel 159 is coupled to the first circumferential end of the inner separation chamber 161 as shown for the cartridge 150 of FIGS. 9-18, inclusion of the inner corner that is further in the first circumferential direction than the inlet channel 159 opening is avoided. Similarly, air that may be trapped in such a corner is also avoided.

[0095] Furthermore, in some embodiments, the inlet channel 159 may be reduced in size and depth compared to the separation area 160. Such a reduction can slow the flow of the sample into the separation area 160 and allow air to be purged from the separation area 160 while it is being filled. Additionally, the reduced size and depth can also help avoid the formation of a sheet of liquid across the cross-section of the separation area 160 that could similarly trap air. For example, in one embodiment, the depth of the inlet channel 159 may be 0.5 mm, while the depth of the inner separation chamber 161 is 2 mm. Thus, the flow of the sample from the inlet channel 159 into the inner separation chamber 161 will not span the entire depth of the inner separation chamber 161, and air will be able to flow around the flow and out of the separation area 160.

[0096] Furthermore, in some embodiments, the cross-sectional area of the inlet channel 159 may be narrower than the cross-sectional area of the reduced diameter portion 163 between the inner separation chamber 161 and the outer separation chamber 162. For example, the inlet channel 159 may have a cross-sectional area of 0.5 mm 2 while the reduced diameter portion 163 has a cross-sectional area of 3 mm 2 . Thus, the volumetric flow rate of the sample into the separation area 160 overwhelms the reduced diameter portion 163 and is less likely to trap air within the outer separation chamber.

[0097] To prevent the entrapment of air within the outer separation chamber 162, in some embodiments, the inner edge 164 of the outer separation chamber 162 extends at an inwardly projecting angle as the inner edge 164 approaches the reduced diameter portion 163 that separates the inner separation chamber 161 from the outer separation chamber 162. Thus, as the outer separation chamber 162 is filled with sample due to the rotation of the cartridge, the air within the outer separation chamber 162 will "float" inwardly to the inner edge 164 and then follow the inner edge 164 to the reduced diameter portion 163. The air will then pass out through the reduced diameter portion 163, through the inner separation chamber 161, and out of the separation area 160.

[0098] 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 confirm that any bubbles in the separation area 160 have a volume of zero 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 bubbles in the separation area 160 and calculate the overall volume of air in the separation area 160. If the calculated volume of air exceeds a predetermined threshold, the controller may be configured to interrupt 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.

[0099] In some embodiments, the separation area 160 and the peripheral channels may include one or more features for precise measurement of the sample and controlled separation of the components of the sample. For example, in some embodiments, the overflow channel 165 may be positioned to enable precise measurement of the amount of sample 152 into the separation area 160. If the amount of sample 152 received within the cartridge 150 exceeds what is required 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.

[0100] Due to the rotation of the cartridge 150 and the centrifugal force on the sample, the separation area 160 is filled from the outer end towards the inner end. Therefore, positioning the opening of the overflow channel 165 at a specific radial position within the inner separation chamber 161 determines the quantity of sample that can be loaded into the separation area 160. For example, as the centrifuge 101 rotates the cartridge 150, the sample will move towards the outer end of the outer separation chamber 162 and generate a filling line that moves inwards as the separation area 160 fills. Once the filling line reaches the radial position of the overflow channel 165, for example, at a radial distance of 17 mm, any additional volume of sample entering the separation area 160 will exit the separation area 160 through the overflow channel 165. Thus, the quantity of sample that will be analyzed can be precisely measured based on the radial position of the overflow channel 165. Separation of Sample Components

[0101] As shown in FIG. 12, after the sample is loaded into the separation area 160, the centrifuge 101 may continue to rotate the cartridge 150 to separate the sample 152 into different components. For example, the centrifuge 101 may rotate the cartridge 150 such that the more concentrated components of the sample are sent outward and the less concentrated components remain radially inward. In some embodiments, the speed of the centrifuge 101 may be increased to separate the components of the sample 152. 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 of 2,000 rpm / second. Upon reaching 1,000 rpm, the centrifuge 101 may further accelerate the cartridge 150 to a rate of 7,000 rpm at 5,000 rpm / second and hold that rate for 90 seconds to separate the components. In another embodiment, the centrifuge 101 may skip the initial transfer rotation speed and proceed directly from 0 rpm to a separation speed of 10,000 rpm at an acceleration of 2,000 rpm / second. The separation step may occur at a rotational speed of 1,000 rpm to 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 breakage. The duration of the separation may be performed within the range of 10 seconds to 5 minutes.

[0102] In some embodiments, the sample 152 may be whole blood, and the continuous rotation of the cartridge 150 may separate red blood cells from plasma as depicted in FIG. 12. 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 more concentrated red blood cells are pushed radially outward while the less concentrated plasma moves radially inward into the plasma compartment 161.

[0103] The angled inner edge 164 of the outer separation chamber 162 can assist in separating the components of the sample in a manner similar to promoting the removal of air from the outer separation chamber 162 as described above. As the centrifuge 101 rotates the cartridge 150, the more concentrated components will move outward and the less concentrated components will move inward. Thus, similar to the air flow path within the outer separation chamber 162 during the filling process, the lighter components of the sample will move inward and then follow the angled inner edge 164 of the outer separation chamber 162 until they reach the reduced diameter portion 163 and pass into the inner separation chamber 161.

[0104] In some embodiments, the 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. The controller 140 may further be configured to analyze the image and determine the filling level of the more concentrated components of the sample within the separation area 160. In some embodiments, the controller 140 is configured to confirm that a more concentrated component of the sample has moved outward from a pre-determined filling level. The controller may similarly be configured to continue the analysis in response to such a confirmation.

[0105] For example, when the sample is whole blood, the controller 140 may be configured to analyze the image and determine the filling level of red blood cells within the separation area. If the filling level of the red blood cells is outside a pre-determined radius, the controller 140 may be configured to continue the analysis. On the other hand, if the filling level of the red blood cells is inside the pre-determined radius, the controller 140 may be configured to send a control signal to the centrifuge 101 to further separate the components of the blood sample and continue to rotate the cartridge. For example, the image may be captured and analyzed at 90 seconds of separation time. If the level of red blood cells is, for example, inward of a threshold distance of 22 mm from the center of rotation, the controller 140 may be configured to capture an additional image, send a control signal, and continue to rotate for an additional 30 seconds before re-evaluating the level of red blood cells. In some embodiments, the duration or speed of this additional control signal may be based on the identified filling level of the red blood cells. Alternatively, the controller 140 may be configured to interrupt the analysis. 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 the analysis. Therefore, identifying the filling level of red blood cells makes it possible to determine the quality of the plasma transferred for further analysis.

[0106] 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.

[0107] Still further, in some embodiments, the controller 140 may be configured to analyze an image of a 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. One of ordinary skill in the art will readily appreciate that for a given chamber geometry, rotation rate, and rotation time, blood with a lower hematocrit level will exhibit a separation line at a larger radius than blood with a higher hematocrit level. For a given cartridge geometry and rotation parameters, different hematocrit levels can be 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 the values stored within the controller to determine the hematocrit level of the initiated sample after a predetermined rotation time. Further, the controller 140 may be configured to proceed with the analysis in response to confirming that the hematocrit level falls below a pre-determined threshold. Transfer of supernatant

[0108] As shown in FIG. 13, a portion of the sample 152 may be removed from the separation area 160 through a siphon 167 extending from the separation area 160. The siphon 167 leads to a second chamber, such as the mixing chamber 175, and is 1 mm 2It may be in the form of a microfluidic channel with a cross-sectional area. The siphon 167 may include a first section 168 extending from the separation area 160, a vertex 169, and a second section 170 extending from the vertex 169 to the mixing chamber 175. The first section 168 of the siphon 167 extends from the siphon inlet 171 in a direction having a radially inward component away from the inner separation chamber 161 toward the vertex 169. Further, the second section 170 extends from the vertex 169 to the siphon outlet 172 which is further radially outward than the siphon inlet 171 of the siphon. For example, the siphon inlet 171 may be at a radial position of 21 mm from the center of rotation, while the siphon vertex may be at 16 mm from the center of rotation, and the siphon outlet 172 may be at a radial distance of 30 mm from the center of rotation. Other radial distances may be selected to suit the needs of the application as long as the siphon outlet 172 is at a larger radial distance than the siphon inlet 171 and the vertex 169 is at a radial distance less than both the siphon inlet 171 and the siphon outlet 171. Thus, the vertex 169 is the radially innermost point of the siphon 167 and the siphon outlet 172 is radially outward compared to the siphon inlet 171. Therefore, since the rotation of the centrifuge generally drives the sample radially outward, once a portion of the sample passes across the vertex 169, the siphon 167 will drive a portion of the sample from the inner separation chamber 161 to the mixing chamber 175.

[0109] In some embodiments, the siphon may be primed, i.e., a portion of the sample may be strongly moved over the vertex so as to initiate the sucking action through capillary action. In other words, the capillary force may draw the sample into the first section 168 of the siphon 167 and over the vertex 169 until the sucking action draws additional fluid from the inner separation chamber 161. The cross-sectional area of the siphon 167 is smaller, for example, about 0.1 mm 2 ~ about 0.3 mm 2 or about 0.2 mm 2It may be. In other embodiments, the siphon 167 may be primed through the use of a pump that draws the sample into the siphon 167 until the sample passes the apex.

[0110] Furthermore, in some embodiments, the siphon may be primed by acceleration. For example, in one embodiment, after the cartridge 150 has completed the separation step at 7,000 rpm, it is decelerated by the centrifuge 101 from 7,000 rpm to 3,000 rpm at 2,000 rpm / second to prepare for the siphon step. While the cartridge 150 is rotating in the first circumferential direction, inertia will propel the sample and cause it to continue moving in that direction. Thus, if the cartridge 150 is rapidly decelerated from 3,000 rpm to 0 rpm, for example, at 8,000 rpm / second, inertia will cause the sample 152 to continue moving in the first circumferential direction, and the sample will flow through the first section 168 of the siphon 167 and through the apex 169 that is radially outward of the fill level of the separation area 160 due to its extent along the first circumferential direction. At this point, the centrifuge 101 may reverse the direction of rotation to -1,000 rpm at an acceleration of 2,000 rpm / second and hold that speed. The centrifugal force will move the fluid in the channel 170 radially outward toward the siphon outlet 172 that is radially outward of the siphon inlet 171. The separation area 160 will continue to drain until the fill level is radially outward (or "below") the connection where the first section 168 of the siphon 167 opens into the inner separation chamber 161. This method of priming and siphoning is significantly faster than capillary action and / or pump-based priming and siphoning because the entire process can occur in seconds. In some embodiments, the apex 169 is radially inward of the overflow channel 165, which prevents the sample from flowing through the siphon 167 while the separation area 160 is being filled. Other rotational speeds and accelerations can also be used as long as the acceleration is sufficient to push the fluid across the siphon apex 168 and the cartridge 150 continues to rotate and draw fluid from the separation area 160.

[0111] As described above, the first section 168 of the siphon 167 extends in a first circumferential direction and radially inwards. Further, in some embodiments, the shape of the first section 168 of the siphon 167 is shaped to promote priming of the siphon 167 in particular. For example, in some embodiments, a part of the first section 168 at the end connected to the inner separation chamber 161 is substantially parallel to the first circumferential direction, for example within 10 degrees of parallel. The first section 168 gradually curves inwards as it extends towards the apex 169. As described above, in response to the deceleration of the cartridge 150, the sample is pushed in the first circumferential direction. Thus, when the first part of the first section 168 is substantially aligned with the first circumferential direction, the sample flows into the siphon 167 with additional momentum. As a result of this momentum, the sample reaches the apex 169 and flows over it, thereby being able to prime the siphon 167.

[0112] In some embodiments, the position 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 embodiment depicted in FIG. 13, the siphon 167 will transfer a precise amount of sample, such as 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 in the inner separation chamber 161 will drop (i.e., move radially outwards) and be replaced by air from the inlet channel 159 or the overflow channel 165. Once the interface between the sample and the air reaches the first section 168 of the siphon 167, no additional amount of sample will be drawn from the inner separation chamber 161. Thus, the position where the first section 168 opens into the inner separation chamber 161 may be used to define the measured amount of sample that is transferred to the downstream chamber.

[0113] The position of the opening of the first section 168 of the siphon 167 into the inner separation chamber 161 may also be selected to limit the transfer of only certain components of the sample through the siphon 167. For example, in an embodiment where the sample is whole blood and the separation chambers 162, 161 are used to separate red blood cells from plasma, the opening of the first section 168 may be positioned radially inwards from the separated red blood cells. Unintentional inclusion of red blood cells in the sample transferred to the mixing chamber can result in hemoglobin contamination during the mixing process. Therefore, it is advantageous to position the opening of the first section 168 so as to avoid inclusion of red blood cells in the sample transferred through the siphon 167. Thus, if the outer separation chamber 162 is a red blood cell trap configured to receive red blood cells after the separation process, the opening of the first section 168 may be positioned radially inwards from the red blood cell trap and within the plasma container. Similarly, the volume of the outer separation chamber 162 may be selected based on a typical red blood cell volume, for example, a hematocrit level of 52%, 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.

[0114] In some embodiments, the outer separation chamber 162 extends away from the reduced diameter portion 163 in a first circumferential direction. Thus, as the cartridge 150 is decelerated and less concentrated components of the sample are forced through the siphon 167, the more concentrated components are similarly forced away from the reduced diameter portion 163 and the siphon inlet 171 towards the closed end of the outer separation chamber 162. For example, in an embodiment using whole blood, the plasma above the reduced diameter portion 163 is transferred through the siphon 167 and the red blood cells are forced towards the closed end of the red blood cell formed by the outer separation chamber 162.

[0115] As discussed, a large deceleration may be used to prime the siphon. When the cartridge is decelerating, the concentrated components in the outer separation chamber move away from the reduced diameter portion 163 towards the closed end. However, a density gradient may exist within the outer separation chamber where the fluid density is higher towards the more radially outward portions of the outer separation chamber 162. In this case, some backflow may exist in the upper portion of the outer separation chamber where the separated components in the upper portion of the chamber move towards the reduced diameter portion 163. If those components move far enough towards the diameter portion 163, they may be conveyed upward into the upper separation chamber 161 and sucked out of the upper separation chamber 162 into the mixing chamber 176. This can be controlled by rotating longer and further concentrating the concentrated components or by decelerating at a lower rate, but as shown in FIG. 14, it may be advantageous to add a baffle 191 within the lower separation chamber. The baffle 191 may extend substantially through the depth of the outer separation chamber 162 and may be positioned to impede the movement of the concentrated components within the outer separation chamber. The baffle 191 may be spaced from the outer wall of the outer separation chamber 162 to facilitate removal of air during initial filling of the lower separation chamber and to promote separation within the lower separation chamber. The baffle may be circular, oval, square, or rectangular. Additionally, multiple rows of baffles may exist at different radial distances to form a grid. Further, the rows may be circumferentially offset.

[0116] Figure 19 shows an alternative embodiment of the siphon 167 that includes a vent channel 174 at the apex of the bend 169. The vent channel 174 extends inwardly from the apex of the bend 169 toward the center of the cartridge and is used to facilitate the pump-based transfer of the sample from the separation area 160 to the mixing chamber 175. When the manifold 108 is not engaged, the vent channel 174 is open to the atmosphere. When the manifold 108 is aligned on the cartridge 150, the vent may be covered and closed by a seal. In an exemplary method of operating the siphon 167 that includes the vent channel 174, after plasma separation, the manifold 108 is aligned and brought into contact with the cartridge 150. The draw pump 118 draws gas from the cartridge 150 through the outlet port 156 and draws the sample from the separation area 160 through the siphon line 167 into the mixing chamber 175. After a precise predetermined draw volume, the manifold 108 is raised, disconnected from the cartridge 150, and the vent channel 174 is opened to the atmosphere. The centrifuge 101 then rotates the cartridge 150 such that the sample remaining in the siphon line 167 moves along both sides of the siphon line due to centrifugal force and away from the vent channel 174. The vent channel 174 enables the movement of the sample by allowing air drawn through the vent channel to displace the sample within the siphon line 167 as the sample moves toward the mixing chamber 175 and toward the separation area 160 away from the center of rotation. In the absence of the vent channel 174, depending on the rotation of the cartridge, the siphon action would drain the separation area 160 until air reaches the inlet to the siphon line as discussed above. In the vented embodiment of the siphon line 167, the amount of sample transferred to the mixing chamber can be determined by the pump draw volume rather than the geometry of the separation area and the siphon line. Thus, the volume of the sample transferred is selectable rather than fixed. Sample mixing

[0117] From the separation area 160, the plasma moves to a mixing chamber 175 which may have a reagent therein. For example, the mixing chamber 175 may include 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 reagent by rapid acceleration and deceleration of the cartridge 150 while continuing to rotate in a first circumferential direction as shown in FIG. 14 in its entirety.

[0118] In some embodiments, mixing of the plasma with the reagent is facilitated by a mixing ball 176 disposed within the mixing chamber 175. The acceleration and deceleration of the cartridge 150 moves the mixing ball 176 back and forth through the mixing chamber 175 and bounces it off its walls as it rotates in the 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 with acceleration and deceleration at 1,500 rpm / second. This corresponds to a mixing frequency of 5 Hz. The turbulent movement of the mixing ball 176 first rehydrates the paramagnetic capture beads, detection label, control analyte, and control label and releases them into the plasma. The mixing ball 176 further serves to facilitate the binding reaction rate of the target analyte to the paramagnetic capture beads 177 and the detection label. After the mixing step, the target analyte and the detection label may both be attached to the paramagnetic capture beads dispersed throughout the plasma. In some embodiments, rehydration of the reagent and incubation of the target analyte occur in less than 20 minutes, for example, less than 10 minutes or less than 5 minutes.

[0119] In some embodiments, the mixing chamber 175 has geometric features that improve the mixing ability of the mixing ball 176 by varying the direction of the mixing ball 176. For example, in some embodiments, the outer surface of the mixing chamber 175 includes a rough or textured surface to encourage the bouncing back of the mixing ball as it rolls back and forth. Similarly, in some embodiments, the outer surface of the mixing chamber 175 may include radially inward protrusions to "jump" the mixing ball as it passes over the protrusions. Further, in some other embodiments, the ends of the mixing chamber 175 are inclined in a radially inward direction to push the mixing ball inward at the ends of the mixing chamber, reverse the direction of the mixing ball, and pass it back through the mixing chamber in the vicinity of the radially inner side of the mixing chamber. For example, both ends having such an inclination can enable an eight-shaped pattern of the mixing ball as the cartridge is rotated back and forth and moved.

[0120] The term "mixing ball" is used herein with reference to the movement of this feature and not with respect to any particular shape. Thus, the mixing ball 176 can be spherical in some embodiments, but may have another shape in other embodiments. By way of example, the mixing ball 176 can be oval, cubic, or star-shaped. In some embodiments, the surface of the mixing ball is magnetic. The term "non-magnetic" as used herein includes those materials that are neither ferromagnetic nor paramagnetic. Further, in some embodiments, the surface of the mixing ball includes a substance having low reactivity. For example, in some embodiments, the mixing ball 176 may include brass, glass, or Teflon®. Plastics, ceramics, and other hard materials with a density higher than that of the sample may also be used for the mixing ball. In other embodiments, particularly those in which paramagnetic capture beads are not used, the mixing ball 176 may include a ferromagnetic material such as steel. Similarly, in some embodiments, the mixing ball is coated with a substance having low reactivity.

[0121] In some embodiments, the mixing chamber and the peripheral channels include one or more features for retaining a sample within the mixing chamber during the mixing process. For example, in the cartridge 150 shown in FIGS. 9 - 19, both the pre - mixing chamber channel and the post - mixing chamber channel 173 formed by the siphon 167 extend radially inwards from the mixing chamber 175. Thus, centrifugal force presses the sample outwards into the mixing chamber 175 as the cartridge 150 is rotated by the centrifuge 101.

[0122] Similarly, to prevent the movement of the sample out of the mixing chamber by capillary action, at least one of the channels 167, 173 directly connected to the mixing chamber 175 may include capillary breaks 178, 179. For example, in the cartridge 150 as shown in FIG. 14, both the pre - mixing chamber channel 167 and the post - mixing chamber channel 173 include individual capillary breaks 178, 179. The capillary breaks 178, 179 are each formed by a section of the individual channels 167, 173 that expands in a direction leading away from the mixing chamber 175. The expanding cross - sectional area of the capillary breaks 178, 179 results in a reduced capillary force as the sample moves away from the mixing chamber 175. The use of the capillary breaks 178, 179 reduces the effect of capillary action and keeps the culture fluid within the chamber after mixing and incubation of the paramagnetic capture beads, detection labels, control analytes, and control labels. This allows time for the magnet 130 to draw the paramagnetic beads from the suspension without the culture fluid exiting the chamber 175, as will be discussed in more detail below. In the embodiments shown in FIGS. 9 - 19, the capillary breaks are in the form of a rhombus. In other embodiments, other shapes that expand as they protrude away from the mixing chamber 175 are also conceivable as possibilities.

[0123] Furthermore, the use of two capillary breaks can help maintain the balance of 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 the filling lines are located on both sides of the mixing chamber 175 within the capillary breaks 178, 179. Thus, when the sample moves towards one side of the mixing chamber such that the filling line in one of the channels moves radially inwards towards the widened section of an individual capillary break (e.g., 178), the capillary force within that channel will be reduced. At the same time, the filling line in the channel on the opposite side of the mixing chamber 175 should move radially outwards and into the smaller cross-sectional area of the opposing capillary break (e.g., 179) where the capillary force 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 in the same radial position.

[0124] The capillary breaks 178, 179 may also serve as reservoirs and hold a portion of the sample during the initial stages of the mixing process. In some embodiments, the reagents may be stored in a stable dry form within the cartridge 150. For example, the reagents may be lyophilized prior to the analytical method of the present disclosure. In such cases, the mixing of plasma and the lyophilized reagent occurring within the mixing chamber 175 can 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 inwards and outwards from the sample as the mixing process occurs. Thus, the total volume occupied by the sample when first reaching the mixing chamber is larger than that during the later stages of the mixing process when the air is released. The capillary breaks 178, 179 can serve as reservoirs and hold a portion of the sample until the air is released and allowed to escape from the sample.

[0125] In some embodiments, the controller 140 may be configured to capture an image of the mixing chamber 175 or a portion thereof using a process quality control camera after transfer from the separation area 160. The controller 140 may further be configured to analyze the image and determine the fill level of the mixing chamber 175. Knowledge of the precise volume of the sample being analyzed can be useful in determining the accurate concentration of the target analyte. Thus, the controller 140 may be configured to proceed with the analysis in response to determining that the volume within the mixing chamber 175 exceeds a threshold. Further, the controller 140 may be configured to use the volume of the sample being analyzed to normalize the data resulting from the analysis.

[0126] In some embodiments, the volume of a 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 within the pre-mixing chamber channel 167 and the post-mixing chamber channel 173. Accordingly, the controller 140 may be configured to identify the meniscus lines of the sample within both channels from the image captured by the process quality control camera and calculate the volume based on the positions of these meniscus lines. Magnetic Transfer of Sample

[0127] In some embodiments, analyzer 100 may include one or more magnets 130(106) configured to move paramagnetic capture beads, as described in more detail below. As shown in the cross-sectional portion of analyzer 100 illustrated in FIG. 20, each may be coupled to movable stages 132, 133. Magnets 130, 131 may be positioned above or below the cartridge to allow for movement of paramagnetic capture beads 177 from outside the cartridge 150. The linear movement of magnet 130 in the radial and axial directions, in combination with the rotation of cartridge 150 by manifold positioning motor 110, allows magnet 130 to be positioned over any portion of cartridge 150 without the need to move magnet 130 circumferentially. Thus, in some embodiments, stage 133 may use a radial magnet stage 133 to move magnets 130, 131 forward and backward along the radial direction of cartridge 150, and toward and away from cartridge 150 in the axial direction, and may be enabled to introduce or remove the magnetic attraction of paramagnetic capture beads 177 using an axial magnet stage 132. In other embodiments, the movable stage may be operable to move in three dimensions such that it moves over any portion of cartridge 150 without the need for cartridge 150 to be rotated. In some embodiments, the magnet may be an electromagnet, while in other embodiments, the magnet may be a permanent magnet. Further, in some embodiments, the electromagnet can be activated using an AC or 126 current to further facilitate the manipulation of the paramagnetic beads.

[0128] Once the contents of the mixing chamber 175 are thoroughly mixed and the target analyte is attached to the paramagnetic capture beads 177 (as shown in FIG. 14) such that it is dispersed, the magnets 130, 131 may be introduced to move the paramagnetic capture beads 177 through the cartridge 150. When the magnet 130 is installed adjacent to the mixing chamber 175, the cartridge 150 may be rotated back and forth across the magnet 130 to collect the paramagnetic capture beads 177 within the bore, as shown in FIG. 16. In some embodiments, the controller 140 is configured to capture an image of the bead bore after the paramagnetic capture beads are collected using the magnet 130. Further, in some embodiments, the controller 140 is configured to measure the size of the paramagnetic bead bore and proceed with the analysis if the size of the bead bore is within a predetermined range. Otherwise, the controller 140 may identify an error and interrupt the analysis.

[0129] In some embodiments, after the paramagnetic capture beads 177 are immobilized by the magnet 130, the wash buffer 182 may be pumped through the mixing chamber 175 to remove plasma therefrom, as shown in FIG. 16. In some embodiments, during the purge of plasma from the mixing chamber 175, the bore of the paramagnetic capture beads 177 may be held in a particular location on the mixing chamber 175 to avoid dispersion of the bore. For example, the bore may be positioned in a corner of the mixing chamber 175 during the purge of plasma.

[0130] In some embodiments, the wash buffer 182 is delivered to the mixing chamber 175 via the wash chamber 181. The mixing chamber 175 and the wash chamber 181 may be radially offset from each other. Further, in some embodiments, the microfluidic channel between the mixing chamber 175 and the wash chamber 181 extends along a radial line and is 1 mm 2It may have a cross-sectional area. In other words, the microfluidic channel between the mixing chamber 175 and the washing chamber 181 does not extend along the circumferential direction. Therefore, the fluid in the mixing chamber is not forced to flow through the channel from the mixing chamber 175 to the washing chamber 181 during either the acceleration or deceleration of the cartridge 150 during the mixing step. Further, as described above, the post-mixing chamber channel 171506 may include a capillary break 178 that helps to retain the sample within the mixing chamber 175.

[0131] In some embodiments, the wash buffer 182 may be introduced into the cartridge 150 via the manifold 108 using the wash pump 116, and the elution buffer 185 may be pumped into the cartridge 150 via the manifold 108 using the elution pump 117 as shown in FIG. 8. In one embodiment, 150 microliters of the wash buffer 182 may be pumped into the cartridge 150 by the wash pump 116 through the wash supply port 111 of the manifold 108 and through the wash inlet port 154 so as to fill the wash chamber. Similarly, in another embodiment, for example, 25 microliters of the elution buffer 185 may be pumped into the cartridge 150 by the elution pump 117 through the elution supply port 112 of the manifold 108 and through the elution outlet port 155 so as to fill the elution chamber. As described above, the supply ports 111, 112 can be carefully positioned to engage with the individual inlet ports 154, 155 so as to form a sealed connection, respectively.

[0132] In some embodiments, the controller 140 may be configured to capture an image of at least a portion of the cleaning chamber 181 after it has been filled with the cleaning buffer 182. Further, the controller 140 may be configured to analyze the image of the cleaning chamber 181 to confirm the absence of air within the cleaning chamber 181 or to confirm that the volume of any air bubbles within the cleaning chamber 181 is below a predetermined threshold. For example, the controller 140 may be configured to calculate the shape of any air bubbles within the cleaning chamber 181 and to calculate the overall volume of air within the cleaning chamber 181. If the calculated volume of air exceeds the predetermined threshold, the controller may be configured to pump additional fluid therein or to interrupt the analysis. Similarly, the controller may be configured to continue the analysis if the calculated volume of air is below the predetermined threshold or is zero. A similar process can be used for the air within the elution chamber 184. Specifically, the controller 140 may be configured to analyze an image of the elution chamber 184 to confirm the absence of air within the elution chamber 184 or to confirm that the volume of any air bubbles within the elution chamber 184 is below a predetermined threshold. For example, the controller 140 may be configured to calculate the shape of any air bubbles within the elution chamber 184 and to calculate the overall volume of air within the elution chamber 184. If the calculated volume of air exceeds the predetermined threshold, the controller may be configured to interrupt the analysis. Similarly, the controller may be configured to continue the analysis if the calculated volume of air within the elution chamber 184 is below the predetermined threshold or is zero.

[0133] Analyzer 100 may also include a draw pump 118 coupled to cartridge 150 via manifold 108. In particular, cartridge 150 may include a wash outlet port 156 and an elution outlet port 157 connected to draw pump 118 via manifold 108. In particular, wash outlet port 156 may be coupled to wash draw port 113 of manifold 108, and elution outlet port 157 may be coupled to elution draw port 114 of manifold 108. The inlet and outlet ports may form two separate fluid lines through cartridge 150. In particular, wash inlet port 154 and wash outlet port 156 may form a wash line 183. Similarly, elution inlet port 155 and elution outlet port 157 may form an elution line 186 through cartridge 150. The operation of wash pump 116 and draw pump 118 may control the flow of wash buffer 182 through wash line 183, while the operation of elution pump 117 and draw pump 118 may control the flow of elution buffer 185 through elution line 186. Notably, in some embodiments, neither wash buffer 182 nor elution buffer 185 is actually drawn through the respective wash outlet port 156 and elution outlet port 157, but instead, only gas is removed through these outlet ports as a way to control the movement of the individual fluids through wash line 183 and elution line 186. For example, the waste chamber within the cartridge may be large enough that it is not necessary to remove fluid from the cartridge. Further, in some embodiments, wash line 183 and elution line 186 may each be coupled to a separate draw pump rather than both being coupled to a single draw pump. Additionally, a single draw pump may be connected to wash line 183 at one point and alternatively connected to only elution line 186 at a different point.

[0134] In some embodiments, the wash pump 116 and the draw pump 118 are carefully controlled to avoid the wash buffer 182 from entering the detection chamber 184. Further, in some embodiments, the analyzer operates the wash pump 116 and the draw pump 118 to maintain a bubble mass in the connection passage 187 as the wash fluid is drawn into the wash chamber, as shown in FIG. 16. For example, in some embodiments, the controller operates the wash pump 116 and the draw pump 118 at a similar flow rate to transfer the wash buffer along the wash line and avoid the wash fluid from drifting outside the wash line. Similarly, in some embodiments, the elution pump 117 and the draw pump 118 are controlled to avoid the elution buffer 185 from entering the wash chamber 181.

[0135] Further, in some embodiments, the elution buffer and the wash buffer are introduced into the cartridge simultaneously and controlled to avoid secondary contamination. For example, in some embodiments, as the wash buffer 182 and the elution buffer 185 are introduced into the individual wash line 183 and elution line 186, respectively, the wash pump 116, the elution pump 117, and the draw pump 118 are controlled to form air bubbles in the connection passage 187 connecting the wash line 183 and the elution line 186. In particular, in some embodiments, the present connection passage 187 extends between the wash chamber 181 and the detection chamber 184. The air bubbles prevent the mixing of the wash buffer 182 and the elution buffer 185 and form a dam that serves as an "air spring". Further, the air bubbles can be visually monitored to verify that the fluids are not mixed, as will be explained in more detail below. In some embodiments, the present air bubbles are maintained until the target analyte is moved into the detection chamber 184.

[0136] The use of a mass of air or a bubble within the connection passageway obviates the need for a valve to control the flow between the wash chamber and the elution chamber. In some embodiments, the controller 140 may be configured to capture an image of the connection passageway 187 between the wash chamber 181 and the elution chamber 184 to confirm the presence of a mass of air therein. After analyzing the image of the connection passageway 187, if the controller identifies the presence of a mass of air within the connection passageway 187, the controller 140 may be configured to proceed with the analysis. On the other hand, if the controller 140 does not identify a mass of air within the connection passageway 187, the controller may be configured to interrupt the analysis of the present disclosure.

[0137] In some embodiments, at least one of the wash line or the elution line includes an air trap. For example, in some embodiments, the depth of the wash line 183 is increased within the area between the wash inlet port 154 and the wash chamber. This increase in the depth of the wash line 183 provides a space for any air pumped into the wash line to be trapped. For example, in some embodiments, the analyzer holds the cartridge horizontally such that the depth direction is parallel to gravity. Thus, any air within the wash line 183 will float upwardly and into the air trap created by the increased depth of this section of the wash line. The elution line 186 may have a similar air trap in the vicinity of the elution outlet port 155.

[0138] Once the paramagnetic capture beads 177 are collected in the bolus within the mixing chamber 175 as shown in FIG. 15, the magnet 130 may be moved by the movable stage 133 in conjunction with the rotation of the cartridge 150 to convey the bolus of paramagnetic capture beads 177 into the wash chamber 181. In some embodiments, the controller 140 may be configured to capture at least a partial image of the wash chamber 181 after the bead bolus of the paramagnetic capture beads 171 has been transferred to the wash chamber 181. Further, in some embodiments, the controller 140 may measure the size of the paramagnetic bead bolus within the wash chamber 181 and, if the size of the bead bolus within the wash chamber 181 is within a predetermined range, be configured to proceed with the analysis. Otherwise, the controller 140 may identify an error and interrupt the analysis.

[0139] Once the paramagnetic capture beads 177 are disposed within the wash chamber 181, the cartridge 150 may be rotated back and forth to effectively wash the paramagnetic capture beads 177 and remove all contaminants from the sample, except for the target analyte, detection label, and any controls used in the system, as schematically shown in FIG. 17. In some embodiments, the spent wash buffer may be swept out of the wash chamber 181 and a fresh volume of wash buffer 182 may be added to the wash chamber 181 before repeating the wash step. The wash step may be performed several times, for example, 3 times or more.

[0140] In some embodiments, a second magnet 131 may be introduced during the washing step to disperse and re-aggregate the paramagnetic capture beads 177 during a series of steps of the washing operation. In particular, the magnet 130 and the second magnet 131 may be disposed on opposite sides of the washing chamber 181 to disperse and re-aggregate the paramagnetic capture beads 177 as they move across the washing chamber 181. Diffusing the paramagnetic capture beads 177 allows them to be more efficiently washed by the wash buffer when the beads are held together within the bolus. Thus, the time and number of cycles required for the washing step can be reduced compared to conventional washing methods.

[0141] Figures 21 and 22 illustrate two exemplary embodiments of the cleaning operation according to the present invention. Figure 21 illustrates the cleaning operation in which two magnets 130, 131 are moved relative to the cleaning chamber 181 in a sawtooth pattern. In particular, Figure 21 illustrates five discrete locations P1 - P5 that the first magnet 130 and the second magnet 131 occupy during the sawtooth cleaning operation. At position P1, the second magnet 131 is remote from the cleaning chamber 181 while the first magnet 130 is adjacent to the cleaning chamber 181, which causes a bolus to be formed adjacent to the first magnet 130 in the paramagnetic capture beads. The magnets 130, 131 are then axially moved such that the second magnet 131 moves closer to the cleaning chamber 181 while the first magnet 130 moves away from the cleaning chamber 181. In concert with this movement, the cartridge 150 may also be rotated such that the magnets 130, 131 are also laterally repositioned relative to the cleaning chamber 181. As the first magnet 130 moves away from the paramagnetic capture beads 177, the bolus is dispersed into the cleaning solution such that unwanted constituents of the plasma can be separated and washed from the paramagnetic capture beads 177. The dispersion of the paramagnetic capture beads 177 is illustrated in Figure 21 between positions P1 and P2. As the second magnet 131 approaches the cleaning chamber 181, the paramagnetic capture beads 177 are drawn out of suspension and drawn back into a tight bolus. The dispersion and re - agglomeration steps can then be repeated in the opposite direction as the magnets 130, 131 move from position P2 to position P3. Similarly, the process can be continued in a sawtooth pattern for several additional steps.

[0142] Figure 22 illustrates another embodiment of the cleaning operation in which two magnets 130, 131 are moved relative to the cleaning chamber 181 in a square wave pattern. In particular, Figure 22 illustrates nine discrete locations P1 - P9 that the first magnet 130 and the second magnet 131 occupy during the sawtooth cleaning operation. Again, at position P1, the second magnet 131 is remote from the cleaning chamber 181 while the first magnet 130 is adjacent to the cleaning chamber 181, which causes a bolus to form adjacent to the second magnet 131 in the paramagnetic capture beads. The cartridge 150 is then rotated so that the magnets 130, 131 move relative to the cleaning chamber 181. Advantageously, the cartridge 150 may be rotated at a speed sufficient to disperse the paramagnetic capture beads 177 along the surface of the cleaning chamber 181, thereby dispersing the paramagnetic capture beads along the surface of the chamber 181 within the cleaning solution. The magnets 130, 131 are then moved to position P3 such that the second magnet 131 moves closer to the cleaning chamber 181 while the first magnet 130 moves away from the cleaning chamber 181. Again, as the first magnet 130 moves away from the paramagnetic capture beads 177, the bolus is dispersed within the cleaning solution such that unwanted constituents of the plasma can be separated and washed from the paramagnetic capture beads 177. Similarly, as the second magnet 131 approaches the cleaning chamber 181 as shown at position P3, the paramagnetic capture beads 177 are drawn out of the suspension against the cleaning chamber wall.

[0143] The embodiments of the cleaning operation shown in Figures 21 and 22 involve re - aggregating the paramagnetic capture beads into a tight bolus, although in other embodiments, the paramagnetic capture beads may be directed through the cleaning chamber without being tightly coalesced into a bolus during the operation. For example, during the steps of the operation, the beads may be relatively dispersed within the cleaning fluid but may remain moved back and forth along the length of the cleaning chamber by the magnets.

[0144] As described above, in some embodiments, magnet 130 and second magnet 131 are positioned on opposite sides of cartridge 150, e.g., above and below cartridge 150. In other embodiments, magnets 130, 131 are on the same side of cartridge 150 but are disposed on the side opposite the wash chamber 181 in the radial direction. Further, in some embodiments, magnets 130, 131 diffuse paramagnetic capture beads 177 along the length of wash chamber 181. Further, in some embodiments, the distance between first magnet 130 and second magnet 131 is varied using movable stage 132 during the wash step. This relative movement of magnets 130, 131 can promote the perturbation of the bolus of paramagnetic capture beads 177 and improve the washing operation.

[0145] After the washing operation, the paramagnetic capture beads 177 may be recollected using the first magnet 130 and moved through connection passage 187 into the detection chamber 184 filled with elution buffer 185 as shown in FIG. 19. While holding the paramagnetic capture beads 177 using one or more of magnets 130, 131, cartridge 150 may be rotated back and forth to pass the paramagnetic capture beads 177 through detection chamber 184 and elution buffer 185, which removes the 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 conjugate suspension in the elution buffer 185 within detection chamber 184.

[0146] To improve the elution of the target analyte and the label, a magnetic elution operation similar to the washing operation described above may be used. For example, magnets 130, 131 may be moved relative to detection chamber 184 in a particular pattern such as those shown in FIGS. 21 and 22. Controlling the paramagnetic beads in a controlled manner similar to that of the washing operation improves the magnetic elution operation.

[0147] After the dissolution process is performed, the paramagnetic capture beads 177 may be moved outside the detection chamber 184 or to one end of the detection chamber 184 so as to avoid interfering with the optical system 120. The optical system 120 of the analyzer 100 may then be activated to analyze the solution within the detection chamber 184 so as to determine the presence or concentration of the target analyte within the volume of fluid within the detection chamber 184 as described above.

[0148] 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 second electromagnetic radiation source 128 and the second detector 129 of the analyzer may be used to determine the presence of a second target analyte within the sample. In other embodiments, the second electromagnetic radiation source 128 and the second detector 129 may be used to measure the concentration of a control analyte within the cartridge 150. For example, the cartridge 150 may contain a precise known quantity of the control analyte. Thus, the measured concentration of the control analyte may be used as a comparison reference for the target analyte. The measured concentration may then be used to adjust the detected concentration of the target analyte.

[0149] 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 may 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 only detecting 95% of the target analyte within the sample and may accordingly adjust the calculated concentration.

[0150] In some embodiments, the electromagnetic radiation from the first electromagnetic radiation source 121 and the second electromagnetic radiation source 128 is directed to the cartridge using the same objective lens. In fact, in some embodiments, the electromagnetic radiation from the two sources is directed to the same interrogation space. In some embodiments, the first electromagnetic radiation source 121 and the second electromagnetic radiation source 128 emit electromagnetic radiation of different wavelengths, for example, different colors.

[0151] The present disclosure provides systems and methods for the highly sensitive detection and quantification of one or more target analytes, such as markers related to biological states. Singleplex and multiplex assays

[0152] 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 for the more rapid detection and analysis of multiple target analytes using a reduced sample volume and a reduced reagent volume than may be required to perform similar analysis of those target analytes via singleplex assays. Additionally, the multiplexing systems and methods described herein can enable the analysis of a sample containing a target analyte to be compared to a control assay of known concentration.

[0153] To detect and analyze multiple different types of target analytes within a sample, a multiplexing analyzer system can distinguish one type of target analyte from another. This can be accomplished, in part, by labeling different target analytes with different labels having mutually different excitation wavelength bands and / or emission wavelength bands. In some implementations, the different labels have excitation wavelength bands and / or emission wavelength bands with relatively little overlap or no overlap. In other implementations, there may be some degree of overlap between the excitation wavelength bands and / or emission wavelength bands of the labels. Multiplexing can also be achieved by implementing more than one fluid circuit on the same cartridge, where each fluid circuit is spatially distinct and carries reagents for different target analytes. Using different fluid 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

[0154] In an optical system, the electromagnetic radiation source 121 may be set such that the wavelength of the electromagnetic radiation is sufficient to excite a fluorescent label attached to a target analyte. In some embodiments, the electromagnetic radiation source 121 is a laser that emits light within 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 as used in the methods and compositions of the present disclosure can be used without departing from the scope of the present disclosure. The power setting for the laser is generally from 1 mW to 100 mW. However, one of ordinary skill in the art will understand that the laser power can be any setting to achieve an optimal signal-to-noise ratio for the measurement. To do so, the laser power should be set to achieve as many excitation emission cycles as possible during the residence time of the label within the interrogation space. The detector bin time should also be set accordingly. A bin time that is 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. A laser power setting that is too low or too high, or a bin time setting that is too long, will not result in the highest possible signal-to-noise ratio.

[0155] As the interrogation space within the analyzer 100 passes over the labeled target analyte, photons emitted by the fluorescent particles are registered by the detector 122 with a time delay indicative of the time for the interrogation space to pass over the labeled particles. The photon intensity is recorded by the detector 122, the sampling time is divided into bins, and the bins are uniform arbitrary time intervals with a freely selectable time channel width. The number of signals contained in each bin is evaluated. One or more than one of several statistical analysis methods are used to determine when a label or particle is present or when a bin segment contains an artifact. Bin segments containing artifacts are discarded while single bins or bin segments containing labels are counted. The number of labels counted indicates the number of target analytes present in the sample. Query volume

[0156] The query volume can be considered the effective volume of the sample in which the target analyte can be detected when present. Since there are various methods for calculating the query volume of a sample, the simplest way to determine the effective volume (V) of the query volume is to calculate the effective cross-section of the detection volume. Since the detection volume is typically swept through the sample by translating the detection volume through a stationary sample, the volume is typically the result of the cross-sectional area of the detection volume being swept through a distance during the measurement time. As discussed above, the lateral extent of the cross-sectional area of the query volume (perpendicular to the direction of motion of the laser with respect to the sample and perpendicular to the direction of propagation of the laser light) is limited by the numerical aperture at which the laser source is imaged within the sample space. The longitudinal size of the query volume (along the direction of propagation of the laser) is determined by the size of the confocal stop selected. If the sample concentration (C) is known and the number of molecules (N) detected during a certain time period is known, the sample volume consists of the number of molecules detected divided by the concentration of the sample, i.e., V = N / C (where the sample concentration has units of molecules per unit volume).

[0157] For example, in some embodiments of the systems described herein, all detected photons are counted and added in 100 microsecond intervals (photon counting bins). When the molecule of interest is present in a 100 microsecond interval, the count of detected photons is typically significantly higher than the background. Thus, the distance the detection volume has moved relative to the sample is the volume sampled within a single interval, i.e., the appropriate distance to use to calculate the interrogation volume. In this example, if the sample is analyzed over 60 seconds, effectively 600,000 intervals are scanned. When the effective volume is divided by the number of intervals, the resulting volume is essentially the volume of a single interval, i.e., the interrogation volume. Mathematically, the volume of a single interval, i.e., the interrogation volume (Vs), is equal to the number of detected molecules (N) divided by the concentration of the sample (C) multiplied by the number of interval bins (n) (where n represents the number of interval bins during the time the number of molecules N was counted, C·n). For illustrative purposes only, consider that a known standard at 1 femtomolar concentration is run through 600,000 intervals and 20 molecules of the standard are detected. Thus, the interrogation volume Vs is N / (C·n), or 20 / (602.214·6E5), or 55.351μm 3 equal. Thus, in this example, the interrogation spatial volume, which is the effective volume of a single sample corresponding to one photon counting bin, is 55.351μm 3 is. Detector

[0158] In some embodiments, the 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 the photon burst from the fluorescent moiety and convert the amplitude and duration of the photon burst into an electrical signal. Detection devices such as CCD cameras, video input module cameras, and streak cameras can be used to generate an image with a continuous signal. 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

[0159] The apparatuses, kits, and methods of the present disclosure can be used for the sensitive detection and determination of the concentration of several different types of target analytes, such as markers of biological states.

[0160] Examples of molecules or "analytes" that can be detected using the analyzers and related methods of the present disclosure include biopolymers such as proteins, nucleic acids, carbohydrates, and small molecules, both organic and inorganic. In particular, the apparatuses, kits, and methods described herein are useful in the detection of target analytes of proteins and small molecules in biological samples and in the determination of the concentration of such molecules in the sample.

[0161] The molecules detected by the present system and method can be in a state or can be part of a complex, for example, an antibody-antigen complex, or more generally, a protein-protein complex, such as a complex of troponin or a complex of prostate specific antigen (PSA).

[0162] 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 the determination of methods of diagnosis, prognosis, and / or treatment.

[0163] A marker can be any composition and / or molecule, or a complex of a composition and / or molecule, that is associated with, for example, the biological state of an organism (such as a symptom like a diseased or non-diseased state). A marker can be, for example, a small molecule, a polypeptide, a nucleic acid such as DNA and RNA, a lipid such as a phospholipid or a micelle, a cellular component such as a mitochondrion or a chloroplast. The markers contemplated by the present disclosure can be either 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. Although the system of the present disclosure has high potential use in determining the biological state of an organism, it is possible to observe those markers, for example, polypeptides, that are present only at low concentrations, such as those “leached” from diseased tissue. Other highly potentially useful markers or polypeptides can be those associated with a disease, such as those that occur in a tumor host environment. Any suitable marker that provides information regarding a biological state can be used in the methods and compositions of the present disclosure. “Marker,” as the 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.

[0164] Biological states include, but are not limited to, phenotypic states, symptoms affecting an organism, developmental states, age, health, pathology, disease detection, processes, or staging, infections, toxicities, or responses to chemical, environmental, or drug factors (such as drug response phenotype assays, drug toxicity phenotype assays, or drug efficacy phenotype assays).

[0165] As used herein, the term "organism" refers to any living thing consisting of at least one cell. The organism can be as simple as a unicellular organism or as complex as a mammal. The organisms of the present disclosure are preferably mammals. Such mammals can be, for example, humans, or primates (e.g., monkeys, chimpanzees, etc.), domesticated animals (e.g., dogs, cats, horses, etc.), livestock (e.g., goats, sheep, pigs, cows, etc.), or laboratory animals (e.g., mice, rats, etc.). Preferably, the organism is a human. Label

[0166] In some embodiments, the present disclosure provides methods and compositions that include labels for the extremely sensitive detection and quantification of molecules, such as markers.

[0167] Many strategies can be used to label target analytes and enable their detection or discrimination in a mixture of particles. The label can be attached by any known means, including methods that utilize non-specific or specific interactions between the label and the target analyte. The label can provide a detectable signal or can affect the mobility of the particles in an electric field. Labeling can be accomplished directly or through a binding partner.

[0168] In some embodiments, the label consists of a binding partner to the molecule of interest, and the binding partner is attached to a fluorescent moiety. The compositions and methods of the present disclosure can use extremely 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).

[0169] 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 , ALEXA FLUOR® 700 Brilliant Violet such as Brilliant Violet 605 TM molecules (BD Biosciences), and ATTO TM dyes such as ATTO 532 TM (ATTO TECH GmbH). In some embodiments, the dye molecule is an ALEXA FLUOR® 647 dye molecule. Binding partner

[0170] In some embodiments, the binding partner consists of an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0171] The antibody can be specific for any suitable marker. In some embodiments, the antibody is specific for a marker selected from the group consisting of cytokines, growth factors, tumor markers, markers of inflammation, endocrine markers, autoimmune markers, thyroid markers, cardiovascular markers, diabetes markers, markers of infectious diseases, neurological markers, respiratory markers, gastrointestinal markers, musculoskeletal markers, skin diseases, and metabolic markers.

[0172] Any suitable binding partner with the necessary specificity can be used with respect to the molecule to be detected, for example, in the form of a marker. If the molecule, for example, the marker has several different forms, various specificities of the binding partner are conceivable. Suitable binding partners are known in the art and include antibodies, aptamers, lectins, and receptors. A useful and versatile type of binding partner is the antibody.

[0173] A capture binding partner and a detection binding partner pair, for example, a capture and detection antibody pair, can be used in 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 the capture partner immobilized on a solid support, and the other binding partner is typically the 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.

[0174] In some embodiments, it is useful to use antibodies that cross-react with various species as either the capture antibody, the detection antibody, or both. Such embodiments include, for example, the measurement of drug toxicity by determining the release of cardiac troponin into the blood as a marker of heart injury. Cross-reactive antibodies allow toxicity studies to be performed in one species, e.g., a non-human species, and the direct transfer of results to studies or clinical observations in another species, e.g., humans, using the same antibody or antibody pair in the assay reagent, thus reducing variability between assays. Thus, in some embodiments, one or more of the antibodies for use as a binding partner to a marker of the molecule of interest, e.g., a cardiac troponin such as cardiac troponin I, can be a cross-reactive antibody. In some embodiments, the antibody cross-reacts with markers, e.g., cardiac troponin, 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 troponin, from the entire group consisting of human, monkey, dog, and mouse.

[0175] The detailed description of the invention describes various features and functions of the disclosed stem, device, and method with reference to the accompanying drawings. In the drawings, like reference numerals typically identify like components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, the drawings, and the claims are not intended to be limiting. Other embodiments can be utilized and other changes can 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 drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly considered herein.

[0176] Although various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, and the true scope is defined by the following claims. (Embodiment) (Embodiment 1) An analyzer system for measuring the concentration of a target analyte in a sample, a motor, a dock coupled to the motor to be rotated by the operation of the motor, a cartridge held within the dock, receiving the sample, isolating the target analyte of the sample, and including a fluid system configured to collect a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluid system comprising: an inlet chamber, a mixing chamber downstream of the inlet chamber and configured to mix at least a portion of the sample to bind the target analyte to the first label, a wash chamber downstream of the mixing chamber and connected to the mixing chamber by a channel, the wash chamber being radially offset from the mixing chamber so as to prevent flow of the sample into the wash chamber during the mixing process carried out in the mixing chamber, a cartridge including, a first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within a detection chamber of the cartridge, a first detector configured to detect electromagnetic radiation emitted within the interrogation space by the first label when the first 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. (Embodiment 2) The channel extending from the mixing chamber to the cleaning chamber includes a capillary break, and the capillary break has a cross-sectional area that expands in a direction leading away from the mixing chamber, for the analyzer system according to Embodiment 1. (Embodiment 3) The fluid system of the cartridge further includes a separation area disposed between the inlet chamber and the mixing chamber, and the separation area includes a radially inner separation chamber and a radially outer separation chamber connected by a reduced diameter portion, for the analyzer system according to Embodiment 1. (Embodiment 4) A siphon extends from the radially inner separation chamber to the mixing chamber, for the analyzer system according to Embodiment 3. (Embodiment 5) A siphon vent extends from the siphon in the vicinity of the apex of the siphon, for the analyzer system according to Embodiment 4. (Embodiment 6) An analyzer system for measuring the concentration of a target analyte in a sample, a motor, a dock coupled to the motor so as to be rotated by the operation of the motor, a cartridge held within the dock, receiving a sample, isolating the target analyte of the sample, and including a fluid system configured to collect an amount of a first label proportional to the amount of the target analyte in the sample, the fluid system including an inlet chamber, a mixing chamber downstream of the inlet chamber and configured to mix at least a portion of the sample so as to bind the target analyte to the first label, including a cartridge, a processor, and in response to execution by the processor, rotating the motor to rotate the cartridge, intermittently accelerating and decelerating the rotation of a centrifuge to recursively move a mixing ball in the mixing chamber back and forth through the mixing chamber, A controller including a non-transitory computer-readable medium having program instructions stored thereon that cause an implementation of a set of operations, and An analyzer system comprising. (Embodiment 7) The analyzer according to embodiment 6, further comprising a lyophilized reagent disposed in the mixing chamber. (Embodiment 8) The analyzer according to embodiment 6, wherein the mixing ball is non-magnetic. (Embodiment 9) An analyzer system for measuring the concentration of a target analyte in a sample, A motor, A dock coupled to the motor to be rotated by the operation of the motor, A cartridge including a fluid system held in the dock, configured to receive a sample, isolate the target analyte of the sample, and collect an amount of a first label proportional to the amount of the target analyte in the sample, wherein the fluid system A fluid line including a fluid inlet port configured to receive fluid, a first chamber, and a fluid outlet port, A detection chamber, A connection passage between the first chamber and the detection chamber, Including a cartridge, A processor, in response to execution by the processor, Including the step of pumping fluid into the first chamber along the fluid line while maintaining a mass of air in the connection passage, A controller including a non-transitory computer-readable medium having program instructions stored thereon that cause an implementation of a set of operations, and An analyzer system comprising. (Embodiment 10) The analyzer system according to embodiment 9, wherein the fluid line is a cleaning line. (Embodiment 11) The analyzer system according to embodiment 9, wherein the connection passage extends directly from the first chamber. (Embodiment 12) The connection path is the analyzer system according to Embodiment 9 that extends directly to the detection chamber. (Embodiment 13) An analyzer system for measuring the concentration of a target analyte in a sample, a motor, a dock coupled to the motor so as to be rotated by the operation of the motor, a cartridge including a fluid system that is held in the dock, receives a sample, isolates the target analyte in the sample, and collects a quantity of a first label proportional to the quantity of the target analyte in the sample, a fluid line including a fluid inlet port configured to receive fluid, a first chamber, and a fluid outlet port, a detection chamber, a connection path between the first chamber and the detection chamber, and a cartridge, a plurality of paramagnetic beads configured to provide a substrate for the target analyte using the first chamber, a first magnet disposed on a movable stage, a processor, and in response to execution by the processor, a step of facilitating relative movement of the first magnet and the cartridge to draw the paramagnetic beads out of the suspension and into the bolus, the relative movement being facilitated by at least one of moving the first magnet across a first surface or rotating the cartridge, a controller including a non-transitory computer-readable medium having stored thereon program instructions that cause execution of a set of operations, and an analyzer system comprising the same. (Embodiment 14) The controller is further configured to facilitate relative movement of the first magnet and the cartridge to transfer the paramagnetic beads and the target analyte from the first chamber to the second chamber, in the analyzer system according to Embodiment 13. (Embodiment 15) The analyzer system according to Embodiment 14, further comprising a second magnet. (Embodiment 16) The controller is further configured to perform a cleaning operation, the cleaning operation comprising: moving the first magnet away from the first surface of the cartridge so that the paramagnetic beads are dispersed within the second chamber; moving the second magnet towards the second surface of the cartridge so that the paramagnetic beads gather in the vicinity of the second magnet; moving the second magnet away from the second surface of the cartridge so that the paramagnetic beads are dispersed within the second chamber; moving the first magnet towards the first surface of the cartridge so that the paramagnetic beads gather in the vicinity of the first magnet; The analyzer system according to Embodiment 15, comprising: (Embodiment 17) An analyzer system for measuring the concentration of a target analyte in a sample, comprising: a motor; a dock coupled to the motor and configured to be rotated by the operation of the motor; a cartridge held within the dock, configured to receive a sample, isolate the 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 cartridge including a fluid system; a quality control camera configured to capture an image of the cartridge during an analysis operation; a first electromagnetic radiation source configured to provide electromagnetic radiation and form an interrogation space within a detection chamber of the cartridge; a first detector configured to detect electromagnetic radiation emitted within the interrogation space by the first label when the first label is present within the interrogation space; analyzing an image captured by the quality control camera and, in response to the analysis of the image, continuing the analysis operation; identifying the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector. A controller configured as such, and An analyzer system comprising (Embodiment 18) The analyzer system according to Embodiment 17, wherein the controller is configured to further rotate the cartridge in response to the analysis of the image. (Embodiment 19) The analyzer system according to any one of Embodiments 1-18, including a centrifuge, wherein the motor is coupled to the cartridge and configured to rotate the cartridge at a speed of at least 100 rpm to separate components of the sample. (Embodiment 20) The analyzer system according to any one of Embodiments 1-19, further comprising a manifold including a plurality of ports and configured to couple each of the plurality of ports to an individual corresponding port of the cartridge. (Embodiment 21) The analyzer system according to any one of Embodiments 1-18, including a positioning motor, wherein the motor is coupled to the cartridge and configured to pivot the cartridge to align the detection zone of the cartridge with electromagnetic radiation from a first electromagnetic radiation source. (Embodiment 22) The analyzer system according to any one of Embodiments 1-21, further comprising an optical system configured to direct electromagnetic radiation from a first electromagnetic radiation source into the detection chamber of the cartridge and to direct electromagnetic radiation emitted by the label to a detector. (Embodiment 23) The analyzer system according to Embodiment 22, wherein the optical system is a confocal system. (Embodiment 24) The analyzer system according to any one of Embodiments 1-23, wherein all components of the analyzer system are arranged in a common housing, and the dimensions of the common housing are on the order of 1 meter in any direction in any direction. (Embodiment 25) The analyzer system according to any one of Embodiments 1-24, comprising a network interface for receiving control information from a user and outputting analysis data to the user. (Embodiment 26) The analyzer system according to any one of Embodiments 1-25, wherein the cartridge is planar and the chambers within the cartridge are located in a single plane. (Embodiment 27) The analyzer system according to any one of Embodiments 1-26, wherein the cartridge is a disk and the chambers of the cartridge are circumferentially positioned around the disk. (Embodiment 28) The analyzer system according to any one of Embodiments 1-27, wherein the cartridge has no valve. (Embodiment 29) The analyzer system according to any one of Embodiments 1-28, wherein the cartridge is configured to receive a sample in the range of 50 microliters to 1 milliliter. (Embodiment 30) The analyzer system according to any one of Embodiments 1-29, wherein the cartridge contains a reagent stored therein. (Embodiment 31) A cartridge for preparing and containing a sample for measuring the concentration of a target analyte, a fluid system configured to receive a sample, isolate the target analyte of the sample, and collect an amount of a first label proportional to the amount of the target analyte in the sample, a mixing chamber, a first channel in communication with the mixing chamber, a second channel in communication with the mixing chamber, comprising a fluid system, a mixing ball disposed within the mixing chamber, the mixing ball being larger than the first channel and the second channel, a cartridge comprising. (Embodiment 30) The cartridge is a base, a body disposed over a base, and a cover disposed over the body, and comprising, the body including an open path extending therethrough that defines a plurality of chambers of the cartridge. The cartridge according to Embodiment 25. (Embodiment 31) The cartridge is planar, and the chambers within the cartridge are located in a single plane. The cartridge according to Embodiment 30. (Embodiment 32) The cartridge is a disk, and the chambers of the cartridge are circumferentially positioned around the disk. The analyzer system according to any one of Embodiments 30 or 31. (Embodiment 33) The cartridge is valve - less. The analyzer system according to any one of Embodiments 30 - 32. (Embodiment 34) The cartridge is configured to receive a sample in the range of 50 microliters to 1 milliliter. The analyzer system according to any one of Embodiments 30 - 33. (Embodiment 35) The cartridge contains a reagent stored therein. The analyzer system according to any one of Embodiments 30 - 34. (Embodiment 36) A method for detecting the presence of a target analyte in a sample, comprising: introducing the sample into the cartridge, the cartridge including a fluid system for isolating the target analyte of the sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluid system comprising: a first chamber, and a second chamber, and a channel extending from the first chamber to the second chamber, and comprising; the step of binding the target analyte to a substrate consisting of paramagnetic beads; positioning a first magnet near a first surface of the cartridge and adjacent to the first chamber; Promoting relative movement of a first magnet and a cartridge so as to draw paramagnetic beads out of a suspension and into a bore, the relative movement being promoted by at least one of moving the first magnet across a first surface or rotating the cartridge; Directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the cartridge; Receiving, within a first detector, electromagnetic radiation emitted within the interrogation space by a first label if the first label is present within the interrogation space; Using a controller to identify the presence of a target analyte within a sample based on electromagnetic radiation detected by the first detector; A method comprising the above steps. (Embodiment 37) A method of mixing liquids within a cartridge in the form of a flat disc, comprising: Introducing a liquid into a mixing chamber of the cartridge through a channel extending radially inwards from a mixing chamber, the mixing chamber containing mixing balls therein; Rotating the cartridge in a first circumferential direction to press the liquid radially outwards and retain the liquid within the mixing chamber; Intermittently accelerating and decelerating the rotation of the cartridge to move the mixing balls back and forth recursively through the mixing chamber; A method comprising the above steps. (Embodiment 38) The mixing chamber is provided with a lyophilized reagent prior to introduction of the liquid; Mixing resulting from movement of the mixing balls through the mixing chamber releases gas from the lyophilized reagent into the liquid; The method according to Embodiment 37. (Embodiment 39) The method according to Embodiment 38, wherein the released gas moves radially outwards from the mixing chamber. (Embodiment 40) The mixed ball is non-magnetic, the method according to any one of Embodiments 37-39. (Embodiment 41) A method for detecting the presence of a target analyte in a sample, comprising the step of introducing the sample into a cartridge, the cartridge comprising a fluid system for isolating the target analyte of the sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluid system comprising a fluid line comprising a fluid inlet port configured to receive fluid, a first chamber, and a fluid outlet port, a detection chamber, a connection passage between the first chamber and the detection chamber, and comprising the step of, transferring the target analyte to the first chamber, pumping fluid into the first chamber along the fluid line while maintaining a mass of air in the connection passage, transferring the target analyte to the detection chamber, directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the detection chamber of the cartridge, when the first label is present within the interrogation space, receiving, within a first detector, electromagnetic radiation emitted within the interrogation space by the first label, using a controller to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector, and comprising the method. (Embodiment 42) The connection passage extends directly from the first chamber, the method according to Embodiment 41. (Embodiment 43) The connection passage extends directly to the detection chamber, the method according to Embodiment 41 or 42. (Embodiment 44) further comprising the step of transferring the target analyte to the detection chamber, the target analyte being conveyed into the detection chamber by paramagnetic beads transported using a magnet, the method according to any one of Embodiments 41-43. (Embodiment 45) The detection chamber is disposed within an elution line and includes an elution inlet port and an elution outlet port, The method further includes pumping an elution fluid into the elution line to release the target analyte from the paramagnetic beads. The method according to Embodiment 44. (Embodiment 46) The fluid is pumped along the fluid line by feeding the fluid into the fluid line at a fluid inlet port and withdrawing the fluid from the fluid line at a fluid outlet port, according to any of Embodiments 41-45. (Embodiment 47) A method for detecting the presence of a target analyte in a sample, introducing the sample into a cartridge, the cartridge including a fluid system for isolating the target analyte of the sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluid system including a first chamber, a second chamber, a channel extending from the first chamber to the second chamber, comprising; binding the target analyte to a substrate comprising paramagnetic beads; positioning a first magnet near a first surface of the cartridge and adjacent to the first chamber; facilitating relative movement of the first magnet and the cartridge to draw the paramagnetic beads out of suspension and into a bolus, the relative movement being facilitated by at least one of moving the first magnet across the first surface or rotating the cartridge; directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the cartridge; receiving, in a first detector, electromagnetic radiation emitted within the interrogation space by the first label if the first label is present within the interrogation space; Using a controller, identifying the presence of a target analyte in a sample based on electromagnetic radiation detected by a first detector; A method comprising. (Embodiment 48) The method according to embodiment 47, further comprising promoting relative movement of a first magnet and a cartridge so as to transfer paramagnetic beads and a target analyte from a first chamber to a second chamber. (Embodiment 49) The method according to embodiment 47 or 48, further comprising performing a washing operation in the second chamber so as to isolate the target analyte from other components of the sample. (Embodiment 50) The washing operation comprises: Moving the first magnet away from a first surface of the cartridge so that the paramagnetic beads are dispersed within the second chamber; Moving the second magnet towards a second surface of the cartridge so that the paramagnetic beads gather in the vicinity of the second magnet; Moving the second magnet away from the second surface of the cartridge so that the paramagnetic beads are dispersed within the second chamber; Moving the first magnet towards the first surface of the cartridge so that the paramagnetic beads gather in the vicinity of the first magnet; The method according to embodiment 49, comprising. (Embodiment 51) A method for detecting the presence of a target analyte in a sample, comprising: Introducing the sample into a cartridge, the cartridge comprising a fluid system for isolating the target analyte of the sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluid system comprising: An inlet chamber; A separation area connected to the inlet chamber, the separation area comprising an inner separation chamber and an outer separation chamber; A detection chamber downstream of the separation area; Comprising. Transferring a blood sample from the inlet chamber to the separation area; Rotating the cartridge using a centrifuge to move the red blood cells of the blood sample towards the outer separation chamber and the plasma towards the inner separation chamber; Capturing an image of the blood sample within the separation area using a camera; Analyzing the image of the blood sample within the separation area using a controller to determine the position of the red blood cells within the separation area; Transferring plasma from the inner separation chamber to the mixing chamber; Isolating the target analyte from the plasma; Transferring the target analyte to the detection chamber; Directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the detection chamber of the cartridge; Receiving, within a first detector, electromagnetic radiation emitted within the interrogation space by a first label if the first label is present within the interrogation space; Identifying the presence of the target analyte within the sample based on the electromagnetic radiation detected by the first detector using a controller; A method comprising. (Embodiment 52) The method according to embodiment 51, further comprising rotating the cartridge further using a centrifuge to move the red blood cells further towards the outer separation chamber in response to the determined position of the red blood cells. (Embodiment 53) The method according to embodiment 51 or 52, further comprising analyzing an image of the blood sample using a controller to determine the transparency of the plasma within the inner separation chamber after rotating the cartridge using a centrifuge, wherein the step of transferring a portion of the sample from the separation area to the mixing chamber is performed in response to a plasma transparency exceeding a predetermined value. (Embodiment 54) Capturing an image of the plasma within the mixing chamber; Using a controller, analyzing an image of plasma in the mixing chamber and calculating the volume of plasma in the mixing chamber; The method according to embodiment 53, further comprising.

Claims

1. A method for detecting the presence of a target analyte in a sample, the method comprising: introducing the sample into a cartridge, the cartridge including a fluidic system for isolating the target analyte of the sample and collecting a quantity of a first label proportional to the quantity of the target analyte in the sample, the fluidic system comprising: a first chamber; a second chamber; and a channel extending from the first chamber to the second chamber; and binding the target analyte to a substrate comprising paramagnetic beads; positioning a first magnet near a first surface of the cartridge and adjacent to the first chamber; facilitating relative movement of the first magnet and the cartridge to attract the paramagnetic beads out of suspension and into a bolus, the relative movement being facilitated by at least one of moving the first magnet across the first surface or rotating the cartridge; capturing an image of the bead bolus using a camera; analyzing the image of the bead bolus using a controller to determine a size of the bead bolus; directing electromagnetic radiation from a first electromagnetic radiation source to form an interrogation space within the cartridge; receiving in a first detector electromagnetic radiation emitted in the interrogation space by the first label when the first label is present in the interrogation space; using a controller to identify the presence of the target analyte in the sample based on the electromagnetic radiation detected by the first detector; A method comprising:

2. The method of claim 1, further comprising facilitating relative movement of the first magnet and the cartridge so as to transfer the paramagnetic beads and the target analyte from the first chamber to the second chamber.

3. The method of claim 2, further comprising performing a washing operation in the second chamber to isolate the target analyte from other components of the sample.

4. The cleaning operation moving the first magnet away from the first surface of the cartridge so that the paramagnetic beads become dispersed within the second chamber; moving the second magnet toward a second surface of the cartridge such that the paramagnetic beads collect in the vicinity of the second magnet; moving the second magnet away from the second surface of the cartridge so that the paramagnetic beads become dispersed within the second chamber; moving the first magnet toward the first surface of the cartridge so that the paramagnetic beads collect in the vicinity of the first magnet; The method of claim 3, comprising:

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