Methods and systems for high-sensitivity assays and delivery of captured objects

Abstract

Methods and systems for capture object-based assays, including those for measuring the concentration of analyte molecules or particles in a fluid sample, are described. The methods and systems can involve highly sensitive detection of analytes, in some cases employing assay conditions and sample handling that result in capture and detection of a high percentage of analyte molecules or particles in a fluid sample using relatively few capture objects. In some cases, devices and methods for immobilizing capture objects with unexpectedly high efficiency relative to an assay site are also described. Some such devices involve the use of force fields and fluid meniscus forces, alone or in combination, to facilitate or improve capture object immobilization. Techniques for utilizing a relatively high percentage of capture objects in an assay sample are also described, for example, by using the disclosed sample washing techniques, imaging systems, and analytical procedures that can reduce capture object loss.

Description

Technical Field

[0001] (Cross-Reference to Related Applications) This application claims priority pursuant to 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 010,613 entitled "Methods and Systems Related to Highly Sensitive Assays and Delivering Capture Objects" filed on April 15, 2020, and to U.S. Provisional Patent Application No. 63 / 010,625 entitled "Methods and Systems Related to Highly Sensitive Assays and Delivering Capture Objects" filed on April 15, 2020, the entire contents of which are incorporated herein by reference for all purposes.

[0002] (Technical Field) Generally, methods and systems for analyte capture assays, including for measuring the concentration of analyte molecules or particles in a fluid sample, are described.

Background Art

[0003] The ability to accurately measure target analyte molecules (e.g., proteins and nucleic acids) is crucial in many fields, including clinical diagnostics, blood bank testing, research, and biochemical pathway analysis. Assays and associated systems / devices exist for the detection of single target analyte molecules, and they may utilize beads or other capture objects. One category of such assays that generally have high sensitivity is digital enzyme-linked immunosorbent assay ("digital ELISA"). Certain digital ELISA assays involve capturing proteins or other target analytes on microscopic beads (or other capture objects), labeling the target analytes with enzymes, isolating the beads on an array of small wells, and detecting the activity of the enzyme associated with the beads using fluorescence imaging. For example, the spatial localization and / or separation of individual beads on an array allows for the determination of single-molecule signals associated with the beads, enabling measurement at very low values ​​of the number and / or concentration of target analytes. Various other analyte capture-based assays, as well as related systems and apparatus, have also been developed to measure the number and / or concentration of analyte molecules in fluid samples, in which analyte molecules are captured by beads or other capture objects. However, there remains a need for methods, techniques, and systems to improve the sensitivity of such assays. [Overview of the project]

[0004] Methods and apparatus for capture-object-based assays, encompassing the measurement of the concentration of analyte molecules or particles in a fluid sample, are described. The methods and systems utilize assay conditions and sample handling that yield high-sensitivity detection of analytes and, in some cases, result in the capture and detection of a high percentage of analyte molecules or particles in a fluid sample using relatively few capture objects. Apparatus and methods for immobilizing capture objects with unexpectedly high efficiency at the assay site are also described in several examples. Some such apparatuses involve the use of force fields and fluid meniscus forces, either alone or in combination, to facilitate or improve the immobilization of capture objects. Techniques for utilizing a relatively high percentage of capture objects in an assay sample are also described, for example, using disclosed sample washing techniques, imaging systems, and analytical procedures that can reduce capture object loss.

[0005] The subject matter of the present invention may, in some cases, involve products relating to one another, alternative solutions to a particular problem, and / or multiple different uses of one or more systems and / or components.

[0006] In some embodiments, a method for immobilizing a capture object with respect to an assay site is described. In some embodiments, the method includes: delivering a capture object in close proximity to an assay site on a surface; generating a force field in close proximity to a surface that tends to act on the capture object so that the capture object moves toward the surface; flowing a fluid plug containing the capture object in a first direction such that a first-direction retraction meniscus of the fluid plug flows over at least some of the assay sites; flowing the fluid plug in a second different direction such that a second-direction retraction meniscus of the fluid plug flows over at least some of the assay sites; and immobilizing at least some of the capture objects subjected to the steps of flowing the fluid plug in the first direction and / or the second direction with respect to the assay sites.

[0007] In some embodiments, the method includes delivering a capture object in close proximity to an assay site on a surface; generating a force field in close proximity to a surface that tends to act on the capture object so that the capture object moves toward the surface; flowing a fluid plug containing the capture object over at least one of the assay sites; and immobilizing at least some of the capture objects subjected to the flowing step with respect to the assay site, such that at least 20% of the total number of capture objects delivered in close proximity to the assay site are immobilized during the flowing step.

[0008] In some embodiments, a device for immobilizing a capture object with respect to an assay site on the surface of an assay consumable is described. In some embodiments, the device includes a capture object applicator configured to apply a capture object to or in close proximity to the surface of an assay consumable; a force field generator configured, when present, to generate a force field adjacent to and in close proximity to the surface of the assay consumable; a fluid injector configured, when on the surface of the assay consumable, to generate a fluid plug having a first meniscus and a second meniscus, respectively, adjacent to an immiscible fluid; a fluid pump capable of moving the fluid over at least a portion of the surface; and a controller including one or more processors configured to adjust the fluid pump to move the fluid plug bidirectionally over at least a portion of the surface.

[0009] In some embodiments, a device is described for associating a capture object with an assay site on the surface of an assay consumable. In some embodiments, the device includes: a capture object applicator configured to apply a capture object to or in close proximity to the surface of an assay consumable; a force field generator, when present, configured to generate a force field adjacent to and in close proximity to the surface of the assay consumable, wherein the force field is an uneven electric field that can be applied to a dielectric capture object capable of polarizing dielectric force; a fluid injector configured, when on the surface of the assay consumable, to generate a fluid plug having a first meniscus and a second meniscus, respectively, adjacent to an immiscible fluid; a fluid pump capable of moving the fluid over at least a portion of the surface; and a controller including one or more processors configured to adjust the fluid pump to move the fluid plug bidirectionally over at least a portion of the surface.

[0010] In some embodiments, an apparatus for associating an assay site on the surface of an assay consumable with a capture object includes: a capture object applicator configured to apply the capture object to or in close proximity to the surface of the assay consumable; a power source; a conductive solid, when present, in conductive or inductive electrical communication with the power source adjacent to or opposite the surface of the assay consumable; a fluid injector configured to generate a fluid plug; and a controller comprising one or more processors configured to initiate the application of a voltage to at least some of the conductive solid by the power source to (a) generate an uneven electric field in close proximity to the surface, which can be applied to a dielectric capture object capable of polarization of dielectric force; and (b) generate an electric field that moves the fluid plug over at least a portion of the surface.

[0011] In some embodiments, a method for measuring the concentration of analite molecules or particles in a fluid sample is described. In some embodiments, the method includes exposing a solution containing or suspected to contain at least one type of analite molecules or particles to a capture object, each having an affinity for a particular type of analite molecule or particle, wherein the number of capture objects exposed to the solution containing or suspected to contain analite molecules or particles is 50,000 or less; immobilizing a particular type of analite molecule or particle with respect to the capture objects such that at least some of the capture objects associate with at least one particular type of analite molecule or particle from the fluid sample, and a statistically significant proportion of the capture objects do not associate with any particular type of analite molecule or particle from the fluid sample; measuring an index of the number or proportion of capture objects that associate with at least one particular type of analite molecule or particle from the fluid sample; and measuring the concentration of a particular type of analite molecule or particle in the fluid sample, at least in part on the measurement of an index of the number or proportion of capture objects that have been determined to associate with at least one particular type of analite molecule or particle.

[0012] In some embodiments, the method involves exposing a capture object, each having an affinity for a specific type of analite molecule or particle, to a solution containing or suspected to contain at least one type of analite molecule or particle, wherein the number of capture objects exposed to the solution containing or suspected to contain analite molecules or particles is 50,000 or less, and immobilizing analite molecules or particles of a specific type of analite molecule or particle with respect to the capture object so that at least some of the capture objects associate with at least one specific type of analite molecule or particle from the fluid sample. The method includes measuring an index of the number or proportion of captured objects associated with a particular type of analyte molecule or particle from a fluid sample, and measuring the concentration of a particular type of analyte molecule or particle in a fluid sample, at least in part on measuring an index of the number or proportion of captured objects that have been determined to be associated with at least one particular type of analyte molecule or particle, based on measuring an index of the number or proportion of captured objects associated with at least one particular type of analyte molecule or particle from a fluid sample, or measuring the concentration of a particular type of analyte molecule or particle in a fluid sample, at least in part on the measured intensity level of a signal that is an indicator of the presence of multiple particular types of analyte molecules or particles.

[0013] In some embodiments, the method includes: exposing a capture object, each having an affinity for a particular type of analite molecule or particle, to a solution containing or suspected to contain at least one type of analite molecule or particle; immobilizing the particular type of analite molecule or particle with respect to the capture object such that at least some of the capture object associate with at least one particular type of analite molecule or particle from the fluid sample, and a statistically significant proportion of the capture object does not associate with any particular type of analite molecule or particle from the fluid sample; spatially isolating at least 25% of the capture object subjected to the immobilization step to multiple separate locations; processing at least some of the multiple locations subjected to the spatial isolating step to measure an index of the number or proportion of capture objects that associate with at least one particular type of analite molecule or particle from the fluid sample; and measuring the concentration of the particular type of analite molecule or particle in the fluid sample, at least in part on the measurement of the index of the number or proportion of capture objects that have been determined to associate with at least one analite molecule or particle.

[0014] In some embodiments, the method involves exposing a capture object, each having an affinity for a specific type of analite molecule or particle, to a solution containing or suspected to contain at least one type of analite molecule or particle, wherein the number of capture objects exposed to the solution containing or suspected to contain analite molecules or particles is 50,000 or less, and where at least some of the capture objects associate with at least one specific type of analite molecule or particle from the fluid sample, while a statistically significant proportion of the capture objects do not associate with any of the specific types of analite molecules or particles from the fluid sample. The method includes immobilizing analyte molecules or particles; immobilizing at least one binding ligand with respect to a particular type of at least several analyte molecules or particles associated with a capture object; exposing at least one immobilized binding ligand to a precursor labeling agent to convert the precursor labeling agent into a labeling agent which is immobilized with respect to the capture object on which the binding ligand is immobilized; measuring an index of the number or proportion of capture objects containing at least one immobilized labeling agent; and measuring the concentration of a particular type of analyte molecule or particle in a fluid sample, at least in part on measuring an index of the number or proportion of capture objects determined to contain at least one immobilized labeling agent.

[0015] In some embodiments, an apparatus for imaging an array of assay sites on the surface of an assay consumable is described. In some embodiments, the apparatus includes an imaging system comprising a detector and optical system having a fixed field of view larger than the area containing the array of assay sites, and a computer-implemented control system configured to receive information from the imaging system and analyze the entire area containing the array of assay sites, wherein the assay sites have a volume of 10 att liters to 100 picoliters.

[0016] In some embodiments, methods for performing assays to detect analyte molecules or particles in a fluid sample are described. In some embodiments, the method includes providing 1,000 to 200,000 capture objects and preparing capture objects and analite molecules or particles from a fluid sample for detection by performing one or more processes, each of which includes: (1) mixing the capture objects and analite molecules or particles in a liquid to form a capture object suspension; and (2) applying force to the capture object suspension to remove the liquid from the capture object suspension, and applying negative pressure to the capture object suspension via a fluid connection of the capture object suspension to a vacuum source, wherein the application of force tends to move the liquid; and measuring the concentration or proportion of analite molecules or particles in the fluid sample, at least partially based on measuring an index of the number or proportion of capture objects that are determined to be at least 90% of the capture objects of the providing step, wherein the preparation step results in prepared capture objects, of which at least some associate with analite molecules or particles from the fluid sample, and a statistically significant proportion does not associate with either analite molecules or particles, and the total number of prepared capture objects is 90% or more of the capture objects of the providing step.

[0017] In some embodiments, an apparatus for performing an assay is described. In some embodiments, the apparatus includes a sample washer configured to prepare magnetic beads and analyte molecules or particles from a fluid sample for detection; a bead applicator configured to apply magnetic beads to or in close proximity to the surface of an assay consumable whose surface includes a reactor; a magnetic field generator configured to be adjacent to the assay consumable and to generate a magnetic field in close proximity to the surface; a fluid injector configured to generate a fluid plug having a first meniscus and a second meniscus, respectively, adjacent to an immiscible fluid when on the surface of the assay consumable; a fluid pump capable of moving fluid across the surface of the assay consumable; an imaging system including a detector and optical system having a fixed field of view larger than the area defined by the array of reactors; and a controller including one or more processors configured to adjust the fluid pump to move fluid across the surface of the assay consumable.

[0018] In some embodiments, a method is provided for measuring the concentration of analyte molecules or particles in a fluid sample. In some embodiments, the method involves exposing magnetic beads to a solution containing or suspected to contain at least one type of analyte molecules or particles; immobilizing the analyte molecules or particles with respect to the magnetic beads such that at least some of the magnetic beads associate with at least one analyte molecule or particle from the fluid sample, and a statistically significant proportion of the magnetic beads do not associate with any analyte molecules or particles from the fluid sample; removing the solution from at least some portion of the magnetic beads subjected to the immobilization step; delivering the magnetic beads in proximity to a reactor on a surface; and generating a magnetic field in proximity to a surface that tends to act on the captured object to capture it. The method includes moving an object toward the surface, flowing a fluid plug containing magnetic beads so that the retraction meniscus of the fluid plug flows across at least some of the reactor, inserting at least some portion of the magnetic beads into the reactor, imaging the entire reactor after the insertion step, analyzing the entire reactor subjected to the imaging step to measure an index of the number or proportion of magnetic beads associated with analyte molecules or particles from a fluid sample, and measuring the concentration of analyte molecules or particles in the fluid sample, at least in part on measuring an index of the number or proportion of beads determined to associate with at least one analyte molecule or particle.

[0019] In some embodiments, the method involves exposing a capture object to a solution containing or suspected to contain at least one type of analyte molecule or particle; immobilizing the analyte molecule or particle with respect to the capture object such that at least some of the capture object associate with at least one analyte molecule or particle from the fluid sample, and a statistically significant proportion of the capture object does not associate with any analyte molecule or particle from the fluid sample; removing the solution from at least some portion of the capture object subjected to the immobilization step while retaining at least 80% of the capture object subjected to the immobilization step; and, in close proximity to the assay site on the surface, The method includes delivering at least 80% of the captured objects subjected to the removal step, immobilizing at least 20% of the captured objects subjected to the delivery step with respect to the assay site, imaging at least 80% of the assay site, analyzing at least 75% of the assay site subjected to the imaging step to measure an index of the number or proportion of magnetically captured objects associated with analyte molecules or particles from the fluid sample, and measuring the concentration of analyte molecules or particles in the fluid sample, at least in part, based on the measurement of an index of the number or proportion of captured objects determined to associate with at least one analyte molecule or particle.

[0020] In some embodiments, the method is 2 × 10 -18 This involves measuring the concentration of analyte molecules or particles in a fluid sample at a detection level of less than M.

[0021] In some embodiments, a method for immobilizing a capture object with respect to an assay site is described. In some embodiments, the method includes delivering a capture object in close proximity to an assay site on a surface; applying an external force to the capture object attached to the delivery step to reduce the distance between the capture object and the assay site; flowing a fluid plug containing the capture object so that the retracting meniscus of the fluid plug flows across the assay site; and immobilizing the capture object with respect to the assay site by applying a force at least partially contributed by the retracting meniscus.

[0022] In some embodiments, a method for associating a capture object with respect to an assay site is described. In some embodiments, the method includes: delivering a capture object in close proximity to an assay site on a surface; applying an external force to the capture object attached to the delivery step to reduce the distance between the capture object and the assay site, wherein the external force is a dielectrophoretic force; flowing a fluid plug containing the capture object so that the receding meniscus of the fluid plug flows across the assay site; and associating the capture object with respect to the assay site through the application of a force at least partially contributed by the receding meniscus.

[0023] In some embodiments, a method for associating a capture object with an assay site is described. In some embodiments, the method includes: delivering a capture object in close proximity to the assay site on a surface by flowing a fluid plug containing the capture object into the assay site using digital microfluidic technology; reducing the distance between the capture object and the assay site by applying an external dielectrophoretic force by generating an uneven electric field on the capture object attached to the delivery step; and associating the capture object with respect to the assay site by applying a force at least partially contributed by the dielectrophoretic force.

[0024] In some embodiments, a method for immobilizing capture objects with respect to an assay site is described. In some embodiments, the method includes: delivering a fluid containing the capture objects in close proximity to an assay site on a surface; generating a force field in close proximity to a surface that tends to act on the capture objects to move the capture objects toward the surface; applying a lateral force to the capture objects by adjusting the lateral distribution of the force field; and immobilizing at least some of the capture objects with respect to the assay site, at least partially, via the applied lateral force, such that at least 20% of the total number of capture objects delivered in close proximity to the assay site are immobilized during the application step. In some embodiments, a kit is provided. In some embodiments, the kit includes capture objects comprising a binding surface having affinity for analyte molecules or particles, wherein the detection level of a first assay using 5,000 identical capture objects of the kit is at least 50% lower than the detection level of a second assay using 500,000 identical capture objects of the kit, the first assay comprising the step of incubating the capture objects with analyte molecules or particles during a first period, the second assay comprising the step of incubating the capture objects with analyte molecules or particles during a second period, the first period being 100 times longer than the second period, and the first and second assays being carried out under otherwise the same conditions.

[0025] In some embodiments, the kit includes a packaged container for the analyte detection assay, containing 50,000 to 5,000,000 capture objects, each containing a binding surface having affinity for analytes and having an average diameter of 0.1 micrometers to 100 micrometers, and the analyte detection assay is 50 × 10 -18 This can be done at detection levels of M or lower.

[0026] In some embodiments, a composition is provided. In some embodiments, the composition comprises an isolated fluid having a volume of 10 to 1000 microliters, at least one type of analyte molecule or particle present at a concentration of 0.001 aM to 10 pM, and 100 to 50,000 capture objects comprising a binding surface having affinity for at least one type of analyte molecule or particle.

[0027] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting aspects of the invention when considered in conjunction with the accompanying drawings. Where this specification and any reference incorporated herein contain disclosures that conflict and / or are inconsistent, this specification shall prevail. Non-limiting aspects of the present invention are described by reference to the accompanying drawings, which are schematic and not intended to be drawn to a specific scale. Each identical or nearly identical component illustrated in the drawings is typically represented by the same number. For clarity, not all components are labeled in all drawings, and in some embodiments of the invention, not all components are shown where they are not necessary for a person skilled in the art to understand the invention. In the drawings: [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1 is a block diagram showing components of an embodiment of an apparatus for performing at least a portion of an assay, relating to a specific embodiment, which includes at least an assay consumable handler, a capture object applicator, a fluid injector, a fluid pump, and a controller. [Figure 2A-B] Figure 2A is a schematic diagram of an exemplary method for immobilizing a captured object with respect to an assay site on a surface, according to a specific embodiment. Figure 2B is a schematic diagram of an exemplary method for immobilizing a captured object with respect to an assay site on a surface, according to a specific embodiment, in the presence of a force field. [Figure 2C-D]Figure 2C is a schematic diagram of an exemplary method for immobilizing a captured object with respect to an assay site on a surface, in the presence of a force field during the flow of a fluid plug containing a receding meniscus at a certain point in time when the receding meniscus begins to pass through the assay site, according to a particular embodiment. Figure 2D is a schematic diagram of Figure 2C at a later point in time, according to a particular embodiment, when the receding meniscus has passed through the entire assay site. [Figure 2E-F] Figure 2E is a schematic diagram of an exemplary method for immobilizing a captured object with respect to an assay site on a surface in the presence of a force field during the flow of a fluid plug including a receding meniscus, according to a particular embodiment. Figure 2F is a schematic diagram of the flow of a fluid plug including a receding meniscus, according to a particular embodiment. [Figure 2G] Figure 2G is a schematic diagram of an exemplary method for immobilizing a captured object with respect to an assay site on a surface, in the presence of a force field between the flows of multiple fluid plugs, according to a specific embodiment. [Figure 3A] Figures 3A-3B are schematic diagrams of a device for immobilizing a captured object with respect to an assay site on the surface of an assay consumable, which is operably connected to an assay consumable handler, according to a specific embodiment. [Figure 3B] Figures 3A-3B are schematic diagrams of a device for immobilizing a captured object with respect to an assay site on the surface of an assay consumable, which is operably connected to an assay consumable handler, according to a specific embodiment. [Figure 3C-D] Figures 3C-3D are schematic top views of a capture object adjacent to a surface containing an assay site surrounded by a conductive solid network, in the absence of repulsive dielectric force (Figure 3C) and in the presence of repulsive dielectric force (Figure 3D), relating to a specific embodiment. [Figure 4A-B] Figures 4A-4F are schematic diagrams illustrating exemplary assay consumable handlers according to a specific embodiment. [Figure 4C-D] Figures 4A-4F are schematic diagrams illustrating exemplary assay consumable handlers according to a specific embodiment. [Figure 4E-F] Figures 4A-4F are schematic diagrams illustrating exemplary assay consumable handlers according to a specific embodiment. [Figure 5] Figure 5 is a schematic diagram of an apparatus for imaging an array of assay sites on the surface of an assay consumable, according to a specific embodiment. [Figure 6A] Figures 6A-6B are schematic flow diagrams illustrating one embodiment of a capture object-based assay for detecting analyte molecules or particles, relating to a specific aspect. [Figure 6B] Figures 6A-6B are schematic flow diagrams illustrating one embodiment of a capture object-based assay for detecting analyte molecules or particles, relating to a specific aspect. [Figure 7] Figures 7A-7B are schematic diagrams of exemplary top and perspective views of a microfluidic apparatus for use in detecting analyte molecules or particles, relating to a particular embodiment. [Figure 8] Figure 8 is a schematic diagram of a sample washing apparatus according to a specific embodiment. [Figure 9] Figure 9 is a modeled increasing plot of the ratio of captured protein molecules to beads in an assay using 5,000 beads compared to 500,000 beads, assuming 274,000 captured antibodies per bead, as a function of the capture antibody-antigen interaction dissociation constant (KD) for a specific embodiment. [Figure 10] Figure 10 is a schematic diagram of a magnetic meniscus sweeping (MMS) method for loading beads into a microwell array, according to a specific embodiment. [Figure 11] Figure 11 is a plot of AEB against [IL-17A] for two bead counts and two incubation times, relating to a specific embodiment. [Figure 12] Figure 12 is a plot of AEB against [IL-17A] as a function of the number of captured beads during 4h incubation of beads and samples, according to a specific embodiment. [Figure 13] Figure 13 shows plots of AEB against [IL-17A] during a 4-hour incubation of samples with bead counts ranging from 4,530 to 32,000, relating to a specific embodiment. [Figure 14]Figure 14A shows the AEB as a function of sample incubation time at [IL-17A] = 1.2 fM using 15,000 beads for a specific embodiment. Figure 14B is a plot of the AEB against [IL-17A] as a function of sample incubation time using 15,000 beads for a specific embodiment. [Figure 15] Figure 15 is a plot of AEB as a function of sample volume for [IL-17A], using 15,000 beads and an incubation time of 6 hours, relating to a specific embodiment. [Figure 16] Figure 16 shows plots of AEB for [IL-17A] in a standard ELISA (500,000 beads, 100 μL sample, 30 min incubation) and a digital ELISA using a lower number of beads (5,453, 2,726, or 1,363 beads, 200 μL sample, 24 h incubation) according to a specific embodiment. [Figure 17] Figure 17 shows plots of AEB against [IL-17A] using 5,000 beads and a) 100 μL of sample, 6h incubation (white squares), and b) 250 μL of sample, 24h incubation (black circles) according to a specific embodiment. [Figure 18] Figure 18 is a plot of spike recovery of IL-17A from two spiked concentration serum samples as a function of the number of beads, relating to a specific embodiment. [Figure 19] Figures 19A-19B show dispersion plots of [IL-17A] determined using standard digital ELISA or low-bead count digital ELISA in serum and plasma samples relating to specific embodiments. [Figure 20] Figure 20 is a plot showing the correlation between serum and plasma samples quantified using standard digital ELISA and low-bead count / high-efficiency digital ELISA in a specific embodiment. [Figure 21]Figure 21 shows plots of AEB against concentrations of IL-17A, IL-12p70, p24, IFN-α, IL-4, and PSA using a digital ELISA adjusted for low bead counts (white circles) and a standard digital ELISA (black squares) according to a specific embodiment. [Figure 22] Figure 22 is a plot of AEB against the concentration of spiked [IL-12p70] in diluted serum for a standard ELISA (400,000 beads, 100 μL sample, 30-minute incubation) and a digital ELISA adjusted for lower bead counts (5,368, 2,684, or 1,342 beads, 200 μL sample, 24-hour incubation) according to a specific embodiment. [Figure 23] Figure 23 is a plot of AEB against the concentration of p24 spiked in diluted serum for a standard ELISA (300,000 beads, 125 μL sample, 30-minute incubation) and a digital ELISA adjusted for lower bead counts (5,259, 2,625, or 1,313 beads, 125 μL sample, 24-hour incubation) according to a specific embodiment. [Figure 24] Figure 24 is an image of an array of microwells arranged on a magnet, relating to a specific embodiment. [Modes for carrying out the invention]

[0029] Methods and systems for analyte capture-based assays, including the measurement of the concentration of analyte molecules or particles in a fluid sample, are described. The described methods and systems enable highly sensitive detection of analytes (e.g., at femtomole, atomolecular, zeptomolecular, or lower levels), and in some examples, use assay conditions and sample handling techniques that result in the capture and detection of a high percentage of analyte molecules or particles in the assay sample with fewer captured objects compared to typical conventional assays. Apparatus and methods for immobilizing captured objects (e.g., beads) relative to an assay site (e.g., a reactor (e.g., microwells)) are also described, as some examples with unexpectedly high efficiency. Some of such apparatuses involve the use of force fields (e.g., magnetic fields) and fluid meniscus forces, either alone or in combination, to facilitate or improve the immobilization of captured objects. Techniques for utilizing a relatively high percentage of captured objects in the assay sample are also described, using described washing techniques, imaging systems, and analytical procedures that can reduce the loss of captured objects, for example.

[0030] In some embodiments, apparatuses are described comprising assay consumables having a surface containing assay sites, capture object applicators, force field generators, fluid handling components (e.g., fluid injectors and pumps), controllers, and optionally, specific assay consumable handlers, imaging systems, and sample washers (e.g., non-vacuum-based sample washers). The apparatus may be configured to perform high-sensitivity assays (e.g., digital ELISA). In some examples, the apparatus and associated methods are unexpectedly advantageous in some cases because they involve the use of fewer capture objects (e.g., fewer than 50,000, fewer than 10,000, fewer than 5,000, or less) compared to typical conventional assays. The specific methods described, as well as the components and configurations of the associated apparatuses, can provide non-limiting solutions to the challenges associated with the use of such a small number of capture objects. For example, certain disclosed techniques and associated apparatuses relate to ensuring a sufficient number of capture objects to generate a sufficient signal and to capturing a sufficient number of analytes. One exemplary technique relates to facilitating the effective immobilization of capture objects (e.g., insertion of beads), which may be important in regimes with a low number of capture objects described. Some embodiments relate to system configurations and methods that include generating a force field (e.g., a magnetic field) in close proximity to capture objects (e.g., magnetic beads) near the assay site, and flowing a fluid plug containing the capture objects (and the retracted meniscus of the plug) (e.g., bidirectionally) across the assay site. Other techniques described relate to improvements in assay sensitivity, improved image detection, and analysis and sample handling (e.g., liquid removal techniques, sample incubation).

[0031] For example, conventional high-sensitivity assays such as conventional digital ELISA have enabled highly sensitive measurement of analytes that were previously undetectable, but even higher sensitivity (e.g., atomolecular or lower) is considered advantageous and beneficial. For instance, some analytes (e.g., cytokines (e.g., IL-17A, IL-12p70, interferon-α, interferon-γ, IL-1α, IL-1β)) have limited detectability in certain sample media (e.g., blood), requiring higher analytical sensitivity than conventionally available for their quantification. As another example, certain complex sample media (e.g., stool, cerebrospinal fluid) may require dilution with buffers to reduce matrix effects, which can negatively impact detectability, especially for low concentrations of analytes. Improved detectability can also support the early detection of infectious diseases by enabling more sensitive detection of, for example, viral and bacterial proteins or other antigens. In the case of certain capture-object-based assays (e.g., digital ELISA), improved detection sensitivity (e.g., detection level) increases as the number of detectable species immobilized per capture-object increases. In assays using enzyme labeling on beads, such a ratio can be expressed as the average number of enzymes per bead (AEB), and hypothetically, a higher AEB can result in higher sensitivity. For a given sample containing analytes, the number of detectable species per capture object (e.g., AEB) can be increased by reducing the number of capture objects exposed to the sample. However, using fewer capture objects presents several technical obstacles that hinder and make such an approach impractical. For example, existing capture object-based assay techniques detect capture objects with low efficiency, typically analyzing only about 5% of the capture objects used to capture analytes from a sample. With such low efficiency, conventional assays are considered impractical because they do not obtain a sufficient number of capture beads to analyze.Instead, existing techniques have either (a) completely avoided such challenges by using a larger number of capture objects instead, or (b) focused on increasing sensitivity by simply increasing the absolute number of capture objects detected, regardless of the percentage of capture objects detected. The latter approach increases the proportion of assay sites that associate with capture objects by using a large excess of capture objects compared to the number of assay sites (e.g., wells) in the array (e.g., filling as many wells as possible with beads). The specific approaches described herein employ the opposite approach, rather using a relatively small number of capture objects compared to the number of assay sites compared to conventional assays (e.g., fewer than 50,000), and some of these examples focus on analyzing a high percentage of capture objects exposed to the sample. However, such a reduction in the number of capture objects can also create competing difficulties. The use of a small number of capture objects can result in increased Poisson noise in digital ELISA, which can consequently lead to delayed kinetics and a decrease in the analytes captured within a given time frame. However, unexpectedly, certain methods and apparatuses described herein utilize conditions (e.g., sample volume and incubation time) and techniques (e.g., highly efficient immobilization of the captured material) that can result in higher sensitivity as a result of using less captured material, while at the same time avoiding, or at least mitigating, some or all of the competing difficulties described above to a sufficient degree, thereby achieving higher sensitivity compared to typical existing assay techniques.

[0032] Apparatus and methods for immobilizing capture objects with respect to assay sites are described. Some of these methods and apparatus may facilitate capture object-based assays for detecting and / or quantifying analyte molecules, including assays that use relatively fewer capture objects compared to existing assays.

[0033] In some examples, devices for immobilizing capture objects with respect to an assay site are described. The device may be a subcomponent of a larger system comprising an automated device for performing an assay (e.g., for detecting and / or quantifying analyte molecules or particles). Figure 1 shows a schematic of one such non-limiting system 1 comprising a component for immobilizing capture objects. In Figure 1, system 1 may include an optional assay consumable handler 10, which in certain embodiments is configured to be operably coupled to an assay consumable 5 (it may also be detachable, and its presence is optional, as indicated by the dotted line). Such embodiments may be, for example, an automated robotic system. System 1 may comprise a capture object applicator 20, a force field generator 40, a fluid injector 50, and a fluid pump 60. In some embodiments, system 1 comprises one or more controllers 30, each having one or more processors configured to control and operate specific components of the device. For example, the controller 30 comprises one or more processors configured to control and operate the assay consumable handler 10, the capture object applicator 20, the force field generator 40, the fluid injector 50, and the fluid pump 60, thereby enabling a method for immobilizing the capture object with respect to the assay site on the surface of the assay consumable 5.

[0034] In some such examples, the controller 30 is configured to regulate the fluid pump 60 to move the fluid (e.g., fluid plug) bidirectionally across the surface of the assay consumable 5. It should be understood that in some embodiments, a separate assay consumable handler 10 is not required.

[0035] For example, one or more of the above components may be integrated with the assay consumable (e.g., as part of a microfluidic system on a chip). Other components of System 1 may be configured to perform other steps or operations of the assay. For example, the imaging system 70 may include a detector and optical system for imaging the assay site on the assay consumable, and the computer-implemented control system 80 may be configured to receive information from the imaging system and analyze the assay site (e.g., measure the presence of immobilized capture objects and / or analyte molecules or particles with respect to the assay site). In some, but not all, examples of System 1 may further include a sample washer 90 configured to prepare capture objects and analyte molecules (e.g., from a fluid sample) for detection. In other embodiments, such preparation may be performed separately.

[0036] Each of the assay consumable handler, capture object applicator, force field generator, fluid injector, and fluid pump may be connected to the same or different controller (e.g., controller 30) configured to operate the components described herein. The controller may be configured to automate the immobilization of the capture object and / or various stages of the assay method. In certain embodiments, one or more components or their functions shown separately in Figure 1 may be integrated into a single component. For example, in certain cases, two or more functions of the capture object applicator 20, fluid injector 50, and fluid pump 60 may be integrated into a single component of the system. As another example, in certain embodiments, a single computer-implemented control system (e.g., computer-implemented control system 80) may control the operation of the imaging system 70 and perform the functions of the controller 30 described above. Thus, unless specifically indicated, a reference to any one component does not prevent such component from performing other functions of the system. Similarly, unless specifically illustrated or described, a reference to a system comprising separately detailed components does not necessarily require the components to be physically distinct structural elements (for example, multiple components may share the same structural elements, or they may have common structural elements but be configured to function as multiple components in the system as a whole).

[0037] Delivery of captured material to the assay site surface In some embodiments, including the immobilization of the capture object, the capture object is delivered in close proximity to the assay site on the surface. For example, Figure 2A shows a schematic diagram of a capture object 100 delivered in close proximity to an assay site 110 on a surface 120, according to a particular embodiment. Figure 2A illustrates the capture object 100 as beads and the assay site 110 as a reactor (e.g., a well) on the surface 120, but other configurations are also available and are described in detail below. In some cases, the capture object is delivered in close proximity to the assay site via a fluid. The fluid may be in the form of a plug / bolus of any size or volume, in which two immiscible phases pass (at least partially) through the assay site, or it may be a continuous single-phase flow. For example, according to a particular embodiment, Figure 2A shows the delivery of the capture object 100 in a fluid plug 130 over the assay site 110. The capture object may be delivered in close proximity to the assay site (e.g., within 10 mm, 5 mm, 1 mm, 500 μm, 100 μm or less) and positioned relatively close to the assay site, but it is not necessarily required that it be delivered directly to or immobilized on the assay site immediately after delivery. The capture object may be delivered in close proximity to the assay site by any of the following techniques, for example, by manual operation (e.g., pipetting) or via a component of the apparatus (e.g., a capture object applicator, which will be described in more detail below). The delivered capture object can then be immobilized with respect to the assay site. For example, capture object 100 (e.g., beads) can be inserted into the assay site 110. In this context, immobilization of the capture object with respect to the assay site refers to fixing the position of the capture object at the assay site, for example, by inserting the capture object into a well, encapsulating the capture object in a static droplet, or limiting the capture object to a specific area of ​​the surface defining the assay site. Immobilization of the capture object does not necessarily involve attaching the capture object to the assay site (e.g., chemically, mechanically, or otherwise). As described above, efficient and rapid immobilization of the capture object facilitates the use of fewer capture objects than certain existing capture object-based technologies in some examples.

[0038] captured object The capture objects may have a variety of suitable forms. In some cases, the capture objects are configured to be spatially isolated from each other. The capture objects may be provided in a form that allows them to be spatially separated into multiple locations (e.g., assay sites, channels, etc.). For example, the capture objects may include beads (spherical, disk, ring, cube, etc., of any shape), dispersions or suspensions of microparticles (e.g., multiple particles in a fluid suspension), nanotubes, or others. In some embodiments, the capture objects are insoluble or substantially insoluble in the solvent(s) or solution(s) used in the assay. In some cases, the capture objects are non-porous solids or substantially non-porous solids (e.g., essentially poreless), however, in other cases, the capture objects are porous or substantially porous, hollow, partially hollow, etc. They may be non-absorbent, substantially non-absorbent, substantially absorbent, or absorbent. In some cases, the captured object may contain magnetic material to facilitate certain aspects of the assay (e.g., washing steps, immobilization / loading steps).

[0039] The captured object may be of any preferred size or shape. Non-limiting examples of preferred shapes include spherical, cubic, ellipsoidal, annular, and sheet-like shapes. In certain embodiments, the average diameter (if substantially spherical) or average maximum cross-sectional dimension (for other shapes) of the captured object is 0.1 micrometers or more, 1 micrometer or more, 10 micrometers or more, or more. In some embodiments, the average diameter (if substantially spherical) or average maximum cross-sectional dimension (for other shapes) of the captured object is 100 micrometers or less, 50 micrometers or less, 10 micrometers or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the average diameter of the captured object or the maximum one-dimensional dimension of the captured object is 0.1 micrometers to 100 micrometers, 1 micrometer to 100 micrometers, 10 micrometers to 100 micrometers, or 1 micrometer to 10 micrometers. The “average diameter” or “average maximum cross-sectional dimension” of the captured object is the arithmetic mean of the diameter / average maximum cross-sectional dimension of the captured object. Those skilled in the art can measure the average diameter / maximum cross-sectional dimension of a group of captured objects, for example, using laser scattering, microscopy, sieving, or other known techniques. For example, in some cases, a Coulter counter may be used to measure the average diameter of several beads.

[0040] In certain embodiments, the captured object is or includes beads. The beads may be magnetic beads. In some examples where a magnetic field is generated in close proximity to the surface, the magnetic field acts on the magnetic beads, effectively distributing them spatially with respect to the assay site (e.g., by moving them toward the surface in a desired manner). The magnetic properties of the beads can help separate the beads from the liquid, for example, during a washing step. In some embodiments, the magnetic beads are superparamagnetic, while in other embodiments, the magnetic beads are ferromagnetic. As is generally known, superparamagnetic particles are positively magnetic and have high magnetism, while ferromagnetic particles can be magnetized by an external magnetic field and retain their magnetization after the external field is removed. Further descriptions of the superparamagnetic and ferromagnetic particles of the device are provided in Van Reenen, A, de Jong, AM, den Toonder, IM, & Prins, MW (2014) Integrated lab-on-chip biosensing systems based on magnetic particle actuation—a comprehensive review. Lab on a Chip, 14(12), 1966–1986, which is incorporated herein by reference for all purposes. Potentially suitable beads, including magnetic beads, are commercially available from several suppliers. In some embodiments, at least a portion of the capture material delivered in close proximity to the surface containing the assay site associates with at least one analyte molecule or particle. In some such embodiments, at least a portion of the capture material delivered in close proximity to the surface containing the assay site associates with at least one analyte molecule or particle and one or more binding ligands (detailed below).

[0041] Assay site The assay site may have any suitable form. As described above and illustrated in Figures 2A-2G, the assay site (e.g., assay site 110) may be in the form of a reactor on a surface (e.g., surface 120). The reactor may be a well (e.g., a microwell) on the surface and can be formed using any of the various techniques described in more detail below.

[0042] In some embodiments, assay sites can be hydrodynamically isolated from each other. For example, assay sites (e.g., reactors) may have continuous peripheral walls so that when sealed, there is no fluid connection between the reactors. Other forms of assay sites include, but are not limited to, spatially fixed droplets (e.g., water droplets surrounded by an immiscible fluid, such as a water droplet surrounded by an immiscible oil) and surfaces in which hydrophilic regions are surrounded by hydrophobic regions.

[0043] In some embodiments, all assay sites have approximately the same volume. In other embodiments, assay sites may have different volumes. The volume of individual assay sites can be appropriately selected to facilitate any particular assay protocol. For example, in a set of embodiments where it is desirable to limit the number of capture objects immobilized with respect to each assay site, the volume of the assay sites can vary from less than att liters to more than nanoliters, depending on the size and shape of the capture objects, the detection technique and apparatus used, the number and density of assay sites on the surface, and the expected concentration of capture objects delivered to the surface containing the assay sites. In some embodiments, the size of the assay site (e.g., reactor) can be selected so that only a single bead used for analyte capture can be completely contained within the assay site. In some embodiments, the assay site (e.g., reactor) has a volume of 10 att liters or more, 50 att liters or more, 100 att liters or more, 500 att liters or more, 1 femtoliter or more, 10 femtoliters or more, 50 femtoliters or more, 100 femtoliters or more, or more. In some embodiments, the assay site has a volume of 100 picoliters or less, 50 picoliters or less, 10 picoliters or less, 1 picoliter or less, 500 femtoliters or less, or less. Combinations of these ranges are possible. For example, in some embodiments, the assay site (e.g., reactor) has a volume of 10 attoliters or more and 100 picoliters or less, 10 attoliters or more and 50 picoliters or less, or 1 femtoliter or more and 1 picoliter or less.

[0044] In some embodiments, the assay sites are present on the surface as an array. In Figure 2A, for example, assay site 110 may be part of an array arranged on surface 120. The assay sites (e.g., reactors) may be arranged in a regular pattern or distributed randomly. In some cases, the array is arranged as a two-dimensional array on a surface (e.g., a substantially flat surface). However, in some embodiments, the assay sites are aligned along a single dimension. As such an example, in some embodiments, the assay sites are aligned linearly along the surface of a channel (e.g., a microchannel).

[0045] In some embodiments, the assay site is configured such that the immobilized capture objects are arranged on a plane of a surface (e.g., a flat surface of an assay consumable).

[0046] In some such embodiments, the capture objects arranged on a plane of a surface are arranged as an array. However, in some embodiments, the immobilized capture objects are randomly distributed on a surface (e.g., a flat surface of assay consumables), and the resulting arrangement of the immobilized capture objects establishes the location of the assay sites on the surface. In some such embodiments, forces from a force field and / or fluid from a fluid plug can cause and / or accelerate the arrangement of the capture objects on the surface, and the forces from the force field and / or fluid plug can also keep the capture objects in place after the formation of a random distribution on the surface (e.g., to ensure imaging).

[0047] The number of assay sites on a surface can depend on various considerations. In some embodiments where a capture object-based assay uses assay sites (e.g., reactors) to detect / quantify analytes, the number of assay sites can depend on the number of types of analyte molecules or particles and / or binding ligands used, the possible assay concentration range, the detection method, the size of the capture object, and the type of detectable (e.g., labeling agent released into solution, precipitated labeling agent, etc.). In some embodiments, the surface includes a single assay site (e.g., a single reactor for a channel). However, in some embodiments, the surface includes a multitude of assay sites.

[0048] In some embodiments, the number of assay sites on the surface (array or other) is 1,000 or more, 10,000 or more, 100,000 or more, 200,000 or more, and / or 500,000 or less, 1,000,000 or less, or 1,000,000,000 or less, or more.

[0049] Assay consumables The assay sites described may be part of an assay consumable. In one embodiment, Figure 3A shows a schematic cross-sectional view of an assay consumable 5 including a surface 120 containing an assay site 110. Assay consumable 5 shows one set of assay sites (e.g., an array), but the assay consumable may include two or more sets of assay sites (each residing in a separate set of spatially separated chambers). For example, an assay consumable having a surface containing assay sites (e.g., assay consumable 5) may be in the form of a disk. One such disk is the Simoa® disk, commercially available from Quanterix. In some cases, the surrounding area of ​​the surface containing the assay site (e.g., reactor / well) is elevated, so that the assay site / well is contained within a channel on or inside the assay consumable. The channel may be open (e.g., uncovered like a trough) or closed (e.g., sealed like a tube or conduit). The embodiments shown in Figures 3A-3B represent an assay consumable 5 having a closed channel defined by a lower portion 6 and an upper portion 7, the channel having a height 8 at the assay site 110 (defined as the distance between the surface 120 of the assay consumable 5 and the upper surface portion 9). An example of a suitable assay consumable having a surface including the assay site is described in U.S. Patent Application No. 13 / 035472 by Fournier et al., entitled "SYSTEMS, DEVICES, AND METHODS FOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES," filed on 25 February 2011 and published as U.S. Patent Application Publication No. 2012 / 0196774, which is incorporated herein by reference for all purposes.

[0050] In some embodiments, the total number of capture objects delivered in close proximity to the assay sites is less than or equal to the number of assay sites. For example, referring to Figure 2A, the number of delivered capture objects 100 is less than or equal to the number of assay sites 110 on surface 120. While typical existing techniques for immobilizing capture objects (e.g., for capture object-based assays (e.g., digital ELISA)) use a large excess of capture objects relative to the number of assay sites (e.g., 2, 5, or more), certain embodiments of the present invention employ the opposite approach. As will be described in detail below, the use of a small number of capture objects can, counterintuitively, improve assay sensitivity if a sufficient number are detected. In some embodiments, the total number of capture objects delivered in close proximity to the reactor is 100,000 or less, 50,000 or less, 25,000 or less, 10,000 or less, 5,000 or less, 2,000 or less, or less. In some embodiments, a single capture object is delivered in close proximity to the assay site (or a single assay site). However, in some embodiments, the total number of captured objects delivered in close proximity to the assay site is 100 or more, 200 or more, 500 or more, 1,000 or more, or more. Combinations of these ranges are possible. For example, in some embodiments, the total number of captured objects delivered in close proximity to the assay site is 100 or more and 100,000 or less, or 1,000 or more and 50,000 or less. As described above, in some embodiments, the total number of capture objects delivered in close proximity to the assay sites is less than or equal to the number of assay sites. In some embodiments, the ratio of the total number of capture objects delivered in close proximity to the assay sites to the number of assay sites is 1:1 or less, 1:2 or less, 1:3 or less, 1:4 or less, 1:10 or less, 1:20 or less, 1:30 or less, 1:40 or less, and / or as low as 1:50, 1:100, 1:1,000, 1:2,000, or less.

[0051] Force field generation / Force field generator In some embodiments, an external force is applied to a capture object delivered in close proximity to an assay site on a surface. In some such embodiments, a force field is generated in close proximity to the surface containing the assay site(s). In some examples, the force field is generated by a force field generator. As described above, apparatus 1 may include a force field generator 40 (as shown in Figures 1 and 3A-3B). Figure 2B shows one such embodiment in which, according to a particular embodiment, the force field generator 40 generates a force field represented by a vector field 45. The force field in close proximity to the surface may act on a capture object delivered in close proximity to the assay site, causing the capture object to move toward the surface. For example, in Figure 2B, the force field represented by the vector field 45 may act on the capture object 100, causing the capture object 100 to move toward the surface 120 in a direction parallel to the arrow of the vector field 45. In some embodiments, the force field is a magnetic field. For example, in Figure 2B, the captured object 100 may be magnetic (e.g., a magnetic bead), and the magnetic field represented by the magnetic vector field 45 acts on the captured object 100. In another example, the force field may be an electric field, and the captured object may have an electrostatic charge (e.g., by the functionalization of a captured object having a charged portion). In such a case, an applied electric field with a vector line pointing away from the surface moves a negatively charged captured object towards the surface, and an applied electric field with a vector line pointing towards the surface moves a positively charged captured object towards the surface.

[0052] Applying force from a force field to the captured object in the direction of the component toward the surface having the assay site can rapidly reduce the distance between the captured object and the assay site. In this case, the time required to immobilize the captured object relative to the assay site can be reduced. Furthermore, the force field acting on the captured object can help hold the captured object in place and reduce the extent to which other forces (e.g., dynamic fluid forces, sealing steps) move the captured object away from the surface and assay site. Moreover, we have found that the generation of such a force field can have a synergistic effect with one or more other techniques described herein, including those relating to fluid flow.

[0053] magnetic field As described above, in some embodiments, the force field generated in close proximity to the surface comprising the assay site is a magnetic field. The magnetic field can be generated according to the techniques of the known art. For example, a force field generator may comprise a permanent magnet and / or an electromagnet. The permanent magnet may comprise any of the various materials known in the art (e.g., ferromagnetic or ferromagnetic materials). The permanent magnet may include transition metals (e.g., iron, cobalt, nickel, titanium) and their alloys and / or rare earth metals (e.g., neodymium, samarium) and their alloys. An electromagnet generally generates a magnetic field by passing an electric current through a coil (e.g., a solenoid).

[0054] An electromagnet may comprise a coil of a conductive material (e.g., copper, silver) around a ferromagnetic or ferromagnetic core (e.g., iron). In Figure 2B, the force field generator 40 may be a permanent magnet and / or electromagnet located beneath the surface 120 on which the assay site 110 is located. While this configuration exemplifies one where the assay site is located between the force field generator (e.g., magnet) and the delivered capture object, other configurations are also available. For example, in some embodiments, the force field generator 40 may be located above the fluid 130 containing the capture object 100, and the force field generated from the force field generator, represented by a vector field 45, may act repulsively on the capture object, moving the capture object 100 toward the surface 120 containing the assay site 110. In some cases, the magnetic field is generated such that the magnetic field vector of the magnetic field is directed from the surface toward the bottom of the assay site. For example, the vector field 45 in Figure 2B may be in the form of a magnetic vector field, directed from the surface 120 toward the bottom of the assay site 110. Such a configuration of a magnetic vector field can, in some embodiments, act on the captured objects 100 and move them toward the bottom of the assay site 110.

[0055] The magnitude of the magnetic field may depend on the position of the force field generator (e.g., permanent magnet, electromagnet). In some embodiments, the apparatus is configured to place a permanent magnet and / or electromagnet beneath the assay site of the assay consumable, thereby allowing the permanent magnet or electromagnet to generate a magnetic field of a desired magnitude on the surface of the assay consumable. In some embodiments, the magnitude of the magnetic field on the surface of the assay consumable is 0.1–2 Tesla or 0.2–1 Tesla. It has been observed that specific magnet positions relative to the assay consumable (as well as specific magnetic field strengths and radial versus axial distributions) can result in favorable delivery of captured objects to the surface containing the assay site. For example, placing the force field generator (e.g., permanent magnet) too close to the bottom of the assay site may result in a magnetic field that produces pellets of captured objects (e.g., magnetic beads) toward the edges of a series of assay sites on the surface. However, placing the force field generator (e.g., permanent magnet) too far from the bottom of the assay site may result in a magnetic field that produces pellets of beads toward the center of the series of assay sites. In some embodiments, the apparatus is configured to position the force field generator 0 mm to 5 mm from the bottom of the assay area of ​​the assay consumables.

[0056] electric field In some embodiments, the force field generated in close proximity to the surface containing the assay site is an electric field. The electric field can be generated according to the techniques of the known art. For example, the force field generator 40 may comprise one or more conductive solids connected to an electrical circuit in close proximity to the surface 120. Specifically, the force field generator 40 may be configured as a capacitor comprising a first conductive member (e.g., a first metal layer or plate) connected to an electrical circuit located below the assay site 10 and the surface 120, and a second conductive member (not shown) located above the assay site and electrically coupled to a circuit (e.g., a second metal layer or plate) parallel to the first conductive member. The application of a voltage to the electrical circuit can generate an electric field having a vector component perpendicular to that in the direction of the surface containing the assay site, which acts on the captured object (if it carries charge) and moves the captured object toward the surface as described above.

[0057] Dielectrophoresis In some embodiments, the force field generated in close proximity to a surface containing an assay site is a non-uniform electric field. A non-uniform electric field can act on a capture object delivered in close proximity to the assay site (e.g., to, on, within, and / or near the assay site) to generate a dielectrophoretic force that moves the capture object toward and / or along the plane of the surface. Dielectrophoresis refers to the phenomenon in which polarizable dielectric particles (which may be used as capture objects) are subjected to a non-uniform electric field, and the magnitude and sign of the force (e.g., repulsive or attractive with respect to the electric field gradient) depends on many factors, including the electrical properties of the medium and particles, the size and shape of the particles, and the frequency of the electric field (if an alternating current with that frequency is used to generate the non-uniform electric field). The particles do not need to have an electrostatic charge to obtain dielectrophoretic force. In certain embodiments, dielectrophoresis can be used to facilitate the immobilization of a capture object (e.g., beads) relative to an assay site on a surface using the attractive and / or repulsive forces from the non-uniform electric field. The non-uniform electric field can be an alternating current (AC) electric field or a direct current (DC) electric field. The theory and implementation of dielectrophoresis in microfluidic applications are described in Pethig R. “Review article dielectrophoresis: Status of the theory.” Biomicrofluidics.2010;4(2):022811 and Pesch GR, et al., “A review of dielectrophoretic separation and classification of non-biological particles.” Electrophoresis.2021 Jan;42(l-2):134-52, and their entire contents are incorporated herein by reference for all purposes. As described above, in some embodiments, the force field generator 40 comprises one or more conductive solids adjacent to the surface 120 and connected to an electrical circuit.The unequal electric field may be generated from a conductive solid (e.g., an electrode) connected to an electrical circuit adjacent to the surface 120 that generates an unequal electric field at a selected frequency, thereby allowing the captured object to move toward a surface, such as the surface containing the assay site (in some examples, the bottom of the reactor when such assay site is used), or toward a featureless surface formed to randomly distribute the assay site containing the captured object.

[0058] In some embodiments, negative dielectrophoresis is used, where a repulsive effect from an electric field acts on a polarizable dielectric trap (including an uncharged trap), causing it to move toward the assay site on the surface (e.g., toward the surface containing the assay site and / or along the surface toward the assay site). In such embodiments, the conductive solid of the force field generator is located on the opposite side of the surface, and as a result, the trap delivered between the conductive solid and the surface is repelled away from the conductive solid toward the opposite side, and therefore toward the surface (e.g., the surface containing the assay site (e.g., the reactor)). In some embodiments, where the surface containing the assay site is part of a closed channel (e.g., a microfluidic channel), the conductive solid repelling the trap through negative dielectrophoresis is located adjacent to the part of the channel opposite the assay site. As described above, negative dielectrophoresis can be used with a appropriately selected frequency for the electric field, which can be screened by testing various fields in the presence of the trap until a repulsive effect is observed. In some embodiments in which negative dielectrophoresis is used, at least a portion of the conductive solids (e.g., electrodes) are adjacent to a surface (e.g., a surface comprising the assay site) (e.g., directly adjacent). Such conductive solids may form a network of electrodes (e.g., as wires) on the surface surrounding at least a portion of the assay site on the surface. For example, in some embodiments, the assay site is a surface reactor, and at least a portion of the surface area surrounding the reactor comprises conductive solids electrically connected conductively or dielectrically to a force field generator (e.g., a power source). Figure 3C shows a schematic top view of one such embodiment, in which an assay site 110 in the form of a reactor (e.g., a microwell) is provided, with the surface 120 comprising an assay site 110 in the form of a reactor (e.g., a microwell) surrounded by a network of conductive solids 42 in the form of wire electrodes adjacent to the surface 120, which are electrically connected conductively or inductively to a power source 44 via an electrical connection 45, and a capture object 100 in the form of polarizable dielectric beads is located adjacent to the assay site 110. The repulsive force from such conductive solids adjacent to the surface causes a polarizable dielectric trapping object (e.g., in the form of a bead) on the surface but not inserted into the reactor to move along the surface toward the reactor (where the trapping object does not repel).For example, Figure 3D shows that the captured object 100 moves toward the assay site 110 due to repulsive dielectrophoretic forces from the network of conductive solids 42 toward the assay site 110 due to repulsive dielectrophoretic forces, as indicated by arrow 43, (for example, in response to the formation of an uneven electric field by the application of alternating current by the conductive solids 42), resulting in the insertion of the captured object 100 into the assay site 110. In this method, the immobilization of the captured object by insertion into the reactor can be accelerated via repulsive forces toward and / or along the surface due to dielectrophoresis.

[0059] In some embodiments, positive dielectrophoresis is used, where an inductive effect from an electric field acts on the captured object (including an uncharged captured object), causing it to move toward a surface that may contain the assay site (e.g., toward the surface containing the assay site and / or along the surface toward the assay site). In some such embodiments, a conductive solid in conductive or inductive electrical communication with a force field generator (e.g., a power source) is located adjacent to (e.g., directly adjacent to) the surface containing the assay site, and as a result, the captured object, delivered in close proximity to the assay site and attracted to the conductive solid, moves toward the surface containing the assay site (e.g., a reactor). As described above, positive dielectrophoresis can be used with a appropriately selected frequency for the electric field, which can be screened by testing various fields in the presence of the captured object until an inductive effect is observed.

[0060] In some embodiments in which positive dielectrophoresis is used, at least a portion of the conductive solid (e.g., electrodes) is adjacent to the bottom of the assay site on the surface (e.g., directly adjacent). For example, in some embodiments, the assay site is a surface reactor, and at least a portion of the area of ​​the bottom surface of the reactor (e.g., the bottom surface of a microwell) comprises a conductive solid electrically connected conductively or dielectrically to a force field generator (e.g., a power source). The attractive force from such conductive solid at the bottom of the assay site can move a captured object adjacent to the reactor, e.g., a captured object on the surface but not inserted into the reactor, toward and / or along the surface toward the reactor. In this manner, the immobilization of the captured object by insertion into the reactor can be accelerated via the attractive force toward and / or along the surface due to dielectrophoresis.

[0061] Some embodiments of this disclosure for immobilizing a captured object with respect to an assay site may facilitate immobilization using a sequential or parallel combination of a force field from a force field generator (e.g., a magnetic field, an electric field) and a force from the receding meniscus of a fluid plug, while other embodiments may facilitate the assembly of the captured object primarily (or entirely) through the application of a force from an externally applied force field from a force field generator. For example, in some embodiments using digital (e.g., in contrast to substantially continuous flow as described in detail below) microfluidic technology to deliver the captured object in close proximity to an assay site on a surface, the magnitude of the force due to the receding meniscus of the fluid plug may be relatively small and may not be in a direction that facilitates the delivery of the captured object to the assay site. In some such embodiments, the externally applied force field from a force field generator may be the primary or sole reliance for facilitating the delivery of the captured object to the assay site, substantially less than the force generated by the receding meniscus, thereby, in some cases, it would not be necessary to generate a first-direction receding meniscus and a second-direction receding meniscus. For example, in some embodiments, a captured object can be associated with an assay site on a surface by: flowing a fluid plug containing the captured object onto the assay site (e.g., bringing it into contact with and wetting the assay site) using digital microfluidic techniques (e.g., electrowetting and / or electrophoretic techniques on a dielectric); reducing the distance between the captured object and the assay site by generating an uneven electric field and applying external dielectrophoretic force to the captured object attached to the delivery step; and associating the captured object with respect to the assay site through the application of force that relies at least partially on the dielectrophoretic force.In some embodiments, an apparatus is provided for associating a capture object with an assay site on the surface of an assay consumable, wherein the force field generator comprises a power source and a conductive solid (e.g., an electrode) electrically connected to the power source electrically or inductively, adjacent to or opposite the surface of the assay consumable, and the apparatus comprises a controller having one or more processors configured to initiate the application of a voltage by the power source to at least some of the conductive solid to generate an electric field that moves a fluid plug across at least some of the surface of the assay consumable (e.g., to one or more assay sites). The conductive solid that generates the electric field that moves the fluid plug may be adjacent to the surface of the assay consumable (e.g., beneath a dielectric layer). One or more processors may be configured to signal the power source to apply a voltage to at least some of the conductive solid, and then subsequently to signal the power source to apply similar or different voltages to different conductive solids. In some embodiments, one or more processors are configured to initiate the application of a voltage by the power source to at least some of the conductive solid to generate an uneven electric field that can be applied to dielectric capture objects adjacent to the surface that can polarize dielectrophoretic forces. For example, one or more processors may be configured to cause a power source to send a signal that applies a voltage at an alternating frequency to induce dielectrophoresis. Some of these conductive solids that generate the unequal electric field may be the same ones used to cause the movement of a fluid plug (e.g., via a digital microfluidic process) across at least a portion of the surface. However, in other embodiments, one or more processors are configured to use some conductive solids (e.g., adjacent to or opposite the surface) to cause a power source to initiate the application of a voltage to at least a portion of the conductive solids to generate a unequal electric field, and the other conductive solid (e.g., adjacent to the surface) that is receiving a voltage from the power source is used to move a fluid plug across at least a portion of the surface (e.g., using digital microfluidic techniques (e.g., electrowetting in dielectric techniques)).In some such embodiments, the voltage applied to the conductive solid to generate an uneven electric field and to which dielectrophoretic force can be applied is applied with a different magnitude and / or for a different duration than the voltage applied to the conductive solid to cause the movement of the fluid plug. The conductive solid generating the uneven electric field (e.g., an electrode) may be in conductive or inductive electrical communication with the same power source as the conductive solid that induces the movement of the fluid plug over at least a portion of its surface, or with a different power source.

[0062] The force field generator may be a component of a device for immobilizing a captured object. The force field generator may be adjacent to the assay consumable when operably connected to the assay consumable handler. It should be understood that when the first object is adjacent to the second object, one or more intervening objects may be present between the first object and the second object. In some embodiments, the force field generator is directly adjacent to the assay consumable when operably connected to the assay consumable handler, and there are no intervening components between the force field generator and the assay consumable. Referring again to Figure 3A, the device 1 may include a force field generator 40, and the device 1 may have at least one configuration in which the force field generator 40 is adjacent to and below the assay consumable 5 when the assay consumable is present (e.g., operably connected to the assay consumable handler 10). In some such embodiments, the force field generator 40 of the device 1 includes a magnet (e.g., a permanent magnet, an electromagnet). In some such examples, a force field generator (e.g., force field generator 40) is configured to generate a magnetic field that produces a magnetic field vector directed from the surface toward the bottom of the assay site (e.g., assay site 110).

[0063] Field generation procedure The generation of a force field (e.g., magnetic field, electric field) in close proximity to the surface containing the assay site may occur at any of several points in time during the execution of the described method. In some embodiments, the force field is generated before the delivery of the capture object, while in certain embodiments, the force field is generated during the delivery of the capture object, and in some embodiments, the force field is generated after the delivery of the capture object to the surface. For example, Figure 2A shows a capture object 100 of fluid 130 being delivered in close proximity to the assay site 110 in the absence of a force field, but in some embodiments, a force field represented by a vector field 45 may be present before the delivery of the capture object.

[0064] Fluid plug flow In some embodiments, the delivered capture object is contained within a fluid plug. For example, the delivered capture object 100 in Figure 2A may be contained within a fluid plug 130. The fluid plug (or equivalent bolus) used in this disclosure is an isolated volume of fluid that is at least partially in contact with an immiscible phase (e.g., a gas phase or an immiscible liquid phase) other than the solid channel wall(s) or other solid surface with which it is in contact. The fluid plug is not limited to any particular volume or shape. For example, some fluid plugs may be relatively small compared to the size of the channel through which they are contained (e.g., 3 μL or less in some fluid systems suitable for particular embodiments of this disclosure), while other fluid plugs may be relatively large (e.g., 15 μL or more, 30 μL or more, or more). Some fluid plugs may have a shape that substantially conforms to the cross-sectional shape (e.g., having a circular, square, or rectangular cross-section) of the channel through which they flow (despite the meniscus described later), under certain conditions (e.g., when flowing through a channel having a circular, square, or rectangular cross-sectional shape). However, some fluid plugs may have a substantially non-cylindrical shape for at least a portion of their length along the direction of their flow, and may generally have a shape that depends on and conforms to the shape and configuration of the channel through which they flow (e.g., partial passage through channel turns or crossings, changes in the shape or size of the channel along the direction of flow). Some fluid plugs passing through a channel may have a length in the direction of the channel that is substantially larger (e.g., 2, 3, 5, 10, or more) than the typical cross-sectional size of the channel, and may also have a length of any desirable ratio to the total channel length, and in some cases, a length greater than that. In some embodiments, the captured object is delivered in close proximity to the assay site by flowing a fluid plug containing the captured object through the assay site, at least partially, while in other embodiments, the captured object may be delivered in close proximity to the assay site, separately from the fluid plug.For example, the captured object may be deposited in close proximity to the surface by a different fluid, or in the absence of a fluid, following a step of injecting a fluid in close proximity to the surface to form a fluid plug.

[0065] In some embodiments, the fluid plug containing the captured object flows in a first direction. For example, Figure 2C shows a schematic diagram of the fluid plug 130 flowing in a first direction 150. As will be described in detail below, flowing the fluid plug containing the delivered captured object close to the assay site can contribute to the immobilization of the captured object to the assay site (e.g., inserting beads into a reactor). The fluid plug is generally separated from the solid object and / or immiscible fluid by one or more interfaces. The interface between the fluid plug and the immiscible fluid surrounding the fluid plug may form a meniscus, and its shape may depend on the surface tension effect determined by the composition of the fluid plug, the immiscible fluid and / or any solid surface in contact with the fluid plug. In some embodiments, the fluid plug includes a first meniscus and a second meniscus, each adjacent to the immiscible fluid. Referring to Figure 2A, for example, the fluid plug 130 may have a first meniscus 131 adjacent to the first immiscible fluid 134, and a second meniscus 132 adjacent to the second immiscible fluid 135.

[0066] In some embodiments, the fluid plug containing the captured object contains a liquid. For example, the fluid plug may contain water (e.g., as a solvent for an aqueous solution (e.g., a buffer solution)). In some embodiments, the fluid plug contains a solution containing one or more reagents (e.g., a substrate that can react with a binding ligand that can at least partially associate with the captured object). In certain examples, the fluid plug contains an organic liquid (e.g., N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), alcohol (e.g., ethanol or 2-propanol)). Any of the various immiscible fluids may be used in conjunction with the fluid plug. In some embodiments, the immiscible fluid (e.g., immiscible fluid 134 or immiscible fluid 135) is a gas or contains a gas. Exemplary gases include inert gases (e.g., nitrogen gas, argon), non-inert gases (e.g., oxygen gas), or mixtures thereof (e.g., ambient air). In some embodiments, the immiscible fluid contains a liquid that is not miscible with the fluid in the fluid plug. As one example, in some embodiments where the fluid plug contains water (e.g., an aqueous solution), one or more immiscible fluids adjacent to the fluid plug contain oil (e.g., a hydrofluoroether oil).

[0067] In some embodiments, a fluid plug is introduced to a surface containing the assay site (e.g., an assay consumable) via a fluid injector. For example, the apparatus 1 may include a fluid injector 50 configured to generate a fluid plug having a first meniscus and a second meniscus adjacent to an immiscible fluid (e.g., a gas) when it is on the surface of the assay consumable.

[0068] In some examples, a fluid injector is connected to a channel of an assay consumable having a surface containing the assay site. Figures 3A-3B show a fluid injector 50 that is hydrodynamically connected to an assay consumable 5 when operably connected to an assay consumable handler 10, for example, the fluid injector 50 may be further hydrodynamically connected to a fluid pump and a fluid source (e.g., a source of sample or reagent fluid). The fluid injector 50 can inject a fluid plug 130 having a first meniscus 131 and a second meniscus 132. A pump 60 can cause the fluid plug 130 to flow across the surface 120 of the assay consumable 5. For example, in some embodiments, the pump 60 is an air or vacuum pump positioned distal to the fluid plug 130 with respect to the fluid injectors 20 / 50 (shown in Figure 3A), and the pump 60 is configured to provide a source of pressurized air and / or vacuum that generates a differential pressure causing flow in the fluid plug 130 across the surface 120 (e.g., across at least a portion of the assay site 110). In alternative embodiments, the fluid pump 60 may pump a liquid that is immiscible with the fluid plug 130. In certain embodiments, the fluid pump 60 may be hydrodynamically coupled to the fluid injectors, for example via a switchable / controllable fluid connection to port 20, and may facilitate bidirectional fluid movement in the fluid plug 130 by selectively and alternately applying pressure / vacuum to the inlet of the fluid pump 60 relative to the flow path (to the left of the plug 130 as shown in Figure 3A) and to the inlet of the fluid injector 50 relative to the flow path (to the right of the plug 130 as shown in Figure 3A).

[0069] By flowing a fluid plug containing the captured object in a first direction, a first-direction advancing meniscus and a first-direction receding meniscus can be created. Referring to Figure 2C, for example, by flowing a fluid plug 130 containing the captured object 100 in a first direction 150 (defined by an arrow pointing from right to left), a first-direction advancing meniscus 152 adjacent to an immiscible fluid 135 (e.g., air) and a receding meniscus 151 adjacent to an immiscible fluid 134 (e.g., air) are defined. In Figure 3A, the fluid plug 130 flows to the fluid pump 60 to the left (for example, depending on the application of vacuum by the fluid pump 60), the first meniscus 131 becomes a receding meniscus, and the second meniscus 132 becomes an advancing meniscus.

[0070] In some embodiments, the fluid plug flows in a first direction such that the first-direction receding meniscus flows across at least a portion of the assay site on the surface. One example of this is illustrated in Figure 2C, where the first-direction receding meniscus 151 flows across at least a portion of the assay site 110. Flowing the receding meniscus of the fluid plug across at least several assay sites can facilitate the fixation of the captured object in the fluid plug to the assay site. In a particular example, flowing the receding meniscus of a fluid plug containing beads across a reactor (e.g., a well) on the surface can facilitate the insertion of the beads into the reactor. In the context of this disclosure, it has been found that specific operational and dimensional parameters of such flow can contribute to relatively efficient and effective fixation. Some such embodiments involve configuring the flow such that components toward the surface and perpendicular to it (e.g., toward the bottom of the reactor) form a meniscus to which force can be applied to the captured object. In some embodiments, the first-direction receding meniscus flows over at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or more of the assay site during the step of flowing the fluid plug in the first direction. In some embodiments, the first-direction receding meniscus flows over the entire assay site (e.g., the entire fluid plug flows over the assay site on the surface). For example, Figure 2D shows the entire fluid plug 130, including the first-direction receding meniscus 151, flowing over the assay site 110 on the surface 120. After the first-direction receding meniscus has flowed over at least a portion (or all) of the assay site, some of the captured material may be immobilized with respect to the assay site, while some of the captured material may remain in close proximity to the assay site without being immobilized, and other captured material may still be present in the fluid plug. For example, referring again to Figure 2D, the captured object 111 is immobilized with respect to the assay site 110, while the captured object 112 remains adjacent to the assay site 110 without being immobilized, and the captured object 113 remains contained in the fluid plug 130 even after the entire fluid plug 130 has flowed over the assay site 110.

[0071] In some embodiments, the fluid plug containing the captured object flows over at least a portion of the assay site on the surface in a single pass, while in certain embodiments, the fluid plug flows over the assay site multiple times. Some such embodiments may involve reversing the direction of the fluid plug flow. It has been observed in the context of this disclosure that flowing the fluid plug (e.g., including its receding meniscus) over the assay site multiple times may result in unexpectedly efficient immobilization of the captured object with respect to the assay site. In some embodiments, the fluid plug flows in a second, different direction (relative to a first direction). In some cases, the second direction is opposite to the first direction (e.g., different by an angle of 180 degrees). For example, Figure 2E shows a fluid plug 130 flowing in a second direction 160, which is the opposite of the first direction 150 shown in Figure 2C. The flow of the fluid plug in the second direction defines a second-direction advancing meniscus and a second-direction receding meniscus. The embodiment illustrated in Figure 2E shows, for example, how a fluid plug 130 having a second forward-moving meniscus 162 and a second receding-moving meniscus 161 flows. In some cases, the fluid interface defining the first receding-moving meniscus is the same as that defining the second forward-moving meniscus, and the fluid interface defining the first forward-moving meniscus is the same as that defining the second receding-moving meniscus.

[0072] In some embodiments, the fluid plug flows in a second direction such that the second receding meniscus flows over at least a portion of the assay site on the surface. Referring again to Figure 2E, the second receding meniscus 161 flows over at least a portion of the assay site 110. Such flow may result in further immobilization of the captured object with respect to the assay site. For example, referring again to Figure 2E, with the flow of the fluid plug 130 in the second direction 160, the second receding meniscus 161 may contribute to the immobilization of one of the captured objects 114 of the assay site 110. We have found that a second “meniscus sweep” over part or all of the assay site in the context of this disclosure can, in some examples, efficiently and rapidly immobilize the captured object with respect to the assay site, particularly captured objects that were not immobilized during the flow of the first receding meniscus. In embodiments where specific size and operational parameters result in a receding meniscus that applies a force that moves the captured object toward the surface and perpendicular to it, such multiple sweeps of the fluid plug meniscus over part or all of the assay site can result in the immobilization of an unexpectedly large number of captured objects compared to simple flow or even simple bidirectional flow methods. In some embodiments, the second-direction receding meniscus is flowed over at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or more of the assay site during the step of flowing the fluid plug in the second direction. In some embodiments, the second-direction receding meniscus is flowed over the entire assay site (e.g., the entire fluid plug is flowed over the entire assay site on the surface).

[0073] One way in which a fluid plug can be flowed across a surface in the described method is via a fluid pump. In some embodiments, the described apparatus (e.g., Apparatus 1) comprises a fluid pump capable of moving fluid across at least a portion of the surface, and a controller having one or more processors configured to regulate the fluid pump to move the fluid plug bidirectionally across at least a portion of the surface. Referring to Figures 3A-3B, for example, Apparatus 1 may comprise a fluid pump 60 that, when operably connected to an assay consumable handler 10, is in fluid communication with the assay consumable 5, and the fluid pump 60 may be configured to move the fluid plug 130 bidirectionally across the surface 120 of the lower portion 6 of the assay consumable 5, represented by a double arrow 139. The fluid pump can produce bidirectional flow of fluid across the surface in any of a variety of ways. For example, the fluid pump may be configured to alternate between applying a positive differential pressure to the fluid plug (e.g., by pressurizing a gas after the fluid plug) and applying a negative differential pressure (e.g., by applying a vacuum). The fluid pump 60 can be controlled by one or more controllers (e.g., controller 30). For example, the controller may comprise one or more processors programmed to provide operating signals to the fluid pump in an appropriate sequence, or one or more processors capable of receiving input signals from a user to activate the fluid pump. In some embodiments, one or more processors can regulate the fluid pump to flow the fluid plug in a first direction so that the first receding meniscus of the fluid plug flows over part or all of the reactor, and to flow the fluid plug in a second different direction so that the second receding meniscus of the fluid plug flows over part or all of the reactor. In some embodiments, the controller comprises one or more processors capable of regulating the fluid pump to flow fluid bidirectionally by being programmed to operate the fluid pump to provide positive pressure, and then to operate the fluid pump to provide negative pressure.Alternatively, in some embodiments, the controller comprises one or more processors that can cause fluid to flow bidirectionally by regulating a fluid pump, which is programmed to actuate the fluid pump to apply positive (or negative) pressure in a first direction, and then actuate the fluid pump to apply positive (or negative) pressure in a second, different direction.

[0074] Meniscus force and parameters As described above, specific operational (e.g., flow pattern, flow velocity, contact angle) and dimensional (e.g., fluid plug volume, channel size) parameters are determined in the context of this disclosure and thereby affect the immobilization of the captured object via the fluid plug technology. In some cases, the method may be designed so that the forces contributed by the receding meniscus (e.g., a first-direction receding meniscus and / or a second-direction receding meniscus) facilitate or improve the immobilization of the captured object. When a fluid plug is flowed, the meniscus of the plug generally generates flow-induced capillary forces. Figure 2C illustrates exemplary capillary forces as arrows 153 radiating from a first-direction receding meniscus 151 and a first-direction advancing meniscus 152. One way such forces are generated is by flowing the fluid plug such that the receding meniscus applies capillary forces in the direction of the surface containing the assay site and in the direction perpendicular thereto. Referring again to Figure 2C, the fluid plug 130 can be flowed such that the first receding meniscus 151 applies a capillary force of component 155 perpendicular to the surface 120. In embodiments including, for example, the insertion of beads into the reactor, the capillary force acting in the direction of the surface (and perpendicular to the bottom of the reactor) can act on the beads and push them into the wells. For example, a capillary force having component 155 perpendicular to the surface 120 can push the captured object 111 into one of the assay sites 110, immobilizing the captured object 111 as shown in Figure 2C. In some embodiments, such action can result in relatively effective bead insertion. It should be understood that no arbitrary receding meniscus necessarily has capillary forces having components toward the surface and perpendicular thereto, or that any such force contributes to the immobilization of the captured object to a sufficiently large extent. Instead, such forces may require the provision of specific operational and dimensional parameters described in the context of this disclosure. The inventors have determined certain appropriate parameters.Manipulation outside of these parameters can result in a lack of "downward" capillary force from the receding meniscus, and instead of generating forces that contribute to the immobilization of the captured object as described above, it will generate capillary forces that tend to move the captured object mainly in a direction parallel to the surface containing the assay site, or even away from the assay site, thereby impairing the immobilization of the captured object.

[0075] The fluid plug may be flowed using any of the following techniques. For example, in some embodiments, the fluid plug is acted upon by a positive pressure source (e.g., a fluid pump, pipette, or syringe) and / or a negative pressure source (e.g., a negative pressure source, pipette, or syringe). Some such embodiments may include a device (e.g., device 1) configured to apply a positive and / or negative pressure difference to the fluid plug. In Figure 3A, device 1 includes a fluid pump 60 in fluid communication with a fluid plug 130 on the surface 120 of an assay consumable 5 configured to apply such a positive and / or negative pressure difference. In some embodiments, other fluid techniques (e.g., capillary flow, electrowetting (EWOD) on a dielectric, electrophoresis, etc.) may be used. One way in which EWOD technique may be used is by configuring the fluid pump to move the fluid in the channel by applying a potential across two or more electrodes connected to the assay consumable. In some cases, the surface itself may be positioned to generate gravity-induced flow in the fluid plug.

[0076] contact angle One parameter that can contribute to a receding meniscus that promotes or improves the immobilization of a captured object (for example, via capillary forces having components on and perpendicular to the surface containing the captured object) is the contact angle of the receding meniscus during flow. “Receding meniscus contact angle” means the angle between the surface containing the assay site and the receding contact line as the fluid plug flows. As a specific example, Figure 2F shows the contact angle θ between the surface 120 and the receding contact line 156 of the receding meniscus 151 as the fluid plug 130 flows in a first direction 150, according to a particular embodiment. In the context of this disclosure, it has become clear that having a relatively small contact angle of the receding meniscus can contribute to capillary forces from the receding meniscus, thereby promoting the immobilization of the captured object. As will be apparent to those skilled in the art, the contact angle of the receding meniscus of a fluid plug can be measured, for example, using an angle gauge or an equivalent part of an imaging device. The contact angle between flows can be influenced and regulated using various parameters such as the flow pattern (e.g., substantially continuous vs. otherwise), flow velocity, fluid plug composition (e.g., type of liquid), immiscible fluid composition (e.g., type of gas), and surface composition. For example, the strength of the intermolecular interactions between the fluid plug composition and the surface composition can be selected (e.g., based on the polarity of the fluid plug and / or the hydrophobicity / hydrophilicity of the surface) so that a desired contact angle is achieved. In some embodiments, the surface containing the assay site is or contains a hydrophobic material (e.g., a hydrophobic polymer material), and examples of these will be described in more detail later in the context of assay consumables. Certain combinations of parameters (e.g., flow velocity, surface tension, viscosity) may be expressed as a dimensionless quantity, e.g., capillary number (described in more detail later). In some, but not all, embodiments, operations that result in a certain range of such dimensionless quantities (e.g., capillary number) can produce a contact angle of a receding meniscus that results in capillary forces of directionality and magnitude that promote the fixation of captured objects with respect to the assay site.

[0077] In some embodiments, for at least some steps of flowing the fluid plug across the assay site (e.g., in a first direction), the receding meniscus (e.g., a first-direction receding meniscus) has a contact angle with the surface of less than 90 degrees, 60 degrees or less, 45 degrees or less, 30 degrees or less, 15 degrees or less, or less. Such low contact angles can be maintained for the entire duration of the fluid plug flow steps (e.g., flowing in the first direction at a constant contact angle) in certain embodiments. In certain embodiments, for the entire duration of the fluid plug flow across the assay site (e.g., in a first direction), the receding meniscus (e.g., a first-direction receding meniscus) has a contact angle with the surface of less than 90 degrees, 60 degrees or less, 45 degrees or less, 30 degrees or less, 15 degrees or less, or less. Flowing the fluid plug in a second different direction can be performed such that the second-direction receding meniscus also has contact angles within these ranges. For example, this type of flow can be performed using continuous flow techniques.

[0078] This type of flow is in contrast to certain conventional flow techniques used in microfluidic systems (e.g., conventional segmented flow techniques) that can produce changes in the receding meniscus contact angle between different flow segments (e.g., a first contact angle while moving and another second contact angle when the fluid plug is static). In some embodiments, the described apparatus comprises one or more processors configured to adjust a fluid pump (e.g., fluid pump 60 in Figures 3A-3B) to flow the fluid plug so that the contact angle remains within the above range. For example, one or more processors may be programmed to drive the pump to apply appropriate positive and / or negative pressure to the fluid plug (e.g., in the channel) to achieve a flow velocity that yields the above contact angle. Such results can be achieved by adjusting the fluid pump, taking into account the appropriately programmed size of the assay consumables (e.g., channel height) and the surface material properties of the fluid plug and assay consumables (e.g., relative hydrophobic / hydrophilic).

[0079] Flow Pattern As described above, in some embodiments, the fluid plug containing the captured object is flowed through in a substantially continuous flow pattern. As is known in the art, continuous flow refers to a fully formed (e.g., steady-state) flow (e.g., a fully formed laminar flow through a narrow channel having a parabolic velocity profile), in which case the flow is driven primarily by a propulsive force having sufficient uniformity and continuity to create a fully formed flow pattern, such as an external pressure source (e.g., a pump and a vacuum source), capillary force, etc. For example, a positive pressure source on the right side (or a negative pressure source on the left side) of the fluid plug 130 can cause the fluid plug 130 to flow in the first direction 150 in Figure 2C. The fluid pump 60 of the apparatus 1 may supply such positive pressure.

[0080] Substantially continuous flow in a fluid plug is achieved under conditions that establish a fully formed laminar flow in the plug (sufficiently large fluid plug size, sufficiently high flow velocity, and sufficiently continuous thrust). In some such examples, the fluid plug can flow substantially continuously, and with a velocity profile of the fluid plug in a plane parallel to the direction, which is substantially parabolic as a characteristic of a continuous laminar flow profile. Providing a substantially continuous flow pattern that results in a laminar and parabolic flow in the fluid plug is in contrast to flow patterns in other fluid (e.g., microfluidic) systems, such as segmented flow, where small units of fluid in a first phase flow are completely surrounded by an immiscible fluid phase, thereby enabling the establishment of the above-mentioned fully formed flow pattern with a parabolic flow profile, without contact with the flow channel wall. In segmented flow, small droplets of fluid (e.g., water droplets suspended in immiscible oil passing through a channel) translate substantially stationarily within the immiscible fluid in which they are suspended. The substantially continuous flow of the fluid plug also contrasts with digital microfluidics, where small droplets in the fluid are translated over short, fixed distances in the channel for each separate operating event of insufficient duration, uniformity, and / or size (e.g., electrowetting techniques on dielectrics) to form a fully formed flow pattern; that is, in contrast to the continuous propulsion described above (e.g., from a pressure source), which is appropriate in the particular embodiments disclosed.In many conventional microfluidic systems and techniques, the substantially continuous flow format of a laminar parabolic flow is inconvenient and is considered unsuitable or impractical compared to other techniques such as segmented flow or digital microfluidic techniques due to potential challenges such as Taylor dispersion, solute surface interactions, cross-contamination, and the need for large amounts of reagent and relatively long channel lengths (see, for example, Solvas, XC, & DeMello, A. (2011). Droplet microfluidics: recent developments and future applications. Chemical Communications, 47(7), 1936-1942), which is incorporated herein by reference for all purposes). However, it has become apparent in the context of certain disclosed embodiments that substantially continuous flow of a fluid plug under certain conditions can be effectively used to promote or improve the immobilization of captured objects with respect to an assay site. For example, maintaining a substantially parabolic velocity profile for a fluid plug under laminar flow can result in a substantially parabolic receding meniscus shape. Such a shape can promote the immobilization of captured objects by providing capillary forces with appropriate directionality.

[0081] Channel size In some, but not all, embodiments, the surface through which the fluid plug flows is part of a channel. The channel may be an open channel (e.g., having a bottom and two sides) or a closed channel. For example, referring to Figure 2F, in certain embodiments, surface 120 may be part of a closed channel defined at least partially by surface 120 and upper surface 122. The fluid plug 130 flows through the channel defined by surface 120 and upper surface 122. As described above, the channel may be part of an assay consumable containing an assay site. The size of the channel through which the fluid plug flows can affect the capillary force applied to the captured object by the fluid plug. For example, when the fluid plug 130 flows in a first direction 150, the channel height 148 defined by surface 120 and upper surface 122 can affect the contact angle θ between surface 120 and the receding contact line 156 of the receding meniscus 151. For example, a particular channel height relative to the fluid plug volume may facilitate a substantially continuous layered flow with a parabolic receding meniscus. Such parabolic receding meniscuses may have lower contact angles compared to receding meniscus shapes specific to other flow patterns (e.g., droplets in split and / or digital microfluidic flows). Contact angles such as those described affect the application of force to captured objects and their immobilization to the assay site. In some embodiments, the channel height is relatively large compared to conventional microfluidic techniques. In some embodiments, the channel has a height of 100 micrometers or more, 150 micrometers or more, 200 micrometers or more, 250 micrometers or more, 350 micrometers or more, 400 micrometers or more, 450 micrometers or more, and / or at most 500 micrometers, at most 600 micrometers, at most 800 micrometers, at most 1 mm or more at the assay site.

[0082] flow rate As described above, the flow velocity of the fluid plug across the assay site (e.g., reactor) is a potential operational parameter that can affect the fluid plug behavior and the immobilization of the captured object. In some embodiments, the flow velocity is selected such that the force generated by the receding meniscus (e.g., a first-direction receding meniscus and / or a second-direction receding meniscus) as the fluid plug flows (e.g., in a first direction (or a second direction)) becomes a downward force on the captured object against the surface containing the assay site.

[0083] The downward force may have components oriented toward the surface and perpendicular thereto. The resulting meniscus shape with such a downward force can be characterized as a substantially continuous flow pattern (in contrast to other flow patterns, e.g., those characteristic of turbulence or digital microfluidics). The fluid plug velocity may be selected to produce such a substantially continuous flow. One way the fluid velocity can contribute to the immobilization of the captured object (including, in some examples, relatively efficient and rapid immobilization) is by its effect on the receding meniscus contact angle. In the context of this disclosure, it has become clear that as the fluid plug velocity (e.g., volumetric flow rate) increases, the contact angle of the receding meniscus generally decreases. Furthermore, it has become clear that flowing a fluid plug with a sufficiently high velocity results in a sufficiently low receding meniscus contact angle for capillary forces, which can contribute to the immobilization of the captured object, rather than simply translating it laterally or away from the assay site.

[0084] In some embodiments, the fluid plug is flowed at a velocity of 1 μL / s or more, 2 μL / s or more, 5 μL / s or more, 10 μL / s or more, 15 μL / s or more, 20 μL / s or more, 25 μL / s or more, 30 μL / s or more, or 40 μL / s or more, or greater (e.g., in a first direction, in a second direction). In some embodiments, the fluid plug is flowed at a velocity of 100 μL / s or less, 80 μL / s or less, 60 μL / s or less, 50 μL / s or less, 45 μL / s or less, or less (e.g., in a first direction, in a second direction). Combinations of these ranges are possible. For example, in some embodiments, the fluid plug is flowed at velocities of 1 μL / s or more and 100 μL / s or less, 20 μL / s or more and 100 μL / s or less, or 40 μL / s or more and 50 μL / s or less (e.g., in a first direction, in a second direction). These velocities are contrary to certain conventional fluid plug flow implementations in the field of microfluidics that favor lower velocities. One reason why low velocities (e.g., 10 μL / s or less) are typically used in certain conventional fluid plug / droplet flow techniques related to microfluidics is that droplets are considered more stable at such velocities. For example, in Guan, Y., Li, B., Zhu, M., Cheng, S., & Tu, J. (2019). Deformation, speed, and stability of droplet motion in closed electrowetting-based digital microfluidics. Physics of Fluids, 31(6), 062002, which is incorporated herein by reference for all purposes, it is reported that the plug becomes unstable at high fluid plug velocities. However, surprisingly, from the literature, it has become clear that, in the context of this disclosure, such high flow velocities can improve the speed and efficiency of immobilization of the captured object under selected conditions. In some embodiments, the apparatus described comprises one or more processors configured to regulate a fluid pump (e.g., fluid pump 60 in Figures 3A-3B) to flow the fluid plug over a high flow velocity range, without being bound by the conventions shown above.For example, one or more processors may be programmed to operate a pump to apply appropriate positive and / or negative pressure to the fluid plug (e.g., in the channel) to achieve such a flow velocity (and, for example, the force generated by the meniscus of the fluid plug (e.g., the first or second meniscus) results in a downward force on the captured object with respect to the surface of the assay consumable).

[0085] Plug volume In some embodiments, the fluid plugs are relatively large. While typical conventional microfluidic flow techniques use relatively small droplets to deliver, for example, suspended objects, it has become apparent in the context of this disclosure that fluid plugs with larger volumes can be more stable and, compared to fluid plugs with smaller volumes, can achieve the desired flow patterns described herein. As one example, flowing a relatively small fluid plug (e.g., 3 μL or less) at a relatively high flow velocity (e.g., 40 μL / s) can result in unstable flow in certain environments (e.g., relatively small channels, e.g., maximum cross-sectional size perpendicular to the flow direction of 2 microns or less, 1 micron or less, 500 microns or less, or less). Such unstable flow can manifest, for example, with larger fluctuations in the contact angle. In contrast, flowing a relatively large fluid plug (e.g., relative to the channel size) at a similarly high flow velocity can, surprisingly, result in a stable flow (more suitable for immobilizing the captured object). The volume of the fluid plug, in combination with other factors (e.g., flow velocity and the nature of the propulsive force for the flow), can contribute to achieving the flow pattern discussed here (e.g., a substantially continuous, radial flow). The flow pattern can then be influenced by factors such as the receding meniscus shape and contact angle. In certain examples, the use of a fluid plug with a relatively large volume can allow for a flow velocity high enough to achieve a receding meniscus with contact angles within the ranges described above, while maintaining satisfactory stability. In some embodiments, the fluid plug containing the trapping material (e.g., beads) has a volume of 3 μL or more, 10 μL or more, 15 μL or more, 20 μL or more, 25 μL or more, 30 μL or more, or larger. In some embodiments, the fluid plug containing the trapping material (e.g., beads) has a volume of 100 μL or less, 80 μL or less, 60 μL or less, 40 μL or less, or 35 μL or less. Combinations of these ranges are possible. For example, in some embodiments, the fluid plug containing the captured object (e.g., beads) has a volume of 3 μL or more and 100 μL or less, or 20 μL or more and 50 μL or less.

[0086] Capillary number As described above, certain combinations of parameters described herein (e.g., flow rate, channel size, fluid plug / immiscible fluid composition) can facilitate immobilization of capture objects relative to assay sites. Combinations of several such parameters may be expressed as dimensionless quantities. By way of one non-limiting example, in some aspects, a fluid plug is flowed under conditions that result in a particular range of capillary number. Capillary number (C a ) is a dimensionless quantity representing the ratio of viscous force to surface tension (at a fluid-fluid interface) during fluid flow, and is expressed as follows: [Mathematical formula] wherein μ is the kinematic viscosity of the fluid, V is the velocity of the fluid, and σ is the surface tension at the interface between the fluids, or the interfacial tension between the fluid and a phase that is immiscible therewith (e.g., a gas (e.g., air)). The capillary number during flow may correlate with the contact angle of the fluid and the immiscible phase during flow. Accordingly, operating a system in which fluid plugs are flowed within a particular capillary number regime may result in certain desirable contact angles, for example, those that promote capillary force at a receding meniscus having force components directed downward and toward the assay site, to promote immobilization of capture objects. Selection of appropriate flow rates, fluid plug compositions (e.g., solvent selection) and / or channel structures (e.g., channel height, channel cross-sectional area) can make the component of the meniscus force directed toward the well prominent, and in some instances allow achieving a capillary number that promotes relatively efficient capture object immobilization. In some aspects, the fluid plug has, at 25°C, 1×10 -6 or higher, 2×10 -6 or higher, 5×10 -6 or higher, 1×10 -5 or higher, 2×10 -5 or higher, 5×10 -5 or higher, 1×10 -4 or higher, 2×10 -4 or higher, 5×10 -4 or higher, and / or at most 1×10 -3At most 2 x 10 -3 , at most 5 x 10 -3 Or at most 1 × 10 -2 It flows under the condition that the capillary number is . Combinations of these ranges (e.g., 1 × 10) -6 The above and 1 × 10 -2 Below, 1 x 10 -4 The above and 1 × 10 -3 The following are available. It should be understood that other considerations, whether related to or unrelated to variables expressed as dimensionless parameters such as the capillary number, may also affect the immobilization of the captured object, and that the operations described may not need to be strictly adhered to in certain embodiments.

[0087] Concentration of the captured substance in the fluid plug Each fluid plug can have a relatively small number of suspended capture objects per unit volume of the fluid plug (for example, before the fluid plug is flowed over at least a portion of the assay site). Some such “diluted” fluid plugs may be useful in delivering a relatively small number of capture objects in close proximity to the capture site, while using a relatively large amount of fluid plug as described above (for example, to improve flow stability). This also contrasts with conventional microfluidic loading techniques, which typically use a relatively large number of beads (e.g., more than 200,000) for bead delivery to the assay site. In some embodiments, the number of capture objects in a fluid plug is 50,000 or less, 10,000 or less, 5,000 or less, 1,000 or less, 500 or less, 200 or less, and / or just 150, 100, 50, 10, 5, 1 or less per μL.

[0088] Immobilization of captured object As described above, in some embodiments, at least a portion of the capture object subjected to the step of flowing a fluid plug in a first direction and / or a second direction is immobilized with respect to the assay site. In certain such embodiments, the assay site includes a reactor, and the capture object is a bead, which is immobilized by being inserted into the reactor. Figure 2C shows one such embodiment in which at least a portion of a bead-shaped capture object 100 is inserted into the reactor-shaped assay site 110 as a result of the flow of the fluid plug 130 in the first direction 150. Similarly, the capture object 100 in Figures 3A-3B may be a bead (e.g., a magnetic bead), and the apparatus 1 is configured to insert the bead 100 into the reactor-shaped assay site 110 on the surface 120 of the assay consumable 5. As described above, the force created by the receding meniscus can contribute to the efficient and rapid immobilization of the capture object, which can help to reduce the number of capture objects used in a capture object-based assay. In some embodiments, the assay sites are located at multiple separate locations on a surface (e.g., as an array), and the step of immobilizing at least a portion of the captured material is performed such that at least a portion of the captured material is isolated across multiple separate locations. Some such embodiments may be useful when performing certain types of digital ELISA techniques.

[0089] Synergistic effect between field strength adjustment and fluid flow In the context of this disclosure, it has become clear that (1) generating a force field (e.g., a magnetic field) in close proximity to the assay site, and (2) flowing a receding meniscus of a fluid plug containing the captured object over the assay site, can each contribute individually to the efficient immobilization of the captured object, but the combination of (1) and (2) can exhibit unexpected synergistic effects and improved performance. Although not constrained by any particular theory, it is thought that the generated force field can rapidly localize the captured object in close proximity to the assay site (e.g., near the opening of a reactor). The downward force contributed by the receding meniscus then encounters the captured object relatively close to the assay site, thereby allowing the forces generated by the receding meniscus and the field to efficiently immobilize the captured object. In some cases where a magnetic field is used (e.g., by a permanent magnet present beneath the assay site), magnetic beads may form a chain. In such cases, the receding meniscus can encounter and break up the chain, thereby spreading the magnetic beads and facilitating the insertion of the beads (in the case of a reactor). Furthermore, the combined magnitude of the forces applied by the force vector field and the receding meniscus may increase the tendency to move the captured object toward the assay site.

[0090] In some embodiments, a force field (e.g., a magnetic field) is present for at least some steps of the fluid plug flowing (e.g., in a first direction, in a second direction). However, in some embodiments, the magnitude of the force field is reduced or eliminated before the step of flowing the fluid plug (e.g., in a first direction). As an example, Figures 2C-2E show the fluid plug 130 flowing in a first direction 150 or a second direction 160 while a force field represented by a vector field 45 is present, and some embodiments include removing or reducing the force field before flowing the fluid plug in the first direction 150 and / or the second direction 160. Such adjustment of the force field may be useful in some examples where force-induced phenomena are undesirable, such as chaining of trapped objects that occurs during the flow of a receding meniscus across an assay site. As an example, a magnetic field (e.g., from a permanent magnet and / or electromagnet) may pull magnetic beads toward a surface including a reactor, which may produce some magnetic chaining. The magnitude of the magnetic field may be reduced or completely eliminated, thereby releasing it from the chain. Finally, when the chain of magnetic beads is released, the retracted meniscus of the fluid plug may be circulated over at least a portion (or all) of the assay site to guide at least a portion of the unchained magnetic beads into the reactor. The magnitude of the magnetic field can be reduced, for example, in the case of a permanent magnet, by causing relative movement between the permanent magnet and the surface. For example, referring to Figure 2E, when the force generator 40 is a permanent magnet, the magnitude of the magnetic vector field 45 can be reduced (to, for example, zero) by moving the force field generator 40 in direction 146 and increasing the distance 147 between the force field generator 40 and the bottom of the assay site 110.

[0091] In some cases, the described apparatus can be configured to adjust the magnitude of the force field by causing relative movement between, for example, a force field generator (e.g., a permanent magnet) and an assay consumable having a surface containing the assay site. For example, Figure 3A shows a force field generator 40 in a first position below an assay consumable 5 having a surface 120, and Figure 3B shows a force field generator 40 in a second position at a greater distance from the assay consumable 5. Such an increase in the distance between the force field generator and the assay site may decrease or essentially eliminate the magnitude of the force field at the assay site. Alternatively, or in addition to linear relative movement, lateral and / or rotational movements may be used to increase the distance between the force field generator and the assay site of the consumable. For example, the force field generator may be rotated in a plane such that in a first radial position the force field generator is positioned close to the assay site of the assay consumable, and in a second radial position the force field generator is positioned away from the assay site of the assay consumable. The repositioning (or removal) of the force field generator may be performed manually or, for example, using an automatic moving stage of the device. In some embodiments, Figures 3A-3B show an automatic moving stage 41 which can be controlled, for example, by a controller 30. For example, the magnitude of the magnetic field can be adjusted by the electromagnet by adjusting the magnitude of the current passing through the electromagnet (e.g., reducing it).

[0092] In some embodiments, the magnitude of the force field may be increased at a later point in the process (e.g., after the capture and immobilization of the object). For example, the previously removed magnet may be reintroduced after bead insertion so that the immobilized bead is held in place during a follow-up step (e.g., a sealing step).

[0093] Percentage of captured objects that become fixed in place In some embodiments, a relatively large percentage of the delivered capture material is immobilized (e.g., during the flow steps in the first and / or second directions). Certain existing techniques for immobilizing capture material (e.g., for capture material-based assays (e.g., digital ELISA)) use a large excess of capture material relative to the number of assay sites (e.g., 5, 6 times or more), while certain embodiments herein employ the opposite approach. In the context of this disclosure, in some examples it has become clear that highly sensitive assays (with a low total number of beads, as described later) are possible by delivering a relatively small number of capture material and immobilizing a high percentage of them, while still generating sufficient signal from the capture material for adequate detection. In some embodiments, at least 20%, at least 25%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, at least 99%, or all of the capture material delivered in close proximity to the assay site is immobilized during the flow steps.

[0094] Percentage of captured area In some embodiments, the captured material is immobilized on a relatively small percentage of the assay site on the surface. This method is in contrast to conventional methods, for example, which strive to fill as many array wells as possible with beads (e.g., up to 100% of the wells are filled with beads). In the context of this disclosure, it has become clear that it may be advantageous to ensure that as many captured material is immobilized as possible rather than occupying as many assay sites as possible. One way to do this is to have a significant excess of assay sites with respect to the number of captured material, which results in a state where only a relatively small percentage of the assay site can be immobilized with captured material. In some embodiments, the captured material is immobilized on 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, and / or as little as 1%, as little as 0.5%, as little as 0.1%, as little as 0.01%, or less of the assay site. As illustrated in Figures 2A-2G, the surface 120 may contain 200,000 assay sites 110 in the shape of reactors, or the method may allow only 2,000 capture objects 100 in the shape of beads to be inserted, which means that the capture objects 100 are immobilized on 1% of the assay sites 110.

[0095] Alternative loading methods While the specific embodiments described above relate to immobilizing a captured object with respect to an assay site via a fluid plug flow that generates a force field simultaneously or sequentially, other formats are also available. These other formats can also immobilize the captured object relatively quickly and / or efficiently. For example, in some embodiments, a laterally moving force field is applied to a captured object delivered in close proximity to the assay site. Such a lateral force can facilitate the movement of the captured object throughout the lateral space around the assay site on the surface, increasing the velocity of the captured object interacting with the assay site. One such embodiment includes applying a lateral force to the captured object by adjusting the lateral distribution of the force field and immobilizing at least a portion of the captured object with respect to the assay site via the applied lateral force. Figure 2B shows a specific example of such any embodiment, where the relative lateral movement is made between a force field generator 40 in the form of a permanent magnet and the surface 120, as indicated by arrows 149a and 149b. Such operation of the force field generator 40 can move the magnetic field represented by the magnetic vector field 45 laterally, and according to several embodiments, when they are magnetic, it can act on the captured object 100 (e.g., magnetic beads). This can cause the beads 100 to move laterally with respect to the surface 120, encountering and being inserted into the assay site 110. Adjusting the lateral distribution of the force field can be done while the fluid 130 (e.g., fluid plug) is stationary or while it is in flow.

[0096] In some embodiments, this may include flowing multiple fluid plugs over at least a portion of the assay site. For example, in some embodiments, a first fluid plug containing the captured object is flowed through the assay site, followed by a second fluid plug which is separated from the first fluid plug by an immiscible fluid, where the second plug flows over at least a portion of the assay site, thereby immobilizing at least some of the captured object with respect to the assay site. Figure 2G illustrates one such embodiment in which a first fluid plug 130 containing the captured object 100 flows in a first direction 150 over the assay site 110, and a second fluid plug 230 containing the captured object 100 flows in the first direction 150 after the first fluid plug 130. The first fluid plug 130 and the second fluid plug 230 may be separated by an immiscible fluid 134 (e.g., a gas (e.g., air)). A sequential plug flow method across the assay site allows, in some examples, the use of a plug fluid with a small number of captured objects (e.g., beads), and may or may not involve a force field (e.g., a magnetic field) acting on the captured objects during implementation.

[0097] A device for immobilizing captured objects. As described above, apparatus for immobilizing capture objects with respect to assay sites is also described. Such apparatus may be configured to perform specific methods for immobilization (e.g., with respect to a combination of force field generation and fluid plug flow) as described above. In some embodiments, apparatus configured to perform methods for immobilizing capture objects may be further configured to perform assays (e.g., assays described later) for detecting and / or quantifying analyte molecules or particles of a fluid sample. For example, apparatus for immobilizing capture objects with respect to assay sites may include (e.g., one or more components for preparing capture objects for detection, such as a sample washer, incubation, etc.) or one or more components for detection or analysis (e.g., an imaging system, a computer-implemented control system). In some embodiments, such combinations of components may be integrated in a robotic system, while in other embodiments, some or all of these components for, for example, sample preparation, capture object immobilization (e.g., with respect to assay sites), and image acquisition / analysis may be integrated as a microfluidic system on a single chip.

[0098] Assay consumables handler In some examples, the apparatus includes an assay consumable handler configured to operably connect to an assay consumable having a surface containing an assay site. Figures 3A–3B show schematic diagrams of one such assay consumable handler 10. The assay consumable handler may support and facilitate the handling and / or placement of assay consumables by or within the apparatus.

[0099] Assay consumable handlers may be stationary, movable, or at least partially movable. For example, an assay consumable handler may be operably integrated with a stage, or comprise a stage, where the stage is movable. The stage may be connected to a controller configured to automatically move the stage and / or the assay consumable handler. In certain embodiments, the assay consumable handler may be sized and / or molded to mate with assay consumables. For example, an assay consumable handler may have a recessed area on which assay consumables can be placed and secured. Alternatively, an assay consumable handler may have a substantially flat surface on which assay consumables can be placed. In some embodiments, the assay consumable handler may have fasteners (e.g., snaps, clips, clamps, ring clamps, etc.) to assist in attaching assay consumables to the assay consumable handler, thereby minimizing or eliminating movement between the consumable and the assay consumable handler during at least a certain period of system operation. As another example, an assay consumable handler may utilize a vacuum or aeration system to secure assay consumables. In certain embodiments, to facilitate proper placement and / or prevent the use of improperly configured or counterfeit assay consumables, the assay consumable handler may be equipped with recognition elements complementary to the recognition elements of the assay consumable. For example, the assay consumable may be equipped with multiple notches, and the assay consumable handler may be equipped with multiple complementary indentations. As another example, the assay consumable may be equipped with an RFID chip or barcode reader, and the assay consumable may require the assay consumable and the assay consumable handler to be linked by an authorized RFID chip or barcode in order to prevent an alarm condition from being triggered or the controller from shutting down system operation.

[0100] Non-limiting examples of assay consumable handlers are shown in Figures 4A–4F. Figure 4A shows assay consumable 500 and assay consumable handler 502. The apparatus includes a member (e.g., arm 501) that can move the assay consumable 500 from a first position not connected to the assay consumable handler to a position connected to the assay consumable handler. The assay consumable 500 includes at least one notch or recognition element (e.g., notch 508) that specifically interacts with a key or recognition element (e.g., key 506) on the assay consumable handler 502. The assay consumable handler 502 also includes a plurality of holes 504 that allow for the application of vacuum to the assay consumable. Once the assay consumable is lowered to the appropriate position (e.g., as shown in Figure 5B), the notch 508 aligns with the key 506, and vacuum can be applied to the holes 504, thereby causing the assay consumable 500 to lie flat in a safe position on the assay consumable handler. After loading the assay consumables into the assay consumable handler, the handler may be positioned so that the components of the apparatus (e.g., sample loader, bead loader, sealer, wiper, imaging system, etc.) are in the appropriate locations. Vacuum may be maintained until a desired number of individual groups of assay sites have been analyzed. Figure 4C shows assay consumables connected to the assay consumable handler via a central mounting clamp 510. The central mounting clamp 510 ensures and maintains the flatness of the assay consumables. Figure 4D shows assay consumables connected to the assay consumable handler via a first ring clamp 512 and a second ring clamp 516. The ring clamps are configured and positioned to hold the assay consumables in the assay consumable handler by securing the outer edges of the assay consumables.

[0101] Figures 4E and 4F show another example of an assay consumable handler comprising a handler grasping arm 556, a cross arm 553 (not shown) operably connected to a part of the device, an assay consumable handler stage 555, and an assay consumable attachment 558. An imaging system 560 is also illustrated. In Figure 5E, a single assay consumable 550 is configured to be moved from a stack 552 to the assay consumable stage 555. Arm 556 is positioned at the appropriate position A so that arm 556 is positioned above the stack 552. An assay consumable attachment 558 (e.g., adsorption cup, clip, etc.) is lowered to grasp the assay consumable 550. Arm 556 of the handler is moved along the cross arm 553 from position A in Figure 4E to position B in Figure 4F so that the assay consumable 550 is positioned above the assay consumable stage 555. Figure 4F shows the assay consumable being lowered to connect the assay consumable 550 to the assay consumable stage 555. In this figure, the assay consumable stage 555 is equipped with a hole 554 that communicates with a vacuum source and fluid, so that a vacuum can be applied to the underside of the assay consumable 550 to hold it in place, as described herein (see, for example, Figure 4A (hole 504) for a similar example). In some cases, the assay consumable handler may be equipped with a conveyor belt assembly.

[0102] Object capture applicator In some embodiments, the apparatus includes a capture object applicator. The capture object applicator can function independently, in conjunction with a fluid injector and / or fluid pump, to apply and deliver the capture object to the surface of the assay consumable. Figures 3A–3B illustrate schematic diagrams of the capture object applicator 20 of apparatus 1. Although Figures 1 and 3A–3B depict the capture object applicator 20 as separate components from the fluid injector 50 and / or fluid pump 60, in some embodiments these components are identical (for example, the fluid injector may inject a fluid plug containing the capture object onto the surface 120 of the assay consumable 5 via positive pressure supplied by the fluid pump 60). In other examples, the capture object applicator includes a pipette used to deliver the capture object (e.g., beads) to the inlet of a channel (e.g., a microfluidic channel) and distribute it onto the assay consumable. Other non-limiting examples of object capture applicators include automated pipettes connected to fluid pumps (e.g., syringe pumps, piston-acting pumps, membrane pumps, etc.) and microfluidic injectors. Like other components, object capture applicators may be connected to controllers configured to automatically operate the object capture applicators.

[0103] In some embodiments, the capture object applicator is configured to apply a relatively small number of capture objects to or near the surface of an assay consumable. For example, the capture object applicator may be connected to a fluid injector and / or fluid pump suitable for producing a relatively small volume of fluid containing the capture objects (e.g., beads) or a relatively thin fluid plug containing the capture objects. In some embodiments, the capture object applicator is configured to apply 100,000 or fewer, 50,000 or fewer, 25,000 or fewer, 10,000 or fewer, 2,000 or fewer, 1,000 or fewer, 500 or fewer, 200 or fewer, 100 or fewer, or just 50, just 20, just 10, just 5, or a single capture object to or near the surface of an assay consumable.

[0104] Detection using imaging systems and fixed fields of view Apparatus and methods for imaging and / or analyzing assay sites (e.g., the shape of an array on the surface of assay consumables) are also disclosed. In the context of this disclosure, it has become clear that certain existing techniques for imaging assay sites do not analyze the entire region containing the assay site, but rather only a subset of it. By analyzing only a subset of the assay site (e.g., when measuring the presence or absence of capture objects and / or associated analytes), a smaller absolute number of immobilized capture objects than those actually immobilized are analyzed. In existing assays using a relatively large number of capture objects (e.g., more than 100,000, more than 200,000, or more), such loss of capture objects may be negligible. However, in assays of this disclosure that may use a relatively small number of capture objects (e.g., 50,000 or less, 10,000 or less, 5,000 or less, or less), such loss of capture objects can have a significant impact on the detection of sufficient signals from the capture objects. Some of the described apparatuses are configured to reduce or limit such loss by analyzing the entire region containing the assay site (e.g., an array of assay sites). In some embodiments, an apparatus is provided for imaging an array of assay sites, which may also be part of a whole system for detecting and / or quantifying analytes. For example, according to some embodiments, apparatus 1 in Figure 1 may comprise an imaging system 70 and a computer-implemented control system 80. The imaging system 70 may be configured to capture an image of an array of assay sites on an assay consumable 5, which can be directed to the imaging system 70 via an assay consumable handler 10. However, it should be understood that the presence of a separate assay consumable handler is optional, and in some embodiments, the imaging system and the assay consumable may be interfaced directly without the assay consumable being handled by an assay consumable handler. One such embodiment may comprise an apparatus for imaging an array on a microfluidic chip, which can be manually operably coupled to the imaging system. In some embodiments, the apparatus may be configured so that the imaging system can capture an image of the array without inverting the assay consumable after the capture object has been immobilized with respect to the assay sites on the surface of the assay consumable. For example, after immobilizing the capture object (e.g., inserting beads), the assay consumable handler may manipulate the assay consumable (e.g., via relative rotation or translation) so that it is aligned with the field of view of the imaging system without reversing the assay consumable (e.g., inverting the assay consumable).

[0105] The imaging system may include a detector and optical system. Various types of detector and optical system configurations are available, and exemplary configurations are described in further detail below. The imaging system with the detector and optical system may have a fixed field of view larger than the area containing the array of assay sites. In some such examples, the apparatus may be configured so that the array of assay sites on the assay consumable can be fully positioned within the fixed field of view of the imaging system. Figure 5 shows a schematic diagram of one such embodiment. In Figure 5, the imaging system 70, comprising a detector 71 and an optical instrument 72, is positioned on the assay consumable 5 which is operably connected to an assay consumable handler 10. The imaging system 70 has a fixed field of view 73 which is larger than the area containing the array of assay sites 110 on the surface 120 of the assay consumable 5. In this context, a fixed field of view between the imaging system and the array of sites means that the imaging system captures an image of the array of assay sites for subsequent analysis with virtually no relative movement (albeit very slight movement) between the field of view and the array. Such a fixed-field imaging system can capture an image of the array as a "single shot" (rather than scanning the array across and obtaining a composite of images of multiple images captured in many different relative directions of the detector / optical instrument and the array). In some embodiments, the apparatus includes a computer-implemented control system configured to receive information from the imaging system. In some such embodiments, the computer-implemented control system is configured to analyze the entire region containing the array of assay sites. Referring again to Figure 5, the computer-implemented control system 80 may be configured to receive information from the imaging system 70. The information may relate to an image of the array of assay sites 110 on the surface 120 of the assay consumable 5 (for example, during a detection step in which the analytes of the sample are detected and / or quantified in the assay). In some embodiments, the computer-implemented control system 80 is configured to analyze the entire region containing the array of assay sites 110.Such a configuration may enable the detection of a larger number of immobilized capture objects relative to the assay site than certain existing techniques that analyze only a subset of the captured images. The computer-implemented control system may be further configured to measure an unknown concentration of analyte molecules or particles in the assay sample based on the analysis of the entire array of assay sites. In some embodiments, the computer-implemented control system is configured to analyze a relatively large area. For example, in some embodiments, the computer-implemented control system is configured to analyze an area of ​​at least 2 mm. 2 at least 5mm 2 , at least 10mm 2 , and / or at most 15mm 2 at most 20mm 2 The system is configured to analyze a number of regions, or more. For example, in some embodiments, the computer-implemented control system is configured to analyze at least 100,000 assay sites, at least 200,000 assay sites, at least 500,000 assay sites, or at least 1,000,000 assay sites, or more.

[0106] Various imaging systems that are potentially useful for carrying out specific embodiments of the present invention are known in the prior art and are commercially available. Such systems and components may be configured based on the needs and requirements of a selected assay method performed by the system and technique used to detect analyte molecules and / or particles. For example, in some assays, analyte molecules and / or particles are not directly detectable, and further reagents (e.g., detectable labels) are used as detection aids. In such cases, components of the imaging system are selected to detect such reagents.

[0107] In certain embodiments, the imaging system is configured to optically interrogate the assay site. Sites exhibiting changes in their optical signatures can be identified by conventional optical trains and optical detection systems. Depending on the species being detected and the operating wavelength, optical filters designed for specific wavelengths may be used for optical reference of the location, as will be understood by those skilled in the art.

[0108] In some embodiments where optical referencing is used, the imaging system comprises multiple light sources and / or multiple filters, and the wavelength and / or light source intensity can be adjusted. Examples of light sources include lasers, continuous spectrum lamps (e.g., mercury vapor, halogen, tungsten lamps), and light-emitting diodes (LEDs). For example, in some cases, a first reference of the assay site may be performed using light in a first wavelength range, while a second reference may be performed using light in a second different wavelength range, thereby causing multiple detectable molecules to fluoresce.

[0109] In some embodiments, optical signals from multiple assay sites are captured using a charge-coupled device (CCD) camera. Other non-limiting examples of devices that can be used to capture images include charge injection devices (CIDs), complementary metal-oxide-semiconductor (CMOS) devices, scientific CMOS (sCMOS) devices, time-delay integration (TDI) devices, photomultiplier tubes (PMTs), and avalanche photodiodes (APDs). Cameras for such a variety of devices are commercially available from several manufacturers.

[0110] In one embodiment, the assay consumable comprises an optical fiber bundle, and multiple assay sites in the form of reactors are formed at the ends of the optical fiber bundle.

[0111] In one embodiment, an array of assay sites for the present invention can be used in conjunction with an optical detection system (for example, the system described in U.S. Patent Application Publication No. 2003 / 0027126, which is incorporated herein by reference for all purposes).

[0112] Those skilled in the art will understand that various elements of an imaging system can be adjusted and / or configured to provide good images. For example, in some cases, assay consumables are imaged through a sealant, and therefore the imaging system can be adjusted and / or configured to account for the presence of the sealant in the optical path. As is known to those skilled in the art, a certain thickness of material can result in spherical aberration and loss of array resolution. Therefore, if the sealant is of a thickness that causes such aberration, the optical portion of the imaging system can be designed to compensate for this increased thickness. Designing the optical instrument such that a fluid matching the index of the sealant may be placed between the objective and the assay consumable can ensure that the difference in material between the objective and the sealant does not result in blurring.

[0113] Other examples of imaging system features that can be tuned and / or configured to improve performance include the speed and focusing ability of the imaging system. In some cases, focusing may involve the use of a laser focusing system based on reflections away from the surface of the assay consumable. Laser focusing systems are commercially available. In other cases, the surface of the assay consumable containing the assay site (which may be similar in wavelength and size to the light being processed) may include structures / reference points incorporated into the assay consumable that can be used to focus the image via diffraction, refraction, absorption, reflection, fluorescence, or a combination of these and other optical phenomena.

[0114] As described above, certain embodiments of the system and apparatus include one or more controllers and / or computer-implemented control systems for operating various elements / subsystems of the system and performing data / image analysis, etc. (e.g., controller 30 / computer-implemented control system 80 shown in Figure 1). Any calculation method, step, simulation, algorithm, system, and system element described may be implemented and / or controlled using one or more computer-implemented control systems (e.g., embodiments of computer-implemented control systems described later). The methods, steps, control systems, and control system elements described are not limited to any specific computer system described, and many other different devices may also be used.

[0115] The computer-implemented control system(s) may be part of the image analysis system and / or other automated system components, or may be operablely connected, and in some embodiments, may be configured and / or programmed to control and adjust operating parameters, and to analyze and calculate values ​​(e.g., the concentration of the analyte molecules or particles described above). In some embodiments, the computer-implemented control system(s) may be able to send and receive reference signals for setting and / or controlling the operating parameters of the system device. In other embodiments, the computer-implemented control system(s) may be separate from other system components and / or may be remotely located and may be configured to receive data from one or more remote assay systems of the present invention via indirect and / or portable means, for example, via a portable electronic data storage device (e.g., magnetic disk), or via a connection via a computer network (e.g., the Internet or a local intranet).

[0116] A computer-implemented control system (one or more) may comprise several known components and circuits, such as processing units (i.e., one or more processors), memory devices, input / output devices and interfaces (e.g., interconnection mechanisms), as well as other components, such as transport circuits (e.g., one or more buses), video and audio data input / output (I / O) subsystems, special-purpose hardware, and other components and circuits, as will be described in more detail later. Furthermore, a computer system (one or more) may be a multi-processor computer system, or may comprise multiple computers connected through a computer network.

[0117] For example, a computer-implemented control system (one or more) may have one or more processors, including commercially available processors such as the x86 series (Celeron and Pentium processors, Intel), similar devices from AMD and Cyrix (Motorola, 680X0 series microprocessors), and IBM PowerPC microprocessors. Many other processors are also available, and a computer system is not limited to a specific processor.

[0118] A processor typically runs programs called operating systems, such as Windows NT, Windows 95 or 98, Windows XP, Windows Vista, Windows 7, Windows 10, UNIX, Linux, DOS, VMS, and MacOS, controls the execution of other computer programs, and also provides scheduling, debugging, input / output control, accounting, compilation, storage transfer, data management and storage management, communication control, and related services. Together, the processor and operating system define the computer platform on which application programs written in high-level programming languages ​​are written. Computer implementation control systems are not limited to a specific computer platform.

[0119] A computer-implemented control system (one or more) may include a storage device, which typically comprises a computer-readable and writable non-volatile recording medium, such as magnetic disks, optical disks, flash memory, and tapes. Such recording media may be removable, such as floppy disks, read / write CDs or memory sticks, or permanent, such as hard disk drives.

[0120] Such recording media typically store signals in binary format (i.e., in a format interpreted as a series of 1s and 0s). A disk (e.g., magnetic or optical) typically has several tracks on which such signals can be stored, in binary format (i.e., in a format interpreted as a series of 1s and 0s). Such signals may define a software program (e.g., an application program) or information processed by an application program for execution by a microprocessor.

[0121] The memory systems of a computer-implemented control system (one or more) may also include IC memory elements, which are typically volatile random-access memory (e.g., dynamic RAM (DRAM) or static memory (SRAM)). Typically, during operation, the processor loads programs and data from a non-volatile recording medium into the IC memory elements, thereby enabling the processor to access program instructions and data, typically faster than the non-volatile recording medium can.

[0122] After processing is complete, the processor generally manipulates data in the IC memory element according to program instructions and then copies the manipulated data to a non-volatile recording medium. Various mechanisms for managing data movement between the non-volatile recording medium and the IC memory element are known, and the computer-implemented control system(s) that implement the above methods, steps, system control, and control of system elements are not limited to these. The computer-implemented control system(s) are not limited to a specific storage device.

[0123] At least a portion of such a storage device may store one or more data structures (e.g., lookup tables) or equations (e.g., equations for calibration curves). For example, at least a portion of a non-volatile storage device may store at least a portion of a database containing one or more such data structures. For example, such a database may be any of various types of databases, including, for example, a file system containing a flat file data structure consisting of data units in which one or more data are separated by delimiters, a relational database consisting of data units in which data are stored in tables, an object-oriented database consisting of data units in which data are stored as objects, other types of databases, or a combination thereof. A computer-implemented control system (one or more) may include video and audio data input / output subsystems. The audio portion of the subsystem may include an analog-to-digital (A / D) converter that receives analog audio information and converts it to digital information. The digital information may be compressed using a known compression system for storage on a hard disk for later use. The video portion of a typical input / output subsystem may include many video image compressors / decompressors known in the prior art. Such compressors / decompressors convert analog video information to compressed digital information and vice versa. The compressed digital information may be stored on a hard disk for later use.

[0124] A computer-implemented control system (one or more) may include one or more output devices. Examples of output devices include cathode ray tube (CRT) displays, liquid crystal displays (LCDs), light-emitting diode (LED) displays and other video output devices, printers, communication devices (e.g., modems or network interfaces), storage devices (e.g., disks or tapes), and audio output devices (e.g., speakers).

[0125] A computer-implemented control system (one or more) may include one or more input devices. Examples of input devices include keyboards, keypads, trackballs, mice, pens, and tablets, communication devices as described above, and data input devices such as audio and image capture devices and sensors. A computer-implemented control system (one or more) is not limited to the specific input or output devices described.

[0126] It should be understood that one or more computer-implemented control systems of any type may be used to carry out the various embodiments described. Aspects of the present invention may be carried out in software, hardware, or firmware, or any combination thereof. For example, one or more computer-implemented control systems may include special-purpose hardware, such as specially programmed application-specific integrated circuits (ASICs). Such special-purpose hardware may be configured to carry out one or more of the above-described methods, steps, simulations, algorithms, system control, and system element control, either as part of or as an independent component of the computer-implemented control system(s) described above.

[0127] Computer-implemented control systems (one or more) and their components may be programmable using one or more suitable computer programming languages. Such languages ​​include procedural programming languages ​​(e.g., LabVIEW, C, Pascal, Fortran, and BASIC), object-oriented languages ​​(e.g., C++, Java, and Eiffel), and other languages ​​(e.g., scripting languages ​​or even assembly languages).

[0128] Methods, steps, simulations, algorithms, system controls, and system element controls may be executed using any of a variety of suitable programming languages, including procedural programming languages, object-oriented programming languages, other languages, and combinations thereof, and may also be executed by a computer system. Such methods, steps, simulations, algorithms, system controls, and system element controls may be executed as modules of separate computer programs, or they may be implemented individually as separate computer programs. Such modules and programs may be executed on separate computers.

[0129] Such methods, steps, simulations, algorithms, system controls, and system element controls may be implemented individually or together as computer program products constructed as computer-readable signals on a computer-readable medium (e.g., a non-volatile recording medium, an IC memory element, or a combination thereof). In each of such methods, steps, simulations, algorithms, system controls, or system element controls, such computer program products may include computer-readable signals constructed on a computer-readable medium that define instructions as part of one or more programs that, as a result of being executed by a computer, instruct the computer to perform, for example, a method, step, simulation, algorithm, system control, or system element control.

[0130] Assays (such as assays using a small number of beads and efficient loading) A method (e.g., an assay) for measuring the concentration of analyte molecules or particles in a fluid sample is described below. As described above, it has unexpectedly become clear in the context of this disclosure that highly sensitive (e.g., low detection limits) detection of analytes can be performed in assays using fewer capture objects compared to typical conventional methods (e.g., certain existing digital ELISA techniques). Counterintuitively, it has become clear that, due to the increase in enzyme per bead (AEB), the increase in sensitivity achieved with fewer capture objects relative to the number of analyte molecules or particles can outweigh the potential loss of sensitivity (e.g., due to increased background signal from Poisson noise or inefficient analyte capture). The specific methods and apparatus described for preparing samples and capture objects, distributing (e.g., loading / spatially isolating) the capture objects, and / or detecting / analyzing the capture objects may contribute, individually or cumulatively, to the ability to use such a low number of beads.

[0131] One exemplary assay format / protocol involves exposing a capture object (e.g., beads) configured to capture a specific type of analite molecule or particle to a solution (e.g., a fluid sample) containing or suspected to contain such analite molecules (or particles). At least some analite molecules are immobilized with respect to the capture object. Each capture object may have an affinity for a specific type of analite molecule or particle. Each capture object may include a binding surface having an affinity for at least one type of analite molecule (e.g., a specific type of analite molecule or particle). In some cases, the binding surface may include multiple capture components. As used in the present invention, “capture component” can be any molecule, other chemical / biological entity, or modification of a solid support that can bind to or otherwise capture a target molecule or particle (e.g., an analite molecule), which is specifically attached to the target molecule or particle (e.g., an analite molecule), thereby immobilizing the target molecule / particle with respect to the capture object. Immobilization may occur by association of the analite molecule with the capture component on the surface of the capture object. In the context of immobilizing analyte molecules or particles with respect to a capturing object, "immobilization" means preventing the dissociation or loss of the target molecule / particle by capturing, attaching, binding, or fixing it, but it does not require absolute rest with respect to either the capturing component or the object.

[0132] The number of analyte molecules immobilized with respect to a capture object may depend on the ratio of the total number of analyte molecules in the sample to at least one of the total number, size, and / or surface density of the capture components of the provided capture object. In some embodiments, the number of molecules or particles immobilized with respect to a single capture object may follow a standard Poisson distribution. In some cases, a statistically significant number of capture objects associate with a single analyte molecule or particle from the fluid sample, and a statistically significant number of capture objects do not associate with any analyte molecule or particle from the fluid sample. In some embodiments, the percentage of captured objects that associate with at least one analite molecule (e.g., a particular type of analite molecule or particle) is less than or equal to 99.999%, 99.99%, 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, 0.1%, or less of the total number of captured objects.

[0133] A small number of captured objects exposed to the sample solution In some embodiments, the number of captured objects exposed to a solution containing or suspected to contain analyte molecules or particles is relatively small. As described above, the unconventional use of a relatively small number of captured objects (e.g., during exposure to analyte molecules or particles and / or during downstream analytical and detection steps) can, in some examples, result in an unexpected or unforeseen increase in sensitivity (e.g., level of detection). The specific teachings of this disclosure relating to the efficient handling of captured objects may help to address the challenges in the known art that have discouraged the use of such a small number of captured objects (e.g., in supersensitive digital ELISA assays) related to handling and detecting such a small number of captured objects. In some embodiments, the number of captured objects (e.g., those with affinity for a particular type of analyte molecule or particle) exposed to a solution containing or suspected to contain analyte molecules or particles is 50,000 or less, 7,500 or less, 5,000 or less, 4,000 or less, 3,000 or less, 2,000 or less, or less. In some embodiments, the number of capture objects (e.g., those with an affinity for a particular type of analite molecule or particle) exposed to a solution containing or suspected of containing analite molecules or particles is 100 or more, 200 or more, 500 or more, 1,000 or more, or more. In some embodiments, the number of capture objects (e.g., those with an affinity for a particular type of analite molecule or particle) exposed to a solution containing or suspected of containing analite molecules or particles is 10,000 or less. Combinations of these ranges are possible. For example, in some embodiments, the number of capture objects (e.g., those with an affinity for a particular type of analite molecule or particle) exposed to a solution containing or suspected of containing analite molecules or particles is 100 to 50,000, 100 to 10,000, or 100 to 5,000.

[0134] In some embodiments, compositions with a relatively small number of capture objects and a relatively low concentration of analytes may be used. Such compositions may be prepared during any of the steps of some of the methods described, or may be provided separately. In the context of this disclosure, it has become apparent that compositions with a relatively small number of capture objects may be unexpectedly used in assays for detecting low concentrations of analytes. The preparation of such compositions contradicts conventional common sense, which typically teaches the use of a large number of capture objects (to increase the likelihood of analyte capture or to avoid handling / detection challenges). In some embodiments, the composition is an isolated fluid having a volume of 10 to 1000 microliters, 50 to 500 microliters, or 100 to 350 microliters. Some such compositions have at least one type of analyte molecule or particle present at concentrations of 0.001 atomoles (aM) to 10 picomoles (pM), 0.01 aM to 1 pM, 0.1 aM to 100 femtomoles (fM), or 1 to 10 fM. In some embodiments, the composition comprises 100 to 10,000 or 1,000 to 5,000 capture objects (e.g., beads) having a binding surface having affinity for at least one type of analyte molecule or particle.

[0135] Incubation time In the context of this disclosure, it has become clear that the duration of exposure of a capture object to a solution containing or suspected to contain analyte molecules or particles can affect the degree to which the analyte molecules become immobilized with respect to the capture object. Exposure of the capture object to a relatively long period of time in the solution (e.g., an incubation step) increases the percentage of analyte molecules or particles that become immobilized with respect to the capture object in the solution, surprisingly, even in the presence of relatively small amounts of capture objects (e.g., 10,000 or less, 5,000 or less, or less). It is thought that relatively long exposure times (e.g., incubation) can overcome the dynamic limitations that may arise in the presence of small amounts of capture objects (e.g., in some examples where immobilization is controlled by bimolecular reaction kinetics). In some embodiments, the captured object is exposed to a solution (e.g., a fluid sample) containing or suspected to contain at least one type of analyte molecule or particle for 15 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more and / or at most 18 hours, at most 24 hours, at most 30 hours or more, or longer.

[0136] Sample volume In the context of this disclosure, it has become clear that the volume of the solution (e.g., fluid sample) to which the captured material is exposed can affect the degree to which analyte molecules are immobilized with respect to the captured material. Exposing the captured material to a relatively large volume of solution (e.g., incubation step) may provide one method for using a relatively diluted solution (e.g., dilute sample) with a relatively small amount of captured material (e.g., 50,000 or less, 10,000 or less, 5,000 or less, or less). In some examples, providing a large volume of solution during the exposure step may result in relatively higher assay sensitivity (e.g., compared to equivalent assays using other smaller volumes) by providing more analyte molecules or particles that can be immobilized with respect to the captured material. A larger number of analytes may increase the ratio of detectable species per captured material (e.g., average enzyme per bead) and potentially the assay sensitivity during the assay. In some embodiments, a solution (e.g., a fluid sample) containing or suspected to contain at least one type of analyte molecule or particle has a volume of 50 microliters or more, 100 microliters or more, 200 microliters or more, 300 microliters or more, and / or at most 400 microliters, at most 500 microliters, at most 1 mL or more.

[0137] Spatial isolation and processing of captured objects. In some embodiments, the assay method employs a step of spatially isolating the capture object at multiple separate locations to facilitate detection / quantification. In such embodiments, the isolation is performed such that each location contains zero or one or more analyte molecules or particles from the fluid sample. Furthermore, in some embodiments, each location may be configured in such a way that it can be processed individually. In some embodiments, the concentration of analyte molecules or particles in the fluid sample may be measured by detecting analyte molecules or particles immobilized with respect to a binding surface having affinity for at least one type of analyte molecule or particle (e.g., one specific type of molecule or particle). In certain embodiments, the binding surface may form one of several locations (e.g., assay sites (e.g., wells / reactors)) on a substrate (e.g., a plate, dish, tip, optical fiber end, channel surface, disk, surface of assay consumables, etc.) (e.g., the surface of the assay site (e.g., well / reactor on the substrate)) or be contained within such a location (e.g., the surface of the capture object (e.g., beads), immobilized with respect to the assay site (e.g., well)). At least some locations may be processed, and an index representing the number or proportion of captured objects associated with at least one analyte molecule or particle from the fluid sample may be measured. In some cases, the concentration of analyte molecules or particles in the fluid sample may be measured, at least partially, based on an index representing the number or proportion. In some cases, the concentration measurement may be at least partially based on the number or proportion of locations measured as containing captured objects that have associated with or had associated with at least one analyte molecule or particle. As is known to those skilled in the art, the concentration of analyte molecules or particles in a fluid sample may be measured in a digital analytical method / system that optionally uses Poisson distribution adjustment and / or at least partially based on the measured intensity of the signal.For example, in some embodiments where the index representing the number or percentage of captured objects measured as associated with analyte molecules or particles represents a relatively low percentage (e.g., 80% or less, 70% or less, 50% or less, or less), the concentration of analyte molecules or particles in a fluid sample can be measured at least partially using digital analytical methods (optionally using Poisson distribution adjustment). However, in some embodiments where the index representing the number or percentage of captured objects measured as associated with analyte molecules or particles represents a relatively high percentage (e.g., 50% or more, 60% or more, 70% or more, 80% or more, 90% or more), the index representing the concentration of analyte molecules or particles in a fluid sample can be measured at least partially based on measuring the intensity level of at least one signal (e.g., a fluorescence signal) that indicates the presence of analyte molecules or particles. In some embodiments, the method includes measuring the concentration of analite molecules or particles in a fluid sample based at least partially on an index representing the number or proportion of captured objects measured to be associated with at least one analite molecule or particle from the fluid sample, or at least partially on a measured intensity level of a signal indicating the presence of multiple analite molecules or particles. In certain embodiments, an automated system configured and programmed to assay and measure an index representing the concentration of analite molecules or particles in a fluid sample may first measure an index representing the proportion of captured objects measured as associated with analite molecules or particles (e.g., the proportion of assay sites indicating a positive signaling state, and / or the average intensity level of the captured sites), and then be programmed to automatically (or manually at the prompt of the user) switch the measurement and quantification technique to be used (i.e., using a digital analytical method; optionally using a method based on Poisson distribution adjustment or analog intensity levels).The use, alone or in combination, of such digital and / or “analog” methods for measuring an index representing the concentration of analyte molecules or particles is described, for example, in U.S. Patent Application No. 13 / 037987, filed March 1, 2011 (published October 6, 2011, as U.S. Patent Application Publication No. 2011 / 0245097, Rissin et al., title of the invention, “METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OF MOLECULES OR PARTICLES”), which is incorporated herein by reference in its entirety for all purposes. In some cases, assay methods and / or systems may be automated.

[0138] In some examples, an index representing the number or proportion of captured objects associated with at least one analyte or molecule can be measured at least partially by processing separate locations (e.g., assay sites), while it should be understood that other techniques for measuring such indexes are available. For example, in some embodiments, at least some of the captured objects subjected to the exposure and immobilization steps are processed individually (e.g., by being individually isolated from the remaining captured objects). One non-limiting method for processing captured objects individually without necessarily spatially separating them at multiple separate locations is to flow at least some of the captured objects through a channel (e.g., a microchannel having a maximum cross-sectional size of 1 mm or less in the flow direction, or 500 micrometers or less) and process the flowed captured objects. For example, the captured objects may flow past a detector (e.g., an optical detector) and be processed accordingly.

[0139] In some embodiments, the captured objects (e.g., some of which may optionally associate with at least one analyte molecule or particle) may be provided as separate droplets (e.g., separated using fluid technology (e.g., microfluidic technology)) or as objects contained within the droplets. In such embodiments, the captured objects include droplets suspended in a fluid immiscible with the droplets, or each contained within them. The droplets may be suspended in a fluid immiscible with at least the droplets during the step of dealing with the captured objects individually (e.g., via a detector). In some examples, the droplets may be provided as an array (e.g., spatially separated on a substantially flat surface). However, in some examples, the droplets may be dealt with individually by being flowed through a channel (e.g., a microchannel) and referenced as they flow through the channel. One way in which droplets may be referenced is by passing them to a detector. For example, the detector may be an optical detector. In this embodiment, droplets are temporarily separated from a fixed detection location by, for example, being flowed through a channel to reach such detection location (e.g., during the step being targeted). While droplets may flow in a single file in some examples, a single-file flow is not required in all cases. For example, droplets may be collected in layers, and all droplets may be imaged substantially simultaneously.

[0140] High percentage of spatial isolation of captured objects In some embodiments, a relatively high percentage of the captured material is separated spatially into multiple distinct locations (e.g., assay sites (e.g., reactors)).

[0141] This approach is contrary to the common practice in the field of supersensitive detection, where a relatively small percentage (e.g., less than 20%) of the total number of capture objects exposed to analyte molecules or particles (e.g., those with affinity for one specific type of molecule or particle) are typically separated to distinct locations (e.g., by immobilization relative to the assay site), and a larger excess of capture objects is abandoned. Thus, the common method focuses on immobilizing a high percentage of capture objects to specific locations at the expense of using a large excess of capture objects. Instead, spatially separating a high percentage of capture objects to distinct locations may allow for the use of a relatively small total number of capture objects in the assay, thereby increasing sensitivity in some cases. In some embodiments, at least 25%, at least 30%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, at least 99%, or all of the captured material (e.g., having affinity for one specific molecule or particle) subjected to the exposure and immobilization steps described above is spatially separated into multiple separate locations (e.g., assay sites (e.g., reactors)).

[0142] Specific methods and apparatus employing the spatial separation of analyte molecules or particles are known in the prior art and may be used (with appropriate modifications in accordance with this disclosure), and are described below: U.S. Patent Application Publication 2007 / 0259448 (U.S. Patent Application No. 11 / 707,385, filed February 16, 2007, Walt et al., “METHODS AND ARRAYS FOR TARGET ANALYTE DETECTION AND DETERMINATION OF TARGET ANALYTE CONCENTRATION IN SOLUTION”); U.S. Patent Application Publication 2007 / 0259385 (U.S. Patent Application No. 11 / 707,383, filed February 16, 2007, Walt et al., “METHODS AND ARRAYS FOR DETECTING CELLS AND CELLULAR COMPONENTS IN SMALL DEFINED Title: "VOLUMES"; U.S. Patent Application Publication No. 2007 / 0259381 (U.S. Patent Application No. 11 / 707,384, filed February 16, 2007, titled "METHODS AND ARRAYS FOR TARGET ANALYTE DETECTION AND DETERMINATION OF REACTION COMPONENTS THAT AFFECT A REACTION"); International Publication No. 2009 / 029073 (International Patent Application No. PCT / US2007 / 019184, filed August 30, 2007, titled "METHODS OF DETERMINING THE CONCENTRATION OF AN ANALYTE IN SOLUTION"); U.S. Patent Application Publication No. 2010 / 0075862 (U.S. Patent Application No. 12 / 236484, filed September 23, 2008, titled "HIGH SENSITIVITY DETERMINATION" by Duffy et al. The name of "FOR THE CONCENTRATION OF ANALYTE MOLECULES OR PARTICLES IN A FLUID SAMPLE");U.S. Patent Application Publication No. 2010 / 00754072 (U.S. Patent Application No. 12 / 236,486, filed September 23, 2008, titled "ULTRA-SENSITIVE DETECTION OF MOLECULES ON SINGLE MOLECULE ARRAYS" by Duffy et al.); U.S. Patent Application Publication No. 2010 / 0075439 (U.S. Patent Application No. 12 / 236488, filed September 23, 2008, titled "ULTRA-SENSITIVE DETECTION OF MOLECULES BY CAPTURE- AND-RELEASE USING REDUCING AGENTS FOLLOWED BY QUANTIFICATION" by Duffy et al.); International Publication No. 2010 / 039179 (International Patent Application No. PCT / US2009 / 005248, filed September 22, 2009, titled "ULTRA-SENSITIVE Title: “DETECTION OF MOLECULES OR ENZYMES”); U.S. Patent Application Publication No. 2010 / 0075355 (U.S. Patent Application No. 12 / 236490, filed September 23, 2008, title: Duffy et al. “ULTRA-SENSITIVE DETECTION OF ENZYMES BY CAPTURE-AND-RELEASE FOLLOWED BY QUANTIFICATION”); U.S. Patent Application No. 12 / 731,130 (filed March 24, 2010, published September 1, 2011 as U.S. Patent Application Publication No. 2011 / 0212848, Duffy et al. “ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE Title: "OBJECTS"); International Patent Application No. PCT / US2011 / 026645 (filed on March 1, 2011, titled "ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS" by Duffy et al., published on September 9, 2011 as International Publication No. 2011 / 109364);International Patent Application No. PCT / US2011 / 026657 (filed March 1, 2011, titled "ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS" by Duffy et al., published September 9, 2011 as International Publication No. 2011 / 109372); US Patent Application No. 12 / 731135 (filed March 24, 2010, published September 1, 2011 as US Patent Application Publication No. 2011-0212462 by Duffy et al., titled "ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS"); International Patent Application No. PCT / US2011 / 026665 (filed March 1, 2011, titled "METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE IN The title is "Assays for the Detection of Molecules or Particles," published internationally on September 9, 2011, as International Publication No. 2011 / 109379); U.S. Patent Application No. 12 / 731136 (filed on March 24, 2010, published on September 1, 2011, as U.S. Patent Publication No. 2011 / 0212537, titled "Methods and Systems for Extending Dynamic Range in Assays for the Detection of Molecules or Particles" by Duffy et al.); U.S. Patent Application No. 13 / 035472 (filed on February 25, 2011, published on U.S. Patent Publication No. 2012-0196774, titled "Systems, Devices, and Methods for Ultra-Sensitive Detection of Molecules or Particles" by Fournier et al. The name of "PARTICLES";U.S. Patent Application No. 13 / 037987 (filed March 1, 2011, published October 6, 2011 as U.S. Patent Application Publication No. 2011 / 0245097, titled Rissin et al., "METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OF MOLECULES OR PARTICLES"); all its contents are incorporated herein by reference for all purposes.

[0143] In some embodiments, an index is also measured representing the number or percentage of positions that contain a capture object but do not associate with an analyte molecule or particle, and / or an index is also measured representing the number or percentage of positions that do not contain any capture object. In such embodiments, the measurement of the concentration of analyte molecules or particles in a fluid sample may be at least partially based on the ratio of the number of positions measured as containing a capture object associated with an analyte molecule or particle to the total number of positions measured as containing a capture object that does not associate with an analyte molecule or particle, and / or the measurement of the concentration of analyte molecules or particles in a fluid sample may be at least partially based on the ratio of the number of positions measured as containing a capture object associated with an analyte molecule or particle to the number of positions measured as not containing any capture object, and / or the measurement of the concentration of analyte molecules or particles in a fluid sample may be at least partially based on the ratio of the number of positions measured as containing a capture object associated with an analyte molecule or particle to the number of positions measured as containing a capture object. In yet another embodiment, the measurement of the concentration of analyte molecules or particles in a fluid sample may be at least in part based on the ratio of the number of locations measured as containing the captured object and analyte molecules or particles to the total number of locations being processed and / or analyzed.

[0144] In certain embodiments, at least a portion of the captured material (e.g., at least a portion of which associates with at least one analyte molecule or particle from the fluid sample) is spatially separated into multiple locations (e.g., assay sites (e.g., reactors in array format)). Reactors may be formed in, from, and / or within any suitable material, and in some cases, as detailed below, the reactors may be sealed or formed by fitting a substrate with a sealing material. In particular, in certain embodiments where quantization of capture objects associated with at least one analyte molecule or particle is required, partitioning of capture objects can be performed so that at least some of the reactors (e.g., a statistically significant proportion, e.g., as described in International Patent Application No. PCT / US2011 / 026645 (filed March 1, 2011, Duffy et al., “ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” published September 9, 2011 as International Publication No. 2011 / 109364), incorporated herein by reference for all purposes, contain at least, or in some cases, only one capture object associated with at least one analyte molecule or particle, and at least some of the reactors (e.g., a statistically significant proportion) contain capture objects not associated with any analyte molecule or particle. In certain embodiments, a captured object associated with at least one analyte molecule or particle is quantified, thereby enabling the detection and / or quantification of analyte molecules or particles in a fluid sample by techniques described in further detail herein.

[0145] An exemplary assay method may proceed as follows: A solution containing or suspected to contain analyte molecules or particles is provided. The solution may be a fluid sample (e.g., body fluid or derived from body fluid). Assay consumables containing assay sites (e.g., in an array) are exposed to the solution. In some cases, the analyte molecules or particles are provided in such a manner (e.g., at a concentration) that at least some assay sites contain a single analyte molecule or particle (e.g., in a statistically significant proportion) and a statistically significant proportion of assay sites do not contain any analyte molecules or particles. The assay sites may optionally be exposed to and / or rinsed with various reagents (e.g., using a reagent loader). The assay sites may then optionally be sealed and imaged (for example, using a system or method described in this disclosure or in U.S. Patent Application No. 13 / 035472 (filed February 25, 2011, published as U.S. Patent Application No. 2012 / 0196774, titled "SYSTEMS, DEVICES, AND METHODS FOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES")). The images may then be analyzed (for example, using a computer-implemented control system) to determine the concentration of analyte molecules or particles in a fluid sample, at least in part on the measurement of the number or percentage of assay sites containing analyte molecules or particles, and / or the number or percentage of sites not containing any analyte molecules or particles. In some cases, the analyte molecules or particles are provided in a manner (e.g., by concentration) in which at least some assay sites contain one or more analyte molecules or particles. In such embodiments, the concentration of analyte molecules or particles in a fluid sample can be measured at least partially by the level of intensity of at least one signal representing the presence of multiple analyte molecules or particles at one or more assay sites.

[0146] In some cases, the method optionally involves exposing the fluid sample to beads (e.g., magnetic beads) that have an affinity for a particular type of molecule or particle. The total number of beads (e.g., those with an affinity for a particular type of molecule or particle) may be relatively small (e.g., 50,000 or less) as described above. At least some of the analyte molecules or particles are immobilized with respect to the beads. In some cases, the analyte molecules or particles are provided in a manner (e.g., concentration) in which a statistically significant proportion of beads associate with a single analyte molecule or particle and a statistically significant proportion of beads do not bind to any analyte molecules or particles. At least some of the beads (e.g., those that associate with a single analyte molecule or particle, or those that do not associate with any analyte molecules or particles) can then be spatially separated / isolated and immobilized with respect to the assay site (e.g., assay consumables). The assay site (e.g., including a reactor) can optionally be exposed to and / or rinsed with various reagents. At least some of the assay sites can then be processed to determine the number of assay sites containing analyte molecules or particles. In some cases, the number of assay sites containing beads that are not associated with analyte molecules or particles, the number of assay sites that do not contain beads, or the total number of assay sites being processed can be measured. Some such measurements may then be used to measure the concentration of analyte molecules or particles in a fluid sample. In some cases, two or more analyte molecules or particles may associate with beads, and / or two or more beads may be present at the assay site. In some cases, the analyte molecules or particles are exposed to at least one additional reactive component simultaneously with and / or after spatially separating at least some of the analyte molecules or particles, and these components are immobilized with respect to the assay site. The analyte molecules or particles may be detected directly or indirectly. For direct detection, the analyte molecules or particles may include a directly reference and / or detectable molecule or part (e.g., a fluorescent substance). For indirect detection, an additional component is used to measure the presence of the analyte molecules or particles.

[0147] For example, an analyte molecule or particle (e.g., optionally associated with beads) can be exposed to at least one type of binding ligand. In certain embodiments, the binding ligand may be suitable for direct detection (e.g., the binding ligand contains a detectable molecule or portion) or suitable for indirect detection (e.g., it contains a component that can convert a labeling precursor into a label). Components of the binding ligand may be suitable for direct detection in embodiments in which the component has a measurable property (e.g., fluorescence emission, color, etc.). Components of the binding ligand may facilitate indirect detection, for example, by converting a labeling precursor into a label (e.g., a drug to be detected in an assay). "Labeling precursor" is any molecule, particle, etc. that can be converted into a label when exposed to a suitable converter (e.g., an enzyme component). "Label" is any molecule, particle, etc. that facilitates detection by acting as a detectable using a selected detection technique. In some embodiments, the binding ligand may include an enzymatic component (e.g., horseradish peroxidase, β-galactosidase, alkaline phosphatase, etc.). The first type of binding ligand may or may not be used in conjunction with a further binding ligand (e.g., a second type, etc.).

[0148] Any assay method may utilize two or more types of binding (e.g., a first type of binding ligand and a second type of binding ligand). In one example, the first type of binding ligand can associate with a first type of analyte molecule or particle, and the second type of binding ligand can associate with the first binding ligand. In other examples, both the first and second types of binding ligands can associate with the same or different epitopes of the analyte molecule or particle.

[0149] In some embodiments, the binding ligand or analyte molecule or particle may include an enzymatic component. The enzymatic component may convert a labeling precursor (e.g., an enzyme substrate) into a labeling agent (e.g., a detectable product). The concentration of the analyte molecule or particle in a fluid sample can then be measured, at least in part, by measuring the number or proportion of capture objects associated with the labeling agent (e.g., by correlating the number of positions containing the labeling agent with the number of positions containing the capture objects). Examples of other non-limiting systems or methods for detection include embodiments in which a nucleic acid precursor is replicated into a large number of copies or converted into a nucleic acid that can be immediately detected (e.g., by introducing a detectable portion (e.g., a fluorescent portion)). Some such methods include polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation, and loop-mediated isothermal amplification (LAMP). Such systems and methods are known to those skilled in the art, as described, for example, in "DNA Amplification: Current Technologies and Applications," Vadim Demidov et al., 2004.

[0150] In some embodiments, the binding ligand includes particles. For example, the binding ligand may include particles having a surface that has affinity for the same particular type of analite molecule or particle (e.g., by having molecules having an affinity immobilized on the surface), as the capture object does. In some embodiments, the analite molecule or particle is immobilized with respect to a capture object having a surface that has affinity for that particular analite molecule or particle, and the binding ligand containing particles having affinity for that same analite molecule or particle is immobilized with respect to the immobilized analite molecule or particle, resulting in a complex containing the capture object and the binding ligand associated with each analite molecule or particle. In some embodiments, the first binding ligand is immobilized with respect to the immobilized analite molecule or particle, and the second binding ligand containing particles is immobilized with respect to the immobilized first binding ligand. In some embodiments, the particles associated with the binding ligand can be detected. The particles associated with the binding ligand can be detected by any of a variety of techniques. For example, the detection of the presence of a binding ligand containing particles (and therefore the presence of an immobilized analite molecule or particle) may include detecting the emission of electromagnetic radiation from the particles. As an example, a particle associated with a binding ligand may be excited via light irradiation, and the particle may emit electromagnetic radiation via detectable fluorescence. Quantum dots and semiconductor polymer dots (Pdots) are examples of types of fluorescent particles that can be used. In some embodiments, the particle emits electromagnetic radiation via photon upconversion (two or more low-energy incident photons are absorbed by the particle (e.g., a 1-nanometer-sized nanoparticle) and converted into a single emitted photon with higher energy (short wavelength)). Such upconverting nanoparticles are known and include, for example, nanoparticles containing lanthanides and actinides doped transition metals. In some embodiments, the presence of a binding ligand containing a particle can be detected via electromagnetic radiation scattering (e.g., optical scattering) (e.g., by using a plasmon particle associated with the binding ligand).As one specific example, the plasmon particles may be gold nanoparticles whose light scattering is affected by binding to other species (e.g., analyte molecules or particles). In some embodiments, the binding ligand may associate with magnetic (e.g., superparamagnetic or ferromagnetic) particles, and the detection of the particle's presence may involve a magnetic phenomenon associated with the particle (e.g., detection of a magnetic field from or affected by the magnetic particle). Any of the various types and / or particle sizes may be used, depending, for example, on the detection technique used. The particles may be, for example, nanoparticles with a maximum cross-sectional size of 100 nm or less, or the particles may be larger (e.g., beads with a cross-sectional size of 100 nm to 100 micrometers).

[0151] Other exemplary embodiments of indirect detection are as follows: In some cases, an analyte molecule or particle is exposed to a labeling precursor (e.g., an enzyme substrate), and the enzyme substrate is converted to a detectable product (e.g., a fluorescent molecule) upon exposure to the analyte molecule or particle.

[0152] Assay methods and apparatus may utilize various components, steps, and / or other aspects known and understood by those skilled in the art. For example, a method may further include measuring at least one background signal (e.g., and further including subtracting the background signal from other measurements), a washing step, and so on. In some cases, the assay or system may include the use of at least one binding ligand, as described herein. In some cases, the measurement of the concentration of analyte molecules or particles in a fluid sample is at least partially based on comparison with a calibration curve of the measured parameters. The calibration curve may be derived using a sample containing known concentrations of the target analyte molecules or particles. In some cases, the calibration curve is at least partially formed by determining at least one calibration coefficient.

[0153] In certain embodiments, soluble or suspended precursor labeling agents may be used, which, once converted to labeling agents, become insoluble in liquid and / or immobilized in / near the assay site (e.g., a reactor where the labeling agent is formed). Such labeling agent precursors and labeling agents, as well as their uses, are described in U.S. Patent Application Publication 2010 / 0075862 (Duffy et al., “HIGH SENSITIVITY DETERMINATION OF THE CONCENTRATION OF ANALYTE MOLECULES OR PARTICLES IN A FLUID SAMPLE,” filed September 23, 2008, concurrently pending U.S. Patent Application No. 12 / 236484), which is incorporated herein by reference for all purposes.

[0154] Figure 6A shows an exemplary embodiment of an assay method that may be used in a particular embodiment of the present invention. A capture object 202 is provided (step (A)). In this example, the capture object comprises several beads. The beads are exposed to a fluid sample containing analyte molecules 203 (e.g., beads 202 are incubated with analyte molecules 203). At least some of the analyte molecules are immobilized with respect to the beads. In this example, the analyte molecules are provided in such a manner (e.g., concentration) that a statistically significant proportion of the beads associate with a single analyte molecule, and a statistically significant proportion of the beads do not bind with any analyte molecules. For example, as shown in step (B), analyte molecule 204 is immobilized with respect to bead 205, thereby forming a complex 206, but some beads 207 do not associate with any analyte molecules. As described herein, it should be understood that in some embodiments, two or more analyte molecules may associate with at least some of the beads. At least some of the multiple beads (e.g., those associated with a single analyte molecule or those not associated with any analyte molecule) can then be spatially separated / isolated into multiple separate locations. Multiple locations are illustrated as a substrate 208 having multiple assay sites in the form of wells / reactors 209, as shown in step (C). In this example, each reactor contains zero or one bead. At least some of the reactors can then be dealt with (e.g., optically or via other detection means) to measure the number of locations containing beads associated with analyte molecules. For example, as shown in step (D), the multiple reactors are optically dealt with using a light source 215, where each reactor is exposed to electromagnetic radiation from the light source 215 (represented by arrow 10). The light emitted from each reactor (represented by arrow 211) is measured (and / or recorded) by a detector 215 (equipped in the same system as the light source 215 in this example). An index representing the number or percentage of reactors (e.g., reactor 212) containing beads associated with analyte molecules is measured based on the light detected from the reactor.In some cases, an index may be measured representing the number or percentage of reactors containing beads that are not associated with analyte molecules (e.g., reactor 213), an index representing the number or percentage of wells that do not contain beads (e.g., reactor 214), and / or an index representing the total number of wells to be measured. Such measurement(s) may then be used to determine the concentration of analyte molecules in the fluid sample.

[0155] A non-limiting example of an embodiment in which the capture object associates with two or more analyte molecules is shown in Figure 6B. A capture object 220 is provided (step (A)). In this example, the capture object includes beads. The beads are exposed to a fluid sample containing analyte molecules 221 (e.g., beads 220 are incubated with analyte molecules 221). At least some of the analyte molecules are immobilized with respect to the beads. For example, as shown in step (B), analyte molecules 222 are immobilized with respect to beads 224, thereby forming complex 226. Also illustrated are complex 230, which includes beads immobilized with respect to three analyte molecules, and complex 232, which includes beads immobilized with respect to two analyte molecules. Furthermore, in some cases, some beads may not associate with analyte molecules (e.g., beads 228). The beads from step (B) are exposed to a binding ligand 231. As shown in step (C), the binding ligand associates with some analyte molecules immobilized with respect to the beads. For example, complex 240 comprises beads 234, analyte molecules 236, and binding ligands 238. The binding ligands are provided such that a statistically significant proportion of the beads containing at least one analyte molecule associate with at least one binding ligand (e.g., 1, 2, 3, etc.), and a statistically significant proportion of the beads containing at least one analyte molecule do not associate with any binding ligand. At least some of the multiple beads from step (C) are then spatially separated into multiple separate locations. As shown in step (D), in this example, the locations include assay sites in the form of reactors 241 on the substrate 242. Multiple reactors may be exposed to beads from step (C), such that each reactor contains 0 or 1 bead. To measure an index representing the number or proportion of reactors containing binding ligands (e.g., reactor 243), the substrate may then be analyzed, in which case the number or proportion may correlate with a measured concentration of analyte molecules in a fluid sample.In some cases, an index representing the number or percentage of reactors containing beads but not containing binding ligands (e.g., reactor 244), an index representing the number or percentage of reactors not containing beads (e.g., reactor 245), and / or the total number of reactors being processed / analyzed may be measured. Some such measurements (one or more) may then be used to determine the concentration of analyte molecules in a fluid sample.

[0156] Multiple assays It should be understood that in some embodiments, a single type of analyte molecule or particle is detected / quantified ("single"), while in other embodiments, two or more types of analyte molecules or particles are detected / quantified ("multiple"). Certain methods described regarding the use of a relatively low number of capture objects and / or spatially separating a relatively high percentage of capture objects during analyte exposure may be particularly beneficial in such multiple assays. For example, a conventional multiple assay involving the detection or measurement of concentrations of both a first type of analyte molecule or particle and a second type of analyte molecule or particle may involve the use of a greater number of capture objects than in a single assay. When a relatively large number of capture objects are used for each first and second type of analyte molecule or particle, the additional capture objects used in a multiplex assay can result in a very large total number of capture objects with affinity for any type of analyte molecule or particle. This can make loading and sealing the capture objects at the assay site difficult or impractical due to the formation of a high solid content that cannot be easily extruded from the surface using oil or, consequently, increases aggregation of the capture objects (e.g., in the assay device). However, by using a relatively small number of capture objects with affinity for each type of analyte molecule or particle (e.g., 50,000 or less, 10,000 or less, or less), a small number of capture objects are used in total, thereby allowing the step of sealing the capture objects at the assay site to be done with oil, for example, with little to no aggregation. Furthermore, it is known that signal and binding events for different analytes or particles can make detection of different analytes difficult due to "crosstalk" (e.g., during substantially simultaneous detection of an array of assay sites). In the context of this disclosure, it has become clear that the use of a relatively low number of capture objects can reduce or eliminate such crosstalk (for example, by resulting in greater distance between stationary capture objects).Some such multiplex assays also benefit from improved sensitivity by using a smaller number of capture objects (e.g., beads) for capturing individual analytes.

[0157] In some embodiments, different capture objects may be used for analyte capture of different analyte targets. In some cases, different subgroups of the entire group of capture objects have different binding specificities (e.g., by including surfaces with different binding specificities). In these embodiments, two or more types of analyte molecules may be quantified and / or detected by a single multiplex assay. For example, the above capture objects may be a first capture object each having affinity for a first type of analyte molecule or particle, and the method may further include exposing a solution to a second capture object each having affinity for a second type of analyte molecule. Exposure to a sample containing the first type of analyte molecule and the second type of analyte molecule results in the immobilization of the first type of analyte molecule with respect to the first capture object and the immobilization of the second type of analyte molecule with respect to the second capture object. The first and second capture objects may be coded to be distinguishable from each other (e.g., to facilitate identification during detection) by including different detectable properties. For example, each subgroup of captured objects may have different fluorescence emission, spectral reflectance, shape, spectral absorption, or FTIR emission or absorption. In certain embodiments, each subgroup of the entire group of captured objects contains one or more dyes (e.g., fluorescent dyes) at varying concentration levels, thereby giving each subgroup of captured objects a characteristic signal (e.g., based on the intensity of fluorescence emission). In some embodiments, including spatial separation, after spatially separating the captured objects at multiple locations of detection, the locations containing first captured objects associated with a first type of analyte molecule can be distinguished from the locations containing second captured objects associated with a second type of analyte molecule by detection of different properties. By measuring the number of locations containing each subgroup of captured objects and / or the number of captured objects associated with analyte molecules, it may be possible to measure the concentrations of the first type and the second type of analyte molecules in a fluid sample based at least in part on these numbers.It should be understood that some multiplexing methods may involve the detection of two different types of analyte molecules or particles (e.g., a first type of analyte molecule or particle and a second type of analyte molecule), and some methods may further involve the detection of a larger number of different types of analyte molecules or particles (e.g., a third type of analyte molecule or particle, a fourth type of analyte molecule or particle, and others). Multiplexing assays may involve the detection of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, and / or at most 100, at most 120, at most 150 or more different types of analyte molecules or particles. The total number of capture objects having affinity for any type of analyte molecule or particle used in the assay may be expanded or contracted depending on the number of different types of analyte molecules or particles detected. For example, a single assay may contain 50,000 or fewer total capture objects (each having affinity for a specific type of analyte molecule or particle), while a "dual" assay may contain 100,000 or fewer total capture objects (50,000 or fewer with affinity for a first type of analyte molecule or particle, or 50,000 or fewer with affinity for a second type of analyte molecule or particle). In some multiple assays, the number of capture objects in each subgroup of capture objects (each subgroup having affinity for a different type of analyte molecule or particle) during the exposure step to the solution is 50,000 or fewer, 25,000 or fewer, 10,000 or fewer, 5,000 or fewer, 2,000 or fewer, and / or just 1,000, just 500, just 200, just 100, or fewer. In some embodiments, the total number of captured objects having affinity for any type of analyte molecule or particle is 100,000 or less, 80,000 or less, 60,000 or less, 50,000 or less, 25,000 or less, 10,000 or less, 5,000 or less, and / or just 2,000, just 1,000, just 500, just 200, just 100, or less.

[0158] In some embodiments, multiple locations may be targeted, and / or multiple capture objects and / or species / molecules / particles of interest may be detected substantially simultaneously. As used in this context, “substantially simultaneously” means processing / detecting the locations / capture objects / species / molecules / particles of interest approximately at the same time, i.e., the time spent processing / detecting at least two locations / capture objects / species / molecules / particles of interest overlaps, as opposed to sequential targeting / detection. Simultaneous targeting / detection can be carried out using various techniques, including optical techniques (e.g., CCD or CMOS detectors). According to some embodiments, spatially separating capture objects and analyte molecules or particles into multiple separate, divisible locations facilitates substantially simultaneous detection by enabling the processing of a large number of locations substantially simultaneously. For example, in embodiments where individual analyte molecules or particles are associated with capture objects spatially separated with respect to other capture objects at multiple separate, separately divisible locations during detection, processing multiple separate, separately divisible locations substantially simultaneously allows for the separation of individual capture objects, and therefore individual analyte molecules or particles. For example, in certain embodiments, individual analite molecules / particles of multiple analite molecules / particles are divided across multiple reactors such that each reactor contains zero or only one species / molecule / particle. In some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 96% of all analite molecules or particles, and at least 97%, at least 98%, at least 99%, and at least 99.5%, are spatially separated with respect to other analite molecules or particles.Multiple analyte molecules or particles can be detected substantially simultaneously within a time frame of less than 1 second, less than 500 milliseconds, less than 100 milliseconds, less than 50 milliseconds, less than 10 milliseconds, less than 500 microseconds, less than 100 microseconds, less than 50 microseconds, less than 1 microsecond between 0.5 microseconds, less than 0.1 microseconds, less than 0.01 microseconds, less than 0.001 microseconds, or less. In some embodiments, multiple analyte molecules or particles can be detected substantially simultaneously within a time frame of about 100 microseconds to about 0.001 microseconds, about 10 microseconds to about 0.01 microseconds, or less.

[0159] In some embodiments, the captured object and / or location is inspected optically. The captured object and / or location, exhibiting a change in optical signature, can be identified by conventional optical trains and optical detection systems. Depending on the species being detected (e.g., type of fluorescent substance) and the operating wavelength, optical filters designed for specific wavelengths can be used for the optical inspection of the location. In embodiments where optical reference is used, the system comprises one or more light sources and / or multiple filters, and the wavelength and / or light source intensity can be adjusted. In some embodiments, light signals from multiple locations are captured using a CCD or CMOS camera. In some embodiments of the present invention, the assay site (e.g., reactor) can be sealed, for example, by fitting a substrate and a sealing member (after introducing, for example, the captured object, analyte molecules or particles, binding ligand and / or labeling precursor). Sealing the assay site (e.g., reactor) can ensure that the contents of each assay site cannot escape from the assay site during the remainder of the assay. In some cases, the assay site (e.g., reactor) may be sealed after the addition of the capture material and, optionally, at least one type of labeling precursor, thereby facilitating the detection of analyte molecules or particles. In embodiments using labeling precursors, by sealing the contents into some or each assay site (e.g., reactor), a reaction producing a detectable label can proceed within the assay site (e.g., reactor), thereby resulting in a detectable content of the label retained at the assay site for detection.

[0160] In some embodiments, at least some (e.g., a subset or all) of the assay sites are not sealed (e.g., after introducing capture objects, analyte molecules or particles, binding ligands and / or labeling precursors). In some such examples, the assay's detection signal production process does not produce freely diffusive detectable molecules (e.g., labeling agents), thereby avoiding interference associated with the diffusion of the capture object signal resulting from the labeling agent diffusing away from other capture objects (which could reduce the accuracy of the assay). For example, in some embodiments, the labeling agent originates from a labeling precursor and is immobilized (e.g., via chemical bonding or precipitation) with respect to and / or near the capture object, as described in detail below. Such immobilization of the labeling agent can result in a spatially immobilized detectable signal on or near the signal-generating capture object and does not practically diffuse from the analyte-signal-generating capture object (e.g., those associated with analyte molecules or particles) to the non-analyte-signal-generating capture object (e.g., those not associated with any analyte molecules or particles). In some embodiments, during the assay or a particular step of the assay (e.g., during the processing step), 50% or less, 25% or less, 10% or less, 5% or less, 2% or less, 1% or less, or any part of the assay site is not sealed. Thus, in some embodiments, the apparatus for immobilizing the capture object and / or performing the assay as described herein does not need to include a sealing member.

[0161] Multiple locations (e.g., assay sites) can be formed by various methods or materials. In some embodiments, the multiple locations include assay sites in the form of reactors / wells on a substrate. In some cases, the reactors can be formed as an array of concave forms on a first surface, in some examples. However, in other cases, the reactors can be formed by fitting a substrate, which may have a featureless surface or include concave states arranged on them on a sealing material, with a sealing material containing multiple concave states. Either the device component (e.g., substrate or sealing component) can be fabricated from a suitable material (e.g., elastic polymer material) for sealing assistance. The surface may be hydrophobic or contain hydrophobic portions, or may be fabricated in that manner. Hydrophobicity can, in some examples, reduce leakage of aqueous samples from the reactor (e.g., microwell). In certain embodiments, the reactor may be configured to accept and contain only a single capture object (e.g., a bead).

[0162] In some embodiments, the assay sites (e.g., reactors) may all have approximately the same volume. In other embodiments, the assay sites (e.g., reactors) may have different volumes. The volume of individual assay sites (e.g., reactors) may be appropriately selected to facilitate any particular assay protocol. For example, in a set of assay sites where it is desirable to limit the number of capture objects for the capture of analytes immobilized with respect to each site to a small number, the volume of the assay sites (e.g., reactors) may vary from less than att liters to more than nanoliters, depending on the properties of the capture objects, the detection techniques and equipment used, the number and density of assay sites (e.g., reactors) on the substrate, and the expected concentration of the capture objects in the fluid applied to the substrate containing the wells. In one embodiment, the size of the assay site (e.g., reactor) may be selected such that only a single capture object used for capturing analyte molecules or particles can be completely contained within the assay site (e.g., reactor) (for example, U.S. Patent Application No. 12 / 731,130 filed on 24 March 2010 (published on 1 September 2011 as U.S. Patent Publication No. 2011 / 0212848, titled "ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS"), filed on 1 March 2011 as International Patent Application No. PCT / US2011 / 026645 (published on 9 September 2011 as International Publication No. 2011 / 109364, titled "ULTRA-SENSITIVE DETECTION OF MOLECULES OR See the name "PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS".

[0163] The total number of positions or the density of positions used in the assay (e.g., the number / density of reactors in the array) may depend on the composition and the end-use of the array. As described above, the number of assay sites (e.g., reactors) used may depend on the number of types of analyte molecules or particles or binding ligands used, the expected concentration range of the assay, the detection method, the size of the captured objects, and the type of detectable (e.g., free labeling agents in solution, precipitated labeling agents, etc.). In some embodiments, the number of captured objects exposed to a solution containing or suspected to contain at least one analyte molecule or particle is less than or equal to the number of positions used in the assay (e.g., the number of assay sites on the surface of the array). In some embodiments, the ratio of the number of capture objects exposed to a solution containing or suspected to contain at least one analyte molecule or particle to the number of separate locations (e.g., assay sites) used in the assay is ≤1:1, ≤1:2, ≤1:3, ≤1:4, ≤1:5, ≤1:10, ≤1:20, ≤1:30, ≤1:40, and / or as low as 1:50, ≤1:100, ≤1:1,000, ≤1:2,000, ≤1:5,000, or less.

[0164] Arrays containing approximately 200,000 to several billion assay sites (e.g., reactors) (or the total number of reactors) can be manufactured using a variety of technologies and materials. Increasing the number of assay sites (e.g., reactors in the form of an array) can increase the dynamic range of the assay or enable the parallel analysis of multiple samples or multiple types of analytes. An array may contain 1,000 to 1,000,000 assay sites (e.g., reactors) per sample being analyzed. In some cases, an array may contain more than 1,000,000 assay sites (e.g., reactors). In some embodiments, the array includes 1,000 to approximately 50,000, 1,000 to 1,000,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1,000,000, 100,000 to 500,000, 1,000 to 100,000, 50,000 to 100,000, 20,000 to 80,000, 30,000 to 70,000, and 40,000 to 60,000 assay sites (e.g., reactors). In some embodiments, the array includes 10,000, 20,000, 50,000, 100,000, 150,000, 200,000, 300,000, 500,000, 1,000,000 or more assay sites (e.g., reactors). The assay sites (e.g., reactors) may have volumes within any of the above ranges (e.g., 10 attoliters to 100 picoliters, 1 femtoliter to 1 picoliter).

[0165] Assay sites (e.g., reactors) may optionally be arranged in the form of an array, either on a substantially planar surface or in a non-planar three-dimensional arrangement.

[0166] Assay sites (e.g., reactors) may be arranged in a regular pattern or randomly distributed. In certain embodiments, the array is a regular pattern of sites on a substantially planar surface where the sites can be processed in the XY coordinate plane. In some embodiments, the assay site (e.g., reactor) is formed on and / or within a solid material. For example, the solid material may be a part of the assay consumable described herein. Such solid material may be or contain a hydrophobic material. As will be understood by those skilled in the art, the number of potentially suitable materials that can be formed in the reactor is very large and includes, but is not limited to, glass (including modified and / or functionalized glass), plastics (acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, cyclic olefin copolymers (COC), cyclic olefin polymers (COP), Teflon®, polysaccharides, nylon or nitrocellulose, etc.), elastomers (e.g., poly(dimethylsiloxane) and polyurethane), composite materials, ceramics, silica or silica-based materials (including silicon and modified silicon), carbon, metals, optical fiber bundles, etc. The substrate material may be selected to allow optical detection without significant autofluorescence. In certain embodiments, the assay site (e.g., reactor) may be formed in a soft material.

[0167] Surface reactors (e.g., substrates or sealants) can be formed using a variety of techniques of the known art, including but not limited to photolithography, stamping, molding, and etching. As those skilled in the art will understand, the technique used may depend on the composition and shape of the support material, as well as the size and number of reactors. In certain embodiments, an array of reactors is formed by creating microwells on one end of a fiber optic bundle and utilizing a plane-corresponding surface as a sealant.

[0168] In some embodiments, the assays and methods described may be carried out using commercially available systems (e.g., Simoa HD-1 Analyzer®, Simoa HD-X Analyzer®, and Quanterix SR-X® (Quanterix®, Lexington, Massachusetts)). Also, refer to U.S. Patent Application No. 13 / 035472, “SYSTEMS, DEVICES, AND METHODS FOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES” (Fournier et al.), filed on 25 February 2011 and published as U.S. Patent Application Publication No. 2012 / 0196774, which is incorporated herein by reference. In some examples, modifications to the Simoa HD-1 Analyzer® and Quanterix SR-X® can facilitate the above-described specific methods and systems for generating force fields and fluid plug flows.

[0169] Alternatively, an equivalent configuration of the reactor may be manufactured using other methods and materials that do not utilize the ends of the optical fiber bundle as the substrate. For example, the array may be a substrate manufactured by spot, print or photolithography techniques of the known art (see, for example, International Publications 95 / 25116, 95 / 35505, PCT US98 / 09163, U.S. Patents 5,700,637, 5,807,522, 5,445,934, 6,406,845 and 6,482,593, which are incorporated herein by reference for all purposes). In some cases, as is known to those skilled in the art, the array may be manufactured using molding, embossing and / or etching techniques.

[0170] In some embodiments, multiple locations include multiple non-reactor / well assay sites. For example, in embodiments where a capture object is used, a patterned, substantially flat surface may be used, and the patterned region forms multiple locations. In some cases, the patterned region may comprise a substantially hydrophilic surface substantially surrounded by a substantially hydrophobic surface.

[0171] In certain embodiments, the captured objects (e.g., beads) may be substantially surrounded by a substantially hydrophilic medium (e.g., including water), and the captured objects may be exposed to a patterned surface, causing them to aggregate in patterned areas (e.g., hydrophilic locations on the surface), thereby spatially separating the beads. For example, in one such embodiment, the substrate is or contains a gel or other material that provides sufficient obstruction to the transport of the material (e.g., a barrier to convection and / or diffusion), so that the captured objects and / or precursor labelers and / or labelers used for analyte capture do not move from one location on or within the material to another, preventing interference or crosstalk between spatial locations containing different captured objects in the time frame required to process the locations and complete the assay. For example, in one embodiment, the captured objects are spatially separated by dispersing the captured objects on and / or within a hydrogel material. In some cases, the labeling precursor may already be present in the hydrogel, thereby facilitating the occurrence of localized concentration of the labeler (e.g., by exposure to a binding ligand or analyte molecule carrying an enzyme component). In yet another embodiment, the captured object may be confined to one or more capillaries. In some cases, the captured object may be absorbed into or confined to a porous or fibrous substrate (e.g., filter paper). In some embodiments, the captured object may be spatially separated on a uniform surface (e.g., a planar surface), and the captured object may be detected using a precursor labeling agent that is converted into a substantially insoluble or precipitated labeling agent that remains localized at or near the location where the corresponding captured object is confined. In some cases, a single analite molecule or particle may be spatially separated into multiple droplets; that is, a single analite molecule or particle may be substantially contained within a droplet containing a first fluid, where the droplet may be substantially surrounded by a second fluid that is substantially immiscible with the first fluid.

[0172] Immobilization of labeling agents for captured objects In some embodiments, a labeling precursor is converted into a labeling agent that is immobilized with respect to a capture object. As one example, a freely diffusive precursor agent may be exposed to a binding ligand immobilized with respect to an analyte molecule or particle that is itself immobilized with respect to a capture object (e.g., beads). The freely diffusive precursor can readily undergo a chemical reaction with components of the binding ligand (e.g., an enzymatic component) to form a labeling agent, which, upon its formation or after further chemical or physical transformation and / or translocation (e.g., further chemical reaction and / or precipitation), becomes immobilized with respect to such capture object (e.g., beads) so that the labeling agent does not freely diffuse from the capture object. The immobilized labeling agent can produce a detectable signal (e.g., emission of electromagnetic radiation, e.g., from fluorescence) on the capture object (e.g., on which the labeling agent is associated with at least one analyte molecule or particle. In such embodiments, an indicator representing the number or proportion of capture objects having at least one immobilized labeling agent can then be measured. The concentration of a particular analyte molecule or particle can then be measured, at least in part, based on the measurement of an index representing the number or proportion of captured objects, which are measured as having at least one immobilized labeling agent.

[0173] In the context of this disclosure, it has been recognized that immobilized labels (in contrast to freely diffusive labels) enable simpler sample handling and / or detection schemes. For example, the absence of freely diffusive labels may simplify a method for detecting captured objects because it eliminates the need to seal the captured objects and the label in spatially and fluidly separated assay sites (e.g., sealed reactors (e.g., sealed microwells)), at least in part, because immobilized labels do not diffuse substantially away from the associated captured objects, thus preventing interference with signal detection from captured objects that are not associated with analyte molecules or particles (as described above, resulting in the measurement of an inaccurate indicator of the number or proportion of captured objects associated with analyte molecules or particles, and thus potentially leading to an inaccurate measurement of the concentration of analyte molecules or particles).

[0174] In some embodiments, the process of converting a labeling precursor immobilized with respect to a capture object associated with an analyte molecule or particle into a labeling agent is performed before the spatial separation of the capture object to multiple separate locations (e.g., separate assay sites such as separate reactors). In some embodiments, the process of converting a labeling precursor immobilized with respect to a capture object associated with an analyte molecule or particle into a labeling agent is performed after the spatial separation of the capture object to multiple separate locations (e.g., separate assay sites such as separate reactors or separate locations on a flat surface).

[0175] Labeling agents derived from labeling precursors can be immobilized with respect to any capture object in a variety of ways. For example, a capture object may have a solid surface to which the labeling agent can be immobilized during or after its formation from the labeling agent precursor. Such immobilization may occur through the formation of a chemical bond between the labeling agent and a functional group attached to the capture object (e.g., a functional group attached to the surface of a bead). Such a chemical bond may be a covalent bond. In some embodiments, the immobilization of the labeling agent with respect to the capture object occurs via non-covalent interactions. One such example is an affinity-based specific bonding interaction between the labeling agent and a species (e.g., a biomolecule, a functional group) attached to the surface of the capture object. In some embodiments, detectable moieties are immobilized with respect to the labeling agent after the formation of a chemical bond between the labeling agent and a species associated with the capture object. For example, additional detectable moieties may associate with the immobilized labeling agent during and / or thereafter via covalent or non-covalent interactions (e.g., hybridization or a non-covalent specific affinity association).

[0176] In some embodiments, the labeling agent is immobilized on the surface of the captured object via nonspecific chemical or physical interactions. For example, in some embodiments, the labeling agent is immobilized by binding to or associating with the captured object, thereby forming a substantially insoluble or precipitated species. For example, the labeling agent may be substantially insoluble in the liquid in which the captured object is present, or the labeling agent may be present at a local concentration higher than its solubility limit, thereby causing the labeling agent to precipitate or deposit on the captured object (e.g., as a film or granular precipitate on the surface of the captured object).

[0177] We discuss binding ligands having a component containing horseradish peroxidase (HRP) as a specific set of exemplary examples of several embodiments, including the conversion of a labeling precursor to a labeling agent immobilized on a capture object via the enzymatic component of the binding ligand. HRP is a common enzymatic component for various assays and is known to those skilled in the art. HRP may be the enzymatic component of a binding ligand and / or other binding ligand (which may then be able to associate with an analyte molecule or particle). In non-limiting examples (where the analyte molecule is an antigen), the binding ligand may be an HRP-labeled antibody or a streptavidin conjugate. In some cases, HRP converts a labeling precursor molecule into a labeling agent molecule that is substantially insoluble under operating conditions and precipitates on the capture object. Many examples of labeling precursors are known, typically those used in Western blotting applications (e.g., chloronaphthol and / or diaminobenzidine). In some cases, the precipitate is a darkly colored molecule that can be optically detected. For example, a dark-colored precipitate can be detected using light when the precipitate absorbs light, unlike the surface of a captured object that lacks such a dark-colored precipitate.

[0178] A binding ligand containing an enzymatic component (e.g., HRP) can be used in conjunction with a labeling precursor molecule (e.g., an enzymatic substrate) that can be immobilized when converted to a labeling molecule (e.g., a detectable product) (e.g., through the formation of a chemical bond with a functional group attached to the surface of the capture object). For example, HRP catalyzes the conversion of tyramide to activated tyramide (e.g., as a free radical) that can be immobilized with respect to the material of a particular capture object in the presence of hydrogen peroxide. For example, the capture object may have a surface containing a functional group (e.g., a hydroxyl group, e.g., a phenolic group) that can react with the free radical of activated tyramide to form a covalent bond that attaches the tyramide to the surface of the capture object. The typically short lifetime (<1 ms) of activated tyramide can prevent significant diffusion of activated tyramide from the site of its formation (e.g., in some examples, the labeling radius is limited to 20 nm). In such a method, almost all tyramide molecules tend to be locally immobilized with respect to the capture object associated with the binding ligand having the horseradish peroxidase component. In some embodiments, a labeling precursor, such as a tyramide molecule, can be attached to any type of molecule or particle that facilitates detection. For example, a tyramide molecule can be attached to a dye (e.g., a fluorescent dye). Thus, the presence of a dye immobilized with respect to a capture object (e.g., via an immobilizing labeling agent) can be used to detect the presence of an analyte molecule associated with such a capture object. In some cases, the conversion of tyramide to activated tyramide can make components associated with tyramide detectable (e.g., fluorescence can be emitted by the activation of a non-fluorescent component). Since HRP catalytically activates tyramide molecules, if a sufficient amount of reactant is provided, the HRP component of a single binding ligand immobilized with respect to a capture object (e.g., via an analyte molecule or particle) can generate a large number of activated tyramide molecules (some or all of which may form covalent bonds with the capture object or be immobilized in other ways), which can generate an amplified signal in the capture object.Furthermore, or alternatively, the immobilized tyramide may form a site for immobilizing additional binding ligands containing HRP components that have affinity for tyramide. The further bound HRP components can further activate the tyramide molecules attached to the capture object, further amplifying the signal. For example, tyramide-biotin can be used to label the capture object, followed by labeling with streptavidin conjugated to a dye for fluorescence detection.

[0179] Other exemplary examples of several embodiments, including the conversion of a labeling precursor to a labeling agent immobilized on a capture object via the enzymatic component of the binding ligand, are binding ligands having a phosphatase-containing component. In non-limiting examples where the analyte molecule is the antigen, the binding ligand may be a phosphatase-labeled antibody or a streptavidin conjugate.

[0180] The phosphatase component can be used, for example, to mediate the amplification of enzyme-labeled fluorescence (ELF) signals. In ELF detection, the binding ligand may have an alkaline or acidic phosphatase component, and the labeling precursor contains the ELF 97 phosphate molecule (2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone). Exposure to the phosphatase component can convert the water-soluble ELF97 phosphate molecule, which has a pale blue fluorescence signal, into a water-insoluble ELF97 alcohol with bright yellow-green fluorescence. The water-insoluble ELF97 can function as a labeling agent that forms a fluorescent precipitate (e.g., precipitation of ELF97 alcohol precipitate on the capture object) that can be immobilized with respect to the capture object. Fluorescence from the ELF97 precipitate on (or near) the capture object can indicate that at least one analyte molecule or particle is associated with the capture object.

[0181] Another example of the conversion of a labeling precursor into an immobilized label is the use of rolling circle amplification (RCA). In such embodiments, a binding ligand (e.g., an antibody) containing an oligonucleotide primer can be bound to an analyte molecule or particle (e.g., associated with a capture object (e.g., beads)). Such a binding ligand may be, for example, an antibody having a single-stranded DNA oligonucleotide primer attached to the antibody (e.g., at the end of the antibody's heavy chain). The binding ligand containing the oligonucleotide primer can be exposed to a circular DNA template having a sequence complementary to the primer when immobilized with respect to the capture object. The complementary sequence of the circular DNA template can be replicated by the conversion of an added nucleotide (precursor label) into a copy of the complementary sequence (e.g., if DNA polymerase is present), which is then attached to the binding ligand as a long oligonucleotide (or polynucleotide) chain. Using the circular DNA template, numerous such copies of complementary sequences (e.g., hundreds) can be produced, resulting in a relatively long polynucleotide chain immobilized with respect to the capture object (e.g., via the binding ligand). The resulting single-stranded polynucleotide chain can serve as a label by having detectable regions, such as fluorescent probes, attached to supplemental nucleotides linked to some or all of the replicated nucleotide sequences of the longer polynucleotide chain (in some cases, multiple detectable regions).

[0182] In some embodiments, the captured objects associated with the immobilized label are spatially separated (e.g., by partitioning). In specific cases, the captured objects are partitioned into multiple assay sites in the form of a reactor (e.g., microwells). Such spatial separation can be performed before or after immobilization of the labeling agent. The reactor may be sealed in some embodiments, but may remain unsealed in others. In some embodiments, the captured objects associated with the immobilized label are limited to droplets. In such embodiments, the droplets are spatially separated. In such examples, the droplets are arranged on a flat surface. In such embodiments, the droplets are temporarily separated from the immobilized detection site by, for example, flowing them through a channel to reach the detection site (e.g., during the processing step). In some embodiments, the captured objects associated with the immobilized label are spatially separated across the entire flat surface (forming, for example, an ordered array or random distribution of captured objects depending on the specific format of the assay).

[0183] Concentration of analyte molecules or particles in a fluid sample, and the sensitivity of the assay. The methods and apparatus described may provide techniques for detecting or quantifying analyte molecules or particles in a fluid sample having a relatively low concentration of analyte molecules or particles. In some embodiments, the concentration of molecules or particles in the fluid sample (e.g., a particular type of molecule or particle) is 50 × 10⁻⁶. -15 M or less, 10×10 -15 M or less, 5×10 -15 M or less, 1×10 -15 M or less, 500×10 -18 M or less, 100×10 -18 M or less, 50×10 -18 M or less, 10×10 -18 M or less, 5×10 -18 M or less, 2×10 -18 M or less, and / or 1 × 10 -18 M's lowness, 500 x 10 -21 Low M, 100 x 10 -21 M's lowness, 50 x 10 -21 M's low, 40 x 10 -21M is low, or below.

[0184] The methods and apparatus described herein may provide assays for detecting or quantifying analyte molecules or particles in a fluid sample having a relatively low detection limit (LOD) for the analyte molecules or particles. “LOD of assay” generally means the concentration of analyte molecules or particles at which a signal is produced three standard deviations above the background. In some embodiments, the assay method provides a detection level of analyte molecules or particles (e.g., a specific type of molecule or particle) in a fluid sample of 50 × 10⁻⁶. -15 M or less, 10×10 -15 M or less, 5×10 -15 M or less, 1×10 -15 M or less, 500×10 -18 M or less, 100×10 -18 M or less, 50×10 -18 M or less, 10×10 -18 M or less, 5×10 -18 M or less, 2×10 -18 M or less, and / or 1 × 10 -18 M's lowness, 500 x 10 -21 Low M, 100 x 10 -21 M's lowness, 50 x 10 -21 M's low, 40 x 10 -21 It is characterized by having a low M value or being below that value.

[0185] As those skilled in the art will understand, many types of analyte molecules and particles can be detected and optionally quantified using the methods and apparatus described, and essentially any analyte molecule that can be immobilized with respect to a capture object can potentially be investigated, at least partially, in these methods and apparatus. Specific and more particular targets of potential interest that may include analyte molecules are mentioned below. The list below is illustrative and non-limiting.

[0186] In some embodiments, the analyte molecule is or contains a protein. For example, the analyte molecule may be an enzyme. Non-limiting examples of enzymes include oxidoreductases, transferases, kinases, hydrolases, lyases, isomerases, and ligases. Further examples of enzymes include, but are not limited to, polymerases, cathepsins, calpains, aminotransferases (e.g., AST and ALT), proteases such as caspases, nucleotide cyclases, transferases, lipases, and enzymes associated with heart attacks. When the systems / methods described herein are used to detect viruses or bacterial agents, suitable target enzymes include viral or bacterial polymerases and viral or bacterial proteases or other such enzymes.

[0187] In other embodiments, analyte molecules include enzymatic components. For example, analyte particles can be enzymatic components present on the surface of enzyme-containing cells or extracellular surfaces. Alternatively, analyte particles can be cells that do not have enzymatic components on their surface. Such cells are typically identified using indirect assay methods described later. Non-limiting examples of enzymatic components include horseradish peroxidase, β-galactosidase, and alkaline phosphatase.

[0188] In some embodiments, analyte molecules include biomolecules. Non-limiting examples of biomolecules include hormones, antibodies, cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular lipid membrane antigens and receptors (neuronal, hormonal, nutrient, and cell surface receptors), or their ligands, or combinations thereof. Non-limiting embodiments of proteins include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc. As will be understood by those skilled in the art, there are many usable protein analyte molecules that can be detected or evaluated for binding partners using the present invention. Suitable protein analyte molecules include, but are not limited to, enzymes as described above, immunoglobulins, hormones, growth factors, cytokines (many of which function as ligands for cell receptors), cancer markers, etc. Non-limiting examples of biomolecules include PSA, TNF-α, troponin and p24, IL-17A, IL-12p70, and interferon-α (IFN-α).

[0189] In some embodiments, the analyte molecule is or contains a biomarker. For example, the analyte may be or contain a neurological biomarker. Examples of suitable neurobiological biomarkers include, but are not limited to, tau protein, neurofilamentous light chain (NF-L), glial fibrillary acidic protein (GFAP), and ubiquitin C-terminal hydrolase LI (UCH-L1). In certain embodiments, the analyte molecule is or contains a post-translational modified protein (e.g., phosphate esterification, methylation, glycosylation), and the capture component contains antibodies specific to the post-translational modification. The modified protein may be captured by a capture component containing a number of specific antibodies, and the captured protein may then be further bound to a binding ligand containing a secondary antibody specific to the post-translational modification. Alternatively, the modified protein may be captured by a capture component containing antibodies specific to the post-translational modification, and the captured protein may then be further bound to a binding ligand containing antibodies specific to each modified protein.

[0190] In some embodiments, the analyte molecule is or contains a nucleic acid. The nucleic acid may be captured by a complementary nucleic acid fragment (e.g., an oligonucleotide), and then optionally labeled with a binding ligand containing a different complementary oligonucleotide.

[0191] Suitable analyte molecules and particles include, but are not limited to, small molecules (organic and inorganic compounds), environmental pollutants (pesticides, insecticides, toxins, etc.), therapeutic molecules (therapeutic and abuse drugs, antibiotics, etc.), biomolecules (hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cell membrane antigens and receptors (nerve, hormone, nutrient, and cell surface receptors), or their ligands, etc.), whole cells (prokaryotes (pathogenic bacteria, etc.) and eukaryotic cells (mammalian tumor cells, etc.)), viruses (retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.), spores, and others.

[0192] Fluid samples containing or suspected to contain analyte molecules may originate from any suitable source. In some cases, the sample may include liquids, fluid granular solids, fluid suspensions of solid particles, supercritical fluids, and / or gases. In some cases, the analyte molecules may be separated or purified from their source before measurement, but in certain embodiments, the untreated sample containing the analyte molecules may be tested directly. Sources of analyte molecules may include synthetics (e.g., those produced in a laboratory), environmental substances (e.g., air, soil, etc.), mammals, animals, plants, or any combination thereof. In certain examples, the source of analyte molecules may be substances from the human body (e.g., blood, serum, plasma, urine, saliva, feces, tissues, organs, etc.). The volume of the fluid sample to be analyzed may be any suitable amount from a wide range of volumes, depending on factors such as the number of capture objects used / available, the number of locations used / available, etc. As described above, in some embodiments, relatively large sample volumes are used compared to existing approaches.

[0193] Integrated microfluidic consumables and systems As described above, the apparatus for performing the assay may incorporate some or all of the components described. For example, the apparatus may comprise a sample inlet member and a capture object storage section (e.g., a container, a chamber). The apparatus may further comprise one or more reagent storage sections (e.g., storage sections for solutions containing one or more binding ligands (e.g., containers, chambers)), some of which may contain conversion agents (e.g., enzyme components). In some embodiments, the apparatus may comprise a chamber for exposing the capture object to a sample fluid (e.g., for incubation of the capture object with one or more analyte molecules or particles from the sample fluid). The apparatus may further comprise a sample washer configured to prepare the capture object and analyte molecules or particles for detection from a fluid sample (e.g., through one or more washing steps with a rinse solution). The sample washer may be used to expose the capture object (some of which may be associated with at least one analyte molecule or particle) to one or more binding ligands and / or conversion agents (e.g., enzyme components). In some embodiments, but not all, the apparatus may comprise an assay consumable handler configured to operably connect to assay consumables. In some embodiments, the assay consumable handler and assay consumable are configured to immobilize a capture object using the method described herein. For example, the assay consumable may have a surface having assay sites (e.g., each having a volume between 10 attoliters and 100 picoliters). The assay consumable handler may further comprise a capture object applicator configured to apply a capture object to the surface of the assay consumable or in close proximity to the surface (e.g., near the assay sites on the surface, if present). In such embodiments, the assay consumable handler may further comprise a force field generator configured to generate a force field adjacent to the assay consumable and in close proximity to the surface (e.g., near the assay sites). Furthermore, the assay consumable handler may further comprise a fluid injector configured to generate fluid plugs (e.g., including aqueous solutions) having a first meniscus and a second meniscus, respectively, when an immiscible fluid (e.g., a gas (e.g., air)) is present on the surface of the assay consumable.However, in some embodiments, the captured objects may be investigated without spatial separation at different locations (e.g., assay sites), and the assay consumables may be configured to process captured objects, for example, containing or potentially containing droplets surrounded by an immiscible fluid as described above. In some embodiments, the assay consumable handler comprises a fluid pump capable of moving fluid across the entire surface of the assay consumable. In some embodiments, storage compartments for other reagents and / or components (e.g., labeling precursors (e.g., enzyme substrates) and sealing materials (e.g., liquid sealing materials)) are included in the assay consumable handler and / or assay consumable. In some embodiments including assay sites (e.g., as arrays), the assay consumable handler may be configured to seal the assay sites containing immobilized captured objects and labeling precursors. The assay consumable handler may also comprise an imaging system with detectors and optics for detecting signals from captured objects (e.g., from droplets, etc., from assay sites). In some embodiments, the assay consumable handler further comprises a controller comprising one or more processors configured to regulate the fluid pump to move fluid across the entire surface of the assay consumable. The assay consumable handler may include a computer-implemented control system configured to receive information from the imaging system and measure an indicator representing the concentration of an analyte or molecule. It should be understood that such an integrated device may take the form of, for example, an automated robotic system or a microfluidic system (e.g., some or all of the components reside on a chip).

[0194] An integrated microfluidic device configured to detect / quantify analyte molecules or particles in a fluid sample may take any of the following forms. In some embodiments, certain components described herein may be present in the form of a microfluidic chip in the assay consumable. Figures 7A and 7B show top and perspective views of such embodiments, respectively. According to a particular embodiment, Figures 7A and 7B show an assay consumable 315 in the form of a microfluidic chip. The assay consumable 315 comprises a sample inlet chamber 301, a captured object storage chamber 302, a binding ligand chamber 303, a conversion agent chamber 304, a sample incubation chamber 305, a binding ligand and conversion agent incubation chamber 306, a sample washing chamber 306, a sealing material chamber 308, a detection region 309, and a labeling agent precursor chamber 310. The various chambers and regions of the microfluidic chip may form fluid connections via one or more microfluidic channels shown as solid lines (e.g., solid line 311) in Figures 7A and 7B. Fluid transfer can be performed using certain techniques described in this disclosure (e.g., negative and / or positive pressure differences provided by a fluid pump (e.g., vacuum), capillary flow techniques, electrophoretic techniques, digital microfluidic techniques (e.g., electrowetting on a dielectric)), and can be controlled by appropriate valve configurations and other microfluidic components known in the prior art. One aspect of a suitable assay may include loading a sample fluid into an assay consumable 315 via a sample inlet chamber 301 and flowing the sample fluid from the sample inlet chamber 301 to a sample incubation chamber 305. Capture objects (e.g., beads) loaded into a capture object chamber 302 (e.g., as packaged or manually loaded capture objects) may be prepared to flow into the sample incubation chamber 305 (e.g., via a buffering solution). The incubation step may be performed in the sample incubation chamber 305, where the capture objects may be exposed to analyte molecules or particles from the sample fluid and subjected to an immobilization step, as described in the assays above.Simultaneously or at different times, the solution containing the binding ligand in the binding ligand chamber 304 and the solution containing the conversion agent (e.g., an enzyme component) may flow into the binding ligand and conversion agent incubation chamber 306, respectively, where they may be incubated (and subsequently associated). The captured objects (at least some of which may be associated with at least one analyte molecule or particle) may flow into the sample washing chamber 307, where they may be combined with the incubated binding ligand / conversion agent from chamber 306. In the sample washing chamber 307, excess analyte molecules or particles or other solution components may be removed via one or more rinse solutions (e.g., buffers) and may also be combined with the binding ligand and conversion agent. After preparation, the captured objects may flow into the detection area 309 where they may be investigated. In some embodiments, the captured objects may be immobilized with respect to the assay site on the surface of the assay consumable 315 of the detection area 309 (e.g., using an immobilization method with a fluid plug flow having the force field generator and / or receding meniscus described above). However, in some embodiments (e.g., in certain embodiments where the captured objects are isolated into separate droplets surrounded by an immiscible fluid in the detection region 309), the captured objects can be investigated as an array or as they flow through a channel (e.g., a single file) past an imaging system operably connected to the detection region 309 (not shown). In some embodiments, including the immobilization of the captured objects relative to the assay site in the detection region 309, a labeling precursor from the labeling precursor chamber 310 can be introduced into the detection chamber 309 after the immobilization of the captured objects. Furthermore, in some embodiments, a sealing step can be performed in which a sealant (e.g., a sealing solution) from the sealant chamber 308 is flowed into the detection region 309 after the immobilization of the captured objects, thereby sealing the assay site (e.g., before detection). The imaging system and a computer-implemented control system can then be used to acquire and analyze images and measure an index of the concentration of analyte molecules or particles.In certain embodiments, a microfluidic chip, such as those illustrated in Figures 7A and 7B, may be designed to be mated with a robot-controlled assay consumable handler and thereby operated and driven. In other embodiments, such a microfluidic chip may be used individually and / or operated manually by an operator.

[0195] In some embodiments, the microfluidic chips illustrated in Figures 7A-7B are configured to use dielectrophoretic force from an uneven electric field, as described above, to associate (e.g., immobilize) captured objects with respect to an assay site (e.g., in the detection region 309). In such embodiments, digital microfluidic technology (e.g., electrowetting in dielectric technology) is used to transport the fluid plug to the detection region (e.g., the detection region 309). For example, at least some detection regions or channels of the microfluidic chip may have conductive or dielectric electrical communication with a power source and be adjacent to the surface of the assay consumables with a conductive solid (e.g., an electrode). The application of a voltage to the conductive solid can cause the fluid plug to move (e.g., from conductive solid to conductive solid) across at least some of the surface of the microfluidic chip (e.g., in the assay site of the detection region 309).

[0196] Liquid handling technology Assays based on captured objects as described above may, in some examples, be carried out using preparation steps that can reduce or avoid the loss of captured objects. As noted above, in assays using relatively few captured objects, the loss of captured objects during the assay can be particularly undesirable. In some embodiments, one or more steps of the assay include forming a captured object suspension by mixing (e.g., associating or not associating) the captured objects and analyte molecules or particles in a liquid, followed by the removal of the liquid. These steps may include exposure of the captured objects to an initial fluid sample, exposure of the captured objects to a reagent (e.g., a binding ligand), and / or a washing step. In the context of this disclosure, it has become clear that when carried out using conventional liquid removal techniques, such liquid exposure and removal processes can be a source of captured object loss. Certain liquid removal techniques described herein (e.g., after sample washing) can lead to avoiding or reducing such loss of captured objects.

[0197] In some embodiments, capture objects may be provided. In some embodiments, relatively few capture objects are provided (e.g., 10,000 or less, 5,000 or less, and / or just 2,000, just 1,000, or less). These capture objects and analyte molecules or particles from a fluid sample can be prepared for detection. Preparation for detection may include one or more process steps, including: (1) mixing the capture objects and analyte molecules or particles in a liquid to form a capture object suspension; and (2) applying force to the capture object suspension to remove the liquid from the capture object suspension. In some embodiments, these preparation steps may be carried out in a suitable container including wells, such as a plate (e.g., a 96-well plate, a 384-well plate, etc.), a test tube, or an Eppendorf tube.

[0198] In some embodiments, one of such two-part processes involves exposing the capture object to a fluid sample containing analyte molecules or particles, the solution of which provides a liquid (e.g., an aqueous solvent (e.g., a buffer or sample medium)). The process then requires removing the liquid from the resulting capture object suspension (e.g., allowing the capture object to form pellets, some of which are associated with at least one analyte molecule or particle).

[0199] In some embodiments, one of such two-part processes includes a later step of resuspending a capture object, in which at least some are associated with at least one analyte molecule or particle, in a solution containing a binding ligand, thereby providing a liquid. The process then requires removing the liquid from the resulting capture object suspension (e.g., to form a pellet of capture objects, in which at least some are associated with at least one analyte molecule or particle and at least one binding ligand).

[0200] In some embodiments, one of the two-part processes includes a washing step using a washing solution, thereby providing a liquid with the washing solution. In certain embodiments, the washing solution is selected such that it does not cause a significant change in the composition of the captured object or analyte molecules or particles, and / or impair any specific binding interactions between at least two components of the assay (e.g., the captured object and the analyte molecules or particles). In other cases, the washing solution may be a solution selected to chemically interact with one or more assay components. As will be understood by those skilled in the art, the washing step may be performed at any appropriate point during the described method. For example, the captured object may be washed after being exposed to one or more solutions containing analyte molecules, binding ligands, labeling precursors, etc.

[0201] As another example, after immobilization of analyte molecules or particles with respect to multiple capture objects, the capture objects may be subjected to a washing step to remove any analyte molecules that are not specifically immobilized with respect to the capture objects. In some embodiments of the two-part process that include a washing step, the process then needs to remove the liquid from the washing solution (e.g., aqueous buffer) from the resulting capture object / washing buffer suspension (e.g., to form a washed pellet of capture objects, at least some of which are associated with at least one analyte molecule or particle and / or at least one binding ligand).

[0202] In the context of this disclosure, it has become apparent in several examples that a specific force can be applied to remove the liquid from the two-part process described above in a manner that results in relatively little loss of captured material. In particular, in some embodiments, a force is applied to the captured material suspension, but this force does not involve applying negative pressure to the captured material suspension via a fluid connection of the captured material suspension to a vacuum source, which tends to remove the liquid. A fluid connection of the captured material suspension to a vacuum source which tends to remove the liquid may include automatic or manual pipetting / syringing of the supernatant. However, such methods, including the application of vacuum via a fluid connection, can, in some embodiments, also pull the captured material away from the suspension, resulting in the loss of captured material. In contrast, it has been found that such problems can be avoided by applying other types of force. For example, in some embodiments, centrifugal force is applied to the captured material suspension, and the centrifugal force contributes to the removal of the liquid. In some embodiments, the apparatus described comprises a sample washer configured to apply such force to remove the washing solution from the captured material suspension. For example, referring to Figure 8, the sample washer 90 may be configured to apply centrifugal force to the captured material suspension. The sample washer 90 may be configured to do so by including a force field generator that can generate a force field in close proximity to the captured object, and as the force field acts on the captured object, the captured object resists the movement caused by the force, and the liquid (e.g., washing solution) is removed. As one example, Figure 8 shows a sample washer 90 comprising a container 710 containing liquid 720 containing a captured object 100. Rotation of the container 710 (indicated by arrow 700) generates a centrifugal force 705, which can remove the liquid 720 from the container 710. In some embodiments, a force field generator 740 (e.g., a magnet) may generate a force field (e.g., a magnetic field) represented by a vector field 745, and as the force field acts on the captured object 100 (e.g., magnetic beads), the captured object resists the movement caused by the centrifugal force 705. Systems like the sample washer 90 may be available commercially, such as Blue® Washer (BlueCatBio, Inc.).

[0203] In some embodiments, the sample washer includes a force field generator capable of generating an electric field in close proximity to the captured object. The electric field can act on the captured object. For example, the electric field can act on the captured object so that it resists the movement caused by the force field applied to remove the liquid. The electric field can be used to facilitate other operations on the captured object during sample preparation (e.g., in a microplate, sample washer, etc.), such as mixing, granulation, and / or resuspension (e.g., after granulation of the captured object). In some embodiments, the force field generator is configured to generate an electric field acting on the captured object using dielectrophoresis (e.g., by generating an uneven electric field). The force field generator may be configured so that the electric field can provide attractive or repulsive forces, for example, depending on the frequency of the electric field (i.e., the frequency of the alternating current). Such a configuration allows different dielectrophoretic forces to be applied to the captured object at different points in the sample preparation process (for example, using positively charged dielectrophoresis to resist the movement of the captured object during liquid removal, and using negatively charged dielectrophoresis to facilitate the movement of the captured object when resuspension or mixing is required).

[0204] In some embodiments, the above process for preparing capture objects may be carried out such that at least a portion thereof associates with analyte molecules or particles from a fluid sample, and a statistically significant proportion does not associate with any analyte molecules or particles, but the total number of prepared capture objects is 90% or more, 95% or more, 99% or more, or more of the initially provided capture objects. The prepared capture objects may then be used in downstream steps of the assay described. Some such steps may include measuring the concentration of analyte molecules or particles in the fluid sample, at least in part on an index representing the number or percentage of capture objects determined to be associated with at least one analyte molecule or particle.

[0205] kinetic principles In the present invention, the inventors have revealed, in the context of this disclosure, certain kinetic considerations that may improve the sensitivity of assays for detecting and / or quantifying analytes. In some cases, such kinetic considerations may contribute to assays having the above-mentioned sensitivity range (e.g., 2 atmoles or less or even less). Some considerations relate to the recognition that the sensitivity of an assay may depend on the efficiency with respect to the capture material (i.e., the range of analyte capture) of the analyte in the solution. Such considerations may be particularly important in some embodiments in which relatively few capture materials are used, as analyte capture efficiency deteriorates when there is little or no capture material (and even less capture component).

[0206] The inventors have recognized that, in some embodiments, the affinity of the capturing material (e.g., the affinity of the binding surface containing the capturing component, if present) can affect the extent to which the analyte is captured under certain conditions. Therefore, in some embodiments, though not always, a relatively high affinity for the analyte (e.g., 10) -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 A capture object is used that includes a bonding surface having a dissociation constant of M or less (or less). Furthermore, as described above, it has become clear in the context of this disclosure that a relatively larger sample volume and a relatively longer exposure time of the capture object to the fluid sample may be employed (for example, using the range described above).

[0207] kit The kinetic insights provided above and shown in the examples below enable the selection of capture objects for such highly sensitive assays, such as assays with relatively few capture objects. In some embodiments, a kit is provided for preparing a sample of analyte molecules or particles for detection. The kit may include capture objects comprising a binding surface having affinity for analyte molecules or particles. In some embodiments, the capture objects may be suitable for assays with relatively few capture objects (based on, for example, their affinity for analytes, the density of capture components on their binding surface, or any of various other considerations evident from this disclosure). In some embodiments, a first assay using 5,000 capture objects identical to those in the kit has a detection limit at least 50%, at least 75%, at least 90%, or at least 99% lower than the detection limit of a second assay using 500,000 capture objects identical to those in the kit, under the same conditions except for the length of the respective incubation steps for the first and second assays. In some embodiments, a first assay includes a step of incubating analyte molecules or particles with the capture object for a first time, while a second assay includes a step of incubating analyte molecules or particles with the capture object for a second time, where the first time is substantially longer than the second time (e.g., 100 times longer). "Same conditions except for..." includes conditions such as sample volume, sample source, and detection conditions, but does not include the concentration of the capture object in the sample. While a kit may be characterized by a comparison of detection limits between assays with 500,000 versus 5,000 capture objects, it should be understood that a kit does not necessarily contain the capture object content that is included at these values. For example, a kit may contain as few as 100 capture objects (or fewer) or as many as 5,000,000 capture objects (or more).

[0208] In some embodiments, the kit provided may include a packaged container for the analyte detection assay. Such a packaged container may contain a relatively small amount of capture material.

[0209] The kit can be packaged for any of a variety of assays. In some embodiments, the kit is packaged for assays requiring up to 96 separate experiments (such as those performed by distributing the capture objects equally across the wells of a 96-well plate). In some embodiments, the packaged container contains 50,000 or more, 100,000 or more, 500,000 or more, 1,000,000 or more and / or up to 2,000,000 or up to 5,000,000 capture objects, each containing a binding surface having affinity for an analyte.

[0210] The binding surface of the capture object may contain, for example, a capture component having affinity for the analyte. The capture object may be relatively small (e.g., having a diameter of 0.1 to 100 micrometers). In some embodiments, the analyte detection assay can be performed at a relatively low detection limit. For example, in some embodiments, the analyte detection assay may be 50 × 10⁻⁶ -18 M or less, 50×10 -18 M or less, 10×10 -18 M or less, 5×10 -18 M or less, 2×10 -18 M or less, 5, 1×10 -18 This can be done with a detection limit of M or less, or even lower.

[0211] This specification describes exemplary apparatus for performing specific assays described herein. The apparatus may include a sample washer configured to prepare magnetic beads and analyte molecules or particles from a fluid sample for detection. In some, but not all, examples, the sample washer is configured to remove liquid from the bead suspension (e.g., by applying centrifugal force instead) without applying negative pressure to the bead suspension. The apparatus may further include an assay consumable handler configured to operably connect to an assay consumable having a surface containing a reactor (e.g., each having a volume of 10 to 100 atcoliters). The apparatus may further include a bead applicator configured to apply magnetic beads to or in close proximity to the surface of the assay consumable. In such embodiments, the apparatus may further include a magnetic field generator configured to generate a magnetic field adjacent to the assay consumable and in close proximity to the surface. Furthermore, the apparatus may include a fluid injector configured adjacent to the assay consumable to generate fluid plugs (e.g., including aqueous solutions) having a first meniscus and a second meniscus, respectively, when an immiscible fluid (e.g., a gas (e.g., air)) is on the surface of the assay consumable. In some embodiments, the apparatus includes a fluid pump capable of moving fluid across the entire surface of the assay consumables. The apparatus may also include an imaging system comprising a detector and optics having a fixed field of view larger than the area defined by the reactor array. In some embodiments, the apparatus further includes a controller comprising one or more processors configured to regulate the fluid pump to move fluid across the entire surface of the assay consumables (e.g., in two directions). The apparatus also includes a computer-implemented control system configured to receive information from the imaging system and analyze the entire area including the reactor array.

[0212] In some embodiments, a method is provided for measuring the concentration of analyte molecules or particles in a fluid sample. The method may include exposing magnetic beads to a solution containing or suspected to contain at least one type of analyte molecules or particles. In some embodiments, the method includes immobilizing the analyte molecules or particles with respect to the magnetic beads, wherein at least some of the magnetic beads are associated with at least one analyte molecule or particle from the fluid sample, and a statistically significant proportion of the magnetic beads are not associated with any analyte molecules or particles from the fluid sample. In some cases, the solution is removed from at least some of the magnetic beads subjected to the immobilization step. In some embodiments, the method further includes delivering the magnetic beads in proximity to a reactor on a surface (e.g., an assay consumable). The method may further include generating a magnetic field in proximity to the surface to act on the captured object and moving the captured object toward the surface (e.g., via a permanent magnet or electromagnet). The method may include flowing a fluid plug containing the magnetic beads such that the receding meniscus of the fluid plug flows over at least part (or all) of the reactor. The method may further include inserting at least some magnetic beads into a reactor. Some embodiments include, after the insertion step, imaging the entire reactor, analyzing the entire reactor subjected to the imaging step, and measuring an index representing the number or proportion of magnetic beads associated with analyte molecules or particles from a fluid sample. In some examples, the concentration of analyte molecules or particles in the fluid sample is measured at least in part on an index representing the number or proportion of beads determined to be associated with at least one analyte molecule or particle.

[0213] In some embodiments, a method is provided for measuring the concentration of analyte molecules or particles in a fluid sample, in which a relatively high percentage of the captured objects are retained. In some embodiments, the method comprises exposing the captured objects to a solution containing or suspected to contain at least one type of analyte molecules or particles. The method further comprises immobilizing the analyte molecules or particles with respect to the captured objects, in which at least some of the captured objects associate with at least one analyte molecule or particle from the fluid sample, and a statistically significant proportion of the captured objects do not associate with any analyte molecules or particles from the fluid sample. In some embodiments, the method further comprises retaining at least 80%, at least 90%, at least 95%, at least 99%, or more of the captured objects subjected to the immobilization step, while removing the solution from at least some of the captured objects subjected to the immobilization step. At least 80%, at least 90%, at least 95%, at least 99%, or more of the captured objects subjected to the removal step can then be delivered in close proximity to an assay site on a surface. In some embodiments, the method includes immobilizing at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, or at least 99% of the capture objects subjected to the delivery step with respect to the assay site. The method may further include imaging at least 80%, at least 90%, at least 95%, at least 99%, or all of the assay site. In some embodiments, the method includes analyzing at least 75%, at least 90%, at least 95%, at least 99%, or all of the assay site subjected to the imaging step to measure an index representing the number or proportion of magnetic capture objects associated with analyte molecules or particles from a fluid sample. The method may then include measuring the concentration of analyte molecules or particles in the fluid sample, at least in part on the index representing the number or proportion of capture objects determined to be associated with at least one analyte molecule or particle.

[0214] U.S. Provisional Patent Application No. 63 / 010613, filed on April 15, 2020, with the title of the invention "Methods and Systems Related to Highly Sensitive Assays and Delivering Capture Objects," and U.S. Provisional Patent Application No. 63 / 010625, filed on April 15, 2020, with the title of the invention "Methods and Systems Related to Highly Sensitive Assays and Delivering Capture Objects," are incorporated herein by reference in their entirety for all purposes. The following examples are intended to illustrate specific aspects of the present invention, but not to illustrate the entire scope of the invention. [Examples]

[0215] Example 1 This example describes experimental procedures and modeling results related to the high sensitivity of a capture-object-based assay following a specific embodiment.

[0216] Protein measurement is crucial for the impact on life sciences, basic research, and the advancement of diagnostics and therapeutics. Higher sensitivity (when combined with high specificity) of protein measurement can provide greater diversity in the proteins detected and the samples in which they are detected. In recent years, immunoassays based on the detection of single proteins have emerged as a promising method for greatly improving the sensitivity of protein measurement, enabling the detection of proteins at subfemtomole concentrations. Described in this example is a “digital” immunoassay approach, which is based on the detection of single enzymes by capturing proteins on microscopic superparamagnetic beads, labeling the proteins with enzymes, and confining the products of the bead and enzyme-substrate reaction into individual wells of a femtoliter-sized array. This method is based on the classical enzyme-linked antibody immunosorbent assay (ELISA) but digitizes what is read out by individual enzymes, and thus has become known as digital ELISA. Digital ELISA is picomolar (10⁻¹⁰) -12 M) Below femtomol (~10 -16 Up to M), it was widely used to improve the sensitivity of immunoassays and enable the measurement of new types of proteins. Most notably, digital ELISA enabled the detection of neurological biomarkers in plasma and serum, offering the possibility of a “blood test for the brain” for the first time. Digital ELISA also enabled the measurement of inflammatory cytokines in the blood of healthy and diseased patients and the detection of proteins important for the early and accurate diagnosis of infectious agents.

[0217] While digital ELISA offers a technique for measuring proteins that were previously undetectable, it is clear that there is a need for even higher sensitivity at low atomolecular concentrations. For example, the detection rate of many cytokines in the blood (e.g., IL-17A) is less than 100%, meaning that quantification of these molecules, which are important when monitoring inflammatory conditions and responses to anti-inflammatory therapies, is not necessarily possible in all healthy individuals. Furthermore, biological insights can be gained by quantifying specific post-translational modifications of proteins, which provide greater biological and diagnostic specificity than the parent molecule, but often represent only a small percentage (~1%) of the total concentration of the parent molecule. For example, the detection of proteins in complex samples (e.g., feces and cerebrospinal fluid) can be achieved by diluting high-concentration samples with buffers to suppress the so-called matrix effect. However, since dilution negatively impacts detectability, a more sensitive assay would enable the detection of low amounts of proteins in complex samples. Early detection of infectious diseases is also made possible by high sensitivity to viral and bacterial proteins (e.g., HIV). Higher analytical sensitivity for proteins also enables the detection of small-volume samples (e.g., blood from rodents, finger sticks, and heel sticks from pediatric patients), testing of non-invasive samples that are typically at low concentrations, and rapid assays. The results described in this example and the examples below were performed to increase the sensitivity of digital ELISA.

[0218] A model of assay dynamics developed from a stepwise analysis of assay efficiency suggests a method for improving the sensitivity of digital ELISA. In digital ELISA, superparamagnetic beads coated with capture antibody are incubated with a sample containing the target protein. The protein binds to the capture antibody with high efficiency at a high on rate, and the protein is statistically distributed across the beads according to a Poisson distribution when [protein] < [beads] (this also applies to subfemtomole concentrations). The beads are washed and sequentially incubated with biotinylated detection antibody and streptavidin-β-galactosidase to label the immunocomplex with a single enzyme. The beads are resuspended in enzyme substrate, loaded into a microwell array, sealed with oil, and imaged to measure the percentage of beads associated with at least one enzyme. From this analysis, the average enzyme number per bead (AEB) is measured via a Poisson distribution. The dynamic model of this process predicted that, based on the concentrations of different components, incubation time, and the on-off rates of different bimolecular interactions, AEB (Autonomous Emission Embedding), i.e., sensitivity, would increase as the number of beads decreased, with a desirable bead count of 10,000–50,000 beads for antibody pairs with good affinity. Previous testing of this model was limited to relatively large bead counts (~500,000) due to the low efficiency of analyzing beads with the original digital ELISA (defined as "bead reading efficiency" = "number of beads to analyze" ÷ "number of beads added to the sample").

[0219] Typically, only 5% (about 25,000 beads) of the beads used to capture proteins from the sample are analyzed, resulting in a typical assay background on-bead ratio (f onThis yields approximately 250 positive beads. Due to the low bead readout efficiency, a high number of beads was required to obtain enough positive beads at the detection limit and to avoid excessive Poisson noise. On the other hand, this example and the examples below demonstrate a method with high bead readout efficiency that enables a substantial increase in AEB and assay sensitivity even with a low number of captured beads (~1,000~50,000). With this method, the most sensitive assay is possible with a small number of beads for protein capture and the ability to read out as many of these beads as possible.

[0220] While certain existing methods increase the number of beads being imaged, they have been limited in terms of improving the sensitivity of digital ELISAs. Firstly, conventional approaches use high bead counts (hundreds of thousands to hundreds of millions) and have not considered the use of lower bead counts (<10,000), which have proven advantageous for high-sensitivity assays in the context of this disclosure. Secondly, these approaches adjust their loading to increase the proportion of wells filled with beads and have not considered factors found to be important for assay sensitivity (i.e., bead readout efficiency) in the context of this disclosure. Finally, these conventional approaches focus solely on the bead loading step of the digital ELISA and have not considered other steps in the process that affect the number of analytical beads (e.g., assay steps and image analysis).

[0221] In the present example and the following examples, a method was developed that can improve the sensitivity of digital ELISA and increase the proportion of beads analyzed by using a low input bead count. An automated method for loading magnetic beads into high-efficiency microwell arrays based on Simoa™ disks (from Quanterix) and oil sealing is also described. To improve bead readout efficiency and investigate each step in the assay, a holistic approach was adopted that included examining bead loss during assay steps and image analysis. Based on the improved bead readout efficiency, higher-sensitivity digital ELISAs were developed for many different proteins, and advantages in the detectability of clinical samples were demonstrated.

[0222] experiment: Materials: Capture antibody beads, detection antibodies, streptavidin-β-galactosidase (SβG), resorufin-β-D-galactopyranoside (RGP), wash buffer, sample dilution buffer, microtiter plates, pipette tips, and Simoa™ disks were obtained from Quanterix. Serum and plasma samples from healthy individuals were obtained from bioIVT.

[0223] Assay Steps: Digital ELISA was performed following either a 3-step or 2-step process. For the 3-step assay, samples were diluted in buffer, and the diluted sample or calibrator solution (100-250 μL) was added to each well of a 96-well microtiter plate. A solution containing superparamagnetic beads coated with capture antibody (25 μL) was then added to each well, and the plate was incubated at 30°C on an orbital shaker (Quanterix). The beads in the wells were then washed using a Simoa Washer™ (Quanterix) or a Blue® Washer (BlueCatBio), with a 96-well magnetic manifold used to retain the beads during washing.

[0224] The beads were then sequentially incubated with 100 μL of detection antibody and 100 μL of SβG, with washing between each step. After the process was complete, the bead pellet was left on the plate and dried on a 96-well magnetic manifold. The two-step assay was similar to the three-step assay, except that the detection antibody was added to the sample and bead mixture during all or part of the sample incubation step (instead of a separate detection antibody step). When necessary to measure bead loss, the number of beads was quantified using a Multisizer Coulter counter particle analyzer (Beckman Coulter).

[0225] Detection and data analysis using Simoa (trademark) A 96-well plate containing a dried bead pellet was transferred to an SR-X® reader (Quanterix) and Simoa readout of the assay beads. The SR-X® was used either as is or modified to implement the magnetic meniscus sweep bead loading protocol described below. In the SR-X®, the bead pellet was reconstituted into the RGP using a disposable tip pipette, and the RGP-bead mixture was transferred to the aspiration port on the Simoa® disk, where the beads were aspirated into the entire well array by vacuum. The beads were either fixed or actively loaded into microwells, then sealed with oil, imaged, and analyzed to obtain average enzyme per bead (AEB). AEB as a function of calibrator concentration was fitted using a four-parameter logistic fit (4PL). Sample concentrations were measured by using their AEB values ​​as estimates from these calibration curves. The limit of detection (LOD) of the assay was calculated as the concentration corresponding to a signal 3 standard deviations higher than the assay background, assuming a coefficient of variation (CV) of 10% as the assay background. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were determined as the lower and upper ranges of the calibration curve, respectively, where the coefficient of variation (CV) profiling indicated an inaccuracy of more than 20% in the measured concentration. The dynamic range of the assays in the examples herein is log10 was measured as (ULOQ / LLOQ). In CV profiling, concentration inaccuracy was calculated using total signal noise. Total noise was calculated by combining the CV of a fixed AEB of 7.1% and the Poisson noise CV (from the number of beads analyzed) for each data point on the calibration curve. Concentration inaccuracy was calculated as the CV of concentrations obtained from 4PL fits of the mea...

Claims

1. Each capture object having an affinity for a specific type of analyte molecule or particle is exposed to a solution containing or suspected to contain at least one type of analyte molecule or particle, and the number of capture objects exposed to the solution containing or suspected to contain analyte molecules or particles is 50,000 or less. To immobilize specific types of analyte molecules or particles with respect to the captured objects, such that at least some of the captured objects are associated with at least one specific type of analyte molecule or particle from the fluid sample, and a statistically significant proportion of the captured objects are not associated with any specific type of analyte molecule or particle from the fluid sample. Immobilizing at least one binding ligand with respect to at least some of a specific type of analyte molecule or particle associated with the captured object, Exposing at least one immobilized binding ligand to a precursor label to convert the precursor label to a label, The step of immobilizing the captured object onto an analyte molecule or particle involves spatially isolating at least a portion of the captured object at multiple separate locations, The step of processing at least some of multiple locations subjected to spatial isolation measures an index of the number or proportion of captured objects associated with at least one of a specific type of analyte molecules or particles from a fluid sample, based on a signal related to the labeling agent, Measuring the concentration of a particular type of analyte molecule or particle in a fluid sample, at least in part, based on measuring an index of the number or proportion of captured objects determined to be associated with at least one particular type of analyte molecule or particle, A method for measuring the concentration of analyte molecules or particles in a fluid sample, including [specific components / synthetic elements].

2. Each capture object having an affinity for a specific type of analyte molecule or particle is exposed to a solution containing or suspected to contain at least one type of analyte molecule or particle, and the number of capture objects exposed to the solution containing or suspected to contain analyte molecules or particles is 50,000 or less. To immobilize a specific type of analyte molecule or particle with respect to the captured object, such that at least some of the captured objects are associated with at least one of a specific type of analyte molecule or particle from the fluid sample, Immobilizing at least one binding ligand with respect to at least some of a specific type of analyte molecule or particle associated with the captured object, Exposing at least one immobilized binding ligand to a precursor label to convert the precursor label to a label, The step of immobilizing the captured object onto an analyte molecule or particle involves spatially isolating at least a portion of the captured object at multiple separate locations, The step of processing at least some of multiple locations subjected to spatial isolation measures an index of the number or proportion of captured objects associated with at least one of a specific type of analyte molecules or particles from a fluid sample, based on a signal related to the labeling agent, Measuring the concentration of a particular type of analite molecule or particle in a fluid sample, at least in part on measuring an index representing the number or proportion of captured objects that have been determined to be associated with at least one particular type of analite molecule or particle, based on the measurement of an index representing the number or proportion of captured objects that have been determined to be associated with at least one particular type of analite molecule or particle, or measuring the concentration of a particular type of analite molecule or particle in a fluid sample, at least in part on the measured intensity level of a signal associated with a labeling agent that is an indicator of the presence of multiple particular types of analite molecules or particles, A method for measuring the concentration of analyte molecules or particles in a fluid sample, including [specific components / synthetic elements].

3. Exposing a capture object, each having an affinity for a specific type of analyte molecule or particle, to a solution containing or suspected to contain at least one type of analyte molecule or particle, To immobilize specific types of analyte molecules or particles with respect to the captured objects, such that at least some of the captured objects are associated with at least one specific type of analyte molecule or particle from the fluid sample, and a statistically significant proportion of the captured objects are not associated with any specific type of analyte molecule or particle from the fluid sample. Immobilizing at least one binding ligand with respect to at least some of a specific type of analyte molecule or particle associated with the captured object, Exposure of at least one immobilized binding ligand to a precursor labeling agent, The step of immobilizing the captured object onto an analyte molecule or particle involves spatially isolating at least 25% of the captured object at multiple separate locations, Converting a precursor labeling agent into a labeling agent, The step of spatially isolating multiple locations involves processing at least some of those locations to identify an indicator of the number or proportion of captured objects associated with at least one specific type of analyte molecules or particles from a fluid sample, based on a signal related to the labeling agent. Measuring the concentration of a particular type of analyte molecule or particle in a fluid sample, at least in part, based on measuring an index of the number or proportion of captured objects determined to be associated with at least one analyte molecule or particle, Includes, The number of captured objects exposed to a solution containing or suspected to contain analyte molecules or particles is 50,000 or less. A method for measuring the concentration of analyte molecules or particles in a fluid sample.

4. Each capture object having an affinity for a specific type of analyte molecule or particle is exposed to a solution containing or suspected to contain at least one type of analyte molecule or particle, and the number of capture objects exposed to the solution containing or suspected to contain analyte molecules or particles is 50,000 or less. Immobilizing a specific type of analyte molecule or particle with respect to the captured object, such that at least some of the captured objects are associated with at least one specific type of analyte molecule or particle from the fluid sample, while a statistically significant proportion of the captured objects are not associated with any specific type of analyte molecule or particle from the fluid sample. Immobilizing at least one binding ligand with respect to at least some of a specific type of analyte molecule or particle associated with the captured object, Exposing at least one immobilized binding ligand to a precursor labeling agent to convert the precursor labeling agent into a labeling agent, and immobilizing this with respect to the captured object on which the binding ligand is immobilized, The step of immobilizing the captured object onto an analyte molecule or particle involves spatially isolating at least a portion of the captured object at multiple separate locations, The process involves processing at least some of multiple locations subjected to a spatial isolation step to measure an index of the number or proportion of captured objects containing at least one immobilized label based on a signal associated with the label, Measuring the concentration of a particular type of analyte molecule or particle in a fluid sample, at least in part, based on measuring an index of the number or proportion of captured objects determined to contain at least one immobilized labeling agent, A method for measuring the concentration of analyte molecules or particles in a fluid sample, including [specific components / synthetic elements].

5. The method according to any one of claims 1 to 2 and 4, comprising spatially isolating at least 25% of the captured object subjected to the step of immobilizing it on an analyte molecule or particle to a plurality of separate locations.

6. The method according to any one of claims 1 to 5, comprising spatially isolating at least 30%, at least 50%, at least 75%, at least 90%, at least 95%, or all of the captured object subjected to the step of immobilizing it on an analyte molecule or particle, in a plurality of separate locations.

7. The method is 50 x 10 －１８ The method according to any one of claims 1 to 6, characterized by a detection level for a specific type of analyte molecule or particle of M or less.

8. The method according to any one of claims 1 to 7, wherein the number of captured objects exposed to a solution containing or suspected to contain analyte molecules or particles is 10,000 or less.

9. The method according to any one of claims 1 to 8, wherein the number of captured objects exposed to a solution containing or suspected to contain analyte molecules or particles is 7,500 or less, 5,000 or less, 4,000 or less, 3,000 or less, or 2,000 or less.

10. The method according to any one of claims 1 to 9, wherein the number of captured objects exposed to a solution containing or suspected to contain analyte molecules or particles is 100 or more.

11. The method according to any one of claims 3 and 5 to 10, wherein the ratio of the number of captured objects exposed to a solution containing or suspected to contain analyte molecules or particles to the number of separate locations is 1:1 or less, 1:2 or less, 1:3 or less, 1:4 or less, 1:5 or less, 1:10 or less, 1:20 or less, 1:30 or less, or 1:40 or less.

12. The method according to any one of claims 1 to 11, wherein the exposure step is performed for 15 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, or 12 hours or more.

13. The method according to any one of claims 1 to 12, wherein the solution containing or suspected of containing at least one type of analyte molecule or particle has a volume of 50 microliters or more, 100 microliters or more, 200 microliters or more, or 300 microliters or more.

14. The method according to any one of claims 1 to 13, wherein the percentage of captured objects associated with at least one particular type of analyte molecule or particle is 99.99% or less, 99.9% or less, 99% or less, 98% or less, 95% or less, 90% or less, 80%, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, 0.5% or less, or 0.1% or less of the total number of captured objects.

15. The method according to any one of claims 1 to 14, wherein the captured object comprises a binding surface having affinity for a particular type of analyte molecule or particle.

16. The method according to any one of claims 1 to 15, wherein the captured object includes beads.

17. The method according to claim 16, wherein the beads are magnetic beads.

18. The method according to claim 17, wherein the magnetic beads are superparamagnetic.

19. The method according to claim 17, wherein the magnetic beads are ferromagnetic.

20. The method according to any one of claims 1 to 19, wherein the average diameter of the captured object is 0.1 micrometers to 100 micrometers.

21. The method according to any one of claims 3 and 5 to 20, wherein multiple separate locations include assay sites on the surface.

22. The method according to claim 21, wherein the assay site has an average volume of 10 at liters to 100 picoliters.

23. The method according to any one of claims 21 to 22, wherein the assay site comprises a reactor.

24. The method according to any one of claims 3 and 5 to 22, wherein multiple separate locations are located on a flat surface.

25. The method according to claim 24, wherein the flat surface is a uniform surface.

26. The method according to any one of claims 3 and 5 to 22, wherein multiple separate locations are on the pattern surface.

27. The method according to any one of claims 1 to 23, wherein the analyte molecule or particle is a protein or nucleic acid.

28. The method according to any one of claims 1 to 27, wherein the binding ligand comprises an enzyme component.

29. The concentration of a specific type of analyte molecule or particle in the fluid sample is 50 × 10 －１５ The method according to any one of claims 1 to 28, wherein M is less than or equal to M.

30. The method according to any one of claims 1 to 29, wherein the captured object contains or is contained within a droplet, and is suspended in a fluid that is immiscible with the droplet.

31. The method according to any one of claims 1 to 30, wherein the measurement of an index of the number or proportion of capture objects associated with at least one of a particular type of analyte molecules or particles from a fluid sample is a replication of a nucleic acid precursor.

32. The method according to claim 31, wherein measuring an index of the number or proportion of capture objects associated with at least one of a particular type of analyte molecules or particles from a fluid sample comprises performing one or more of polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation, and loop-mediated isothermal amplification (LAMP).

33. The method according to claim 31, wherein measuring an index of the number or proportion of captured objects associated with at least one of a particular type of analyte molecules or particles from a fluid sample is performed by performing rolling circle amplification (RCA).

34. The method according to any one of claims 1 to 31, further comprising measuring an index of the number or proportion of capture objects associated with at least one of a particular type of analyte molecules or particles from a fluid sample, or measuring an index of the number or proportion of capture objects containing nucleic acids that can be immediately detected.

35. The method according to any one of claims 1 to 34, wherein the concentration of a particular type of analyte molecule or particle in a fluid sample is measured at least partially by comparison with a calibration standard for the measured parameter.

36. The method according to any one of claims 3 and 5 to 35, wherein a plurality of separate locations are identified using optical techniques.

37. The method according to any one of claims 1 to 36, wherein an index of the concentration of analyte molecules or particles in a fluid sample is measured at least partially based on digital analysis.

38. The method according to any one of claims 1 to 36, wherein an indicator of the concentration of analyte molecules or particles in a fluid sample is measured at least partially based on a measurement of the intensity level of at least one indicator of the presence of analyte molecules or particles.

39. The captured object is the first captured object, and a specific type of analyte molecule or particle is the first type of analyte molecule or particle, and the method further, A second capture object, each containing a binding surface having affinity for a second type of analite molecule or particle, is exposed to a solution containing or suspected to contain at least one type of analite molecule or particle, and the number of second capture objects exposed to the solution containing or suspected to contain analite molecules or particles is 50,000 or less. With respect to the second capture object, the second type of analyte molecules or particles are immobilized such that at least some of the second capture objects are associated with at least one second type of analyte molecule or particle from the fluid sample, and a statistically significant proportion of the capture objects are not associated with any of the second type of analyte molecules or particles from the fluid sample. Measuring an index of the number or proportion of captured objects associated with at least one of a second type of analyte molecules or particles from a fluid sample, Measuring the concentration of a second type of analyte molecule or particle in a fluid sample, at least in part, based on measuring an index of the number or proportion of captured objects determined to be associated with at least one second type of analyte molecule or particle, The method according to any one of claims 1 to 36, including the method described in any one of claims 1 to 36.

40. The method according to claim 39, wherein each first capture object comprises a binding surface having affinity for a first type of analyte molecule or particle, and each second capture object comprises a binding surface having affinity for a second type of analyte molecule or particle.

41. The method according to claim 40, wherein the total number of captured objects having a binding surface that has affinity for any type of analyte molecule or particle exposed to a solution containing or suspected to contain at least one type of analyte molecule or particle is 100,000 or less.

42. The method according to any one of claims 1 to 41, wherein the labeling agent is immobilized with respect to the captured object by the formation of a chemical bond between the labeling agent and a functional group attached to the captured object.

43. The method according to claim 42, wherein, after the formation of a chemical bond, the detectable portion is immobilized with respect to the labeling agent.

44. The method according to any one of claims 4 to 41, wherein the labeling agent is immobilized with respect to the captured object by the formation of substantially insoluble or precipitated species associated with the captured object.

45. The method according to any one of claims 1 to 44, wherein the precursor labeling agent is a nucleotide, and the labeling agent immobilized on the captured object is an oligonucleotide or polynucleotide chain formed from nucleotides.

46. The method according to claim 45, wherein a precursor labeling agent is converted into a labeling agent by rolling circle amplification (RCA).

47. The method according to any one of claims 45 to 46, wherein the labeling agent is at least partially immobilized on the surface of the captured object through nonspecific chemical or physical interactions.

48. The method according to any one of claims 1 to 47, wherein a precursor labeling agent is converted to a labeling agent when exposed to an enzyme or enzyme component.

49. The method according to claim 48, wherein the enzyme or enzyme component is associated with at least one binding ligand.

50. Exposing magnetic beads to a solution containing or suspected to contain at least one type of analyte molecule or particle, wherein the number of captured objects exposed to the solution containing or suspected to contain analyte molecule or particle is 50,000 or less. Immobilizing analyte molecules or particles with respect to magnetic beads such that at least some of the magnetic beads are associated with at least one analyte molecule or particle from the fluid sample, and a statistically significant proportion of the magnetic beads are not associated with any analyte molecule or particle from the fluid sample. The fixation step involves removing the solution from at least a portion of the magnetic beads, Immobilizing at least one binding ligand with respect to at least some of a specific type of analyte molecules or particles associated with the magnetic beads, Exposure of at least one immobilized binding ligand to a precursor labeling agent, Delivering magnetic beads in close proximity to the reactor on the surface, This involves generating a magnetic field in close proximity to a surface that tends to act on the captured object, thereby moving the captured object towards the surface. The fluid plug containing magnetic beads is flowed so that the retracted meniscus of the fluid plug flows across at least some of the reactor, Inserting at least a portion of the magnetic beads into the reactor, Converting a precursor labeling agent into a labeling agent, Imaging the entire reactor after the insertion step, The entire reactor attached to the imaging step is analyzed to measure an indicator of the number or proportion of magnetic beads associated with analyte molecules or particles from the fluid sample, based on the signal related to the labeling agent. Measuring the concentration of analyte molecules or particles in a fluid sample, at least in part, based on measuring an index of the number or proportion of beads determined to be associated with at least one analyte molecule or particle, A method for measuring the concentration of analyte molecules or particles in a fluid sample, including [specific components / synthetic elements].

51. Exposure of the captured objects to a solution containing or suspected to contain at least one type of analyte molecule or particle, wherein the number of captured objects exposed to the solution containing or suspected to contain analyte molecules or particles is 50,000 or less. Immobilizing analite molecules or particles with respect to the captured objects such that at least some of the captured objects are associated with at least one analite molecule or particle from the fluid sample, and a statistically significant proportion of the captured objects are not associated with any analite molecule or particle from the fluid sample. The solution is removed from at least a portion of the captured object subjected to the immobilization step, while retaining at least 80% of the captured object subjected to the immobilization step. Immobilizing at least one binding ligand with respect to at least some of a specific type of analyte molecule or particle associated with the captured object, Exposure of at least one immobilized binding ligand to a precursor labeling agent, To deliver at least 80% of the captured material subjected to the removal step to the assay site on the surface, At least 20% of the captured material attached to the delivery step is immobilized with respect to the assay site, Converting a precursor labeling agent into a labeling agent, Image at least 80% of the assay site, Analyze at least 75% of the assay site attached to the imaging step to measure an index of the number or proportion of magnetically trapped objects associated with analyte molecules or particles from the fluid sample, based on the signal associated with the labeling agent. Measuring the concentration of analyte molecules or particles in a fluid sample, at least in part, based on measuring an indicator of the number or proportion of captured objects determined to be associated with at least one analyte molecule or particle, A method for measuring the concentration of analyte molecules or particles in a fluid sample, including [specific components / synthetic elements].

52. The concentration of analyte molecules or particles in the fluid sample is 2 × 10⁻⁶ －１８ The method according to any one of claims 1 to 47, wherein the detection level is less than M.

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