Kinetic immunoassay and enrichment systems and methods

Abstract

A microfluidic chip with an array of pillars for directing flow of beads is used to analyze reaction kinetics, to enrich for a target analyte, or to perform reactions. A stream may be continuously drawn from the reaction volume into the microfluidic chip. The bead is attached to a primary affinity reagent. The reaction volume has an analyte. The primary affinity reagent binds to the analyte. A secondary affinity reagent with a label binds to the analyte, creating a sandwich of bead, analyte, and label. The binding reactions occur over time in the microfluidic chip. The beads may be imaged after traversing a laminar wash buffer, and the signal intensity is measured. Each bead provides a kinetic monitoring of the immunoassay over the reaction time at which the bead is removed from the reaction media. Embodiments may include enriching a sample for analytes by binding the analytes to beads.

Description

KINETIC IMMUNOASSAY AND ENRICHMENT SYSTEMS AND METHODSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S. Non-Provisional Application No. 18 / 079,986, filed December 13, 2022, titled “KINETIC IMMUNOASSAY SYSTEMS AND METHODS”, U.S. Non-Provisional Application No. 18 / 473,464, filed September 25, 2023, titled “KINETIC IMMUNOASSAY SYSTEMS AND METHODS”, U.S. Non- Provisional Application No. 18 / 313,183, filed May 5, 2023, titled “MULTI-STEP KINETIC IMMUNOASSAY SYSTEMS AND METHODS”, and U.S. Provisional Application No. 63 / 584,582, filed September 22, 2023, titled “TARGET ENRICHMENT METHODS AND SYSTEMS”, all of which are hereby incorporated by reference in their entirety.BACKGROUND

[0002] Typical immunoassay technology carries out affinity binding reactions reported by a label. The label measurement indicates the number of bound complexes after a combination of binding steps and wash procedures. The wash procedures aim to remove excess label to avoid misrepresentation of the reaction label measurement.

[0003] FIG. 1 shows an example of a typical heterogenous immunoassay. At stage 104, affinity agents, like antibodies (e.g., antibody 108) are attached to a surface. Analytes (e.g., antigen 112) are in solution and may be captured by the affinity agents, like the antibody. The surface is washed to remove unbound analytes.

[0004] At stage 116, a labeling complex, often an antibody carrying a label (e.g., antibody 120 with label 124) is introduced. Labeling antibodies bind to analytes that are bound to the affinity agents, like an antibody that are attached to the surface. The surface is washed again to remove unbound labeling complexes.

[0005] At stage 128, a signal from the label is measured. A quantity of labels is determined from the signal. A quantity of antigens is determined from the signal.

[0006] Immunoassay technology measuring protocols currently available interrupt the binding reaction several times to prepare the sample for label read out due to washing steps to remove unreacted labels. The measurement is only representative of the number of labelled molecules having reacted up to the point when the reaction was interrupted. To repeat measurements for a specific reaction time, the incubation time of the reaction and each wash procedure should be identical, which requires a rigorous procedure to achieve repetitive and robust results. Calibration reactions may also need to be carried out simultaneously to benchmark the label measurement.

[0007] Interrupting the binding reaction to fix the label intensity for read out results in a measurement representing a single point in time during the reaction. Unless the reaction is incubated for several hours to reach reaction equilibrium, the point at which the reaction is interrupted takes place during a non-equilibrium binding kinetic phase of the reaction. The single point measurement is impacted by the quality of the mixing, washing procedures, temperature, and other factors and can show great variability.

[0008] Nucleic acid molecules, such as DNA and RNA, are essential macromolecules that encode genetic information and play crucial roles in the functioning of living organisms. The ability to selectively enrich specific nucleic acid sequences from a complex mixture of genetic material has become increasingly important in modern molecular biology and biomedical research. This is due to the need for high-throughput sequencing, accurate diagnostics, and a deeper understanding of genetic variation and gene expression patterns.

[0009] Conventional methods for enriching nucleic acid molecules often involve timeconsuming and labor-intensive processes, such as size-based separation, PCR amplification, or hybridization-based capture. These methods have limitations in terms of specificity, scalability, and efficiency, making them less suitable for handling large and diverse sets of nucleic acid sequences.

[0010] Embodiments described herein allow for the measurement of analyte concentration in body fluids or buffer solutions down to, if required, very low concentrations and limits of detection. Other embodiments described herein allow for real-time measurement of binding reactions accurately and efficiently. Still other embodiments described herein allow for the capture of analytes and efficient washing of the captured analytes, which can be followed by dissociation and collection of the analytes with or without immediate analysis. Embodiments described herein address these and other challenges and improvements.BRIEF SUMMARY

[0011] Embodiments of the present invention include a microfluidic chip with an array of structures (e.g., pillars). Such a microfluidic chip may be used for measuring reaction kinetics. Another embodiment includes the measurement of an analyte concentration in body fluids or buffer solutions down to, if required, low concentrations and limits of detection. A nano / microliter stream may be continuously drawn from a reaction volume / reactor into the array of structures in the microfluidic chip. A plurality of beads may be in the reaction volume. Each bead may be attached to a primary affinity reagent. The reaction volume may also have an analyte. The primary affinity reagent may bind to the analyte in the reaction volume. A secondary affinity reagent with a fluorescent label may bind to the analyte, creating a sandwich of bead, analyte, and label either in the reaction volume or along an output path from the reaction volume. The primary and secondary affinity reagents may be antibodies. The analyte may be an antigen, nucleic acid molecule, or other compound.

[0012] A washing buffer removes non-specific interactions (unbound labels and antigens) from the bead surface after the beads are extracted from the reaction volume into the array of structures. The beads may be imaged after traversing a laminar wash buffer, and the signal intensity is measured. The beads may be drawn from the binding reaction in the reaction volume into the array of structures continuously over the duration of the reaction. When the beads arrive at the point of imaging and / or signal measurement, each bead has the same “time of flight” from leaving the reaction media through wash, and imaging. This means that each bead measurement represents the progress of the immunoassay at the time of the specific bead leaving the reaction volume. Each bead provides a kinetic monitoring of the immunoassay over the reaction time at which the bead is removed from the reaction media. A kinetic profile of the reaction can be established. The microfluidic chip can be used to detect low concentrations of a target analyte.

[0013] The microfluidic chip may include a first reactant reservoir (e.g., reaction volume) at one end of the area of the array of structures. The first reactant reservoir can hold a plurality of beads in a liquid and optionally other reagents able to react with the beads in the first reactant reservoir. The bead mixture can enter the array of structures from the reactant reservoir. In addition to the first reactant reservoir, one or more additional reservoirs can be added, all of them are in fluidic communication with the array of structures of the microfluidic chip. These additional reservoirs can hold washing solutions and / or otherreagents, including a second plurality of beads. The washing solutions and / or other reagents can enter the array of structures simultaneously or sequentially with the reaction mixture from the first reactant reservoir. Once the beads enter the array of structures, the beads are displaced laterally while flowing though the array of structures, and the beads intersect with other fluids entering the structure of arrays from the additional reservoirs. When washing solutions enter the array of structures, washing of the beads may occur and reagents that have not reacted with the beads may be removed by the washing solution. If in addition reagents enter the array of pillars from the additional reservoirs, one or more reactions can occur between the bead and the reagent while the beads intersect the flow path of the reagents to form a reaction product. The reaction between the components and the bead can result from covalent or electrostatic forces. Optionally, the reaction product can intersect with a third flow path with still other reagents for additional reactions or a flow path with washing solution. In some embodiments, the bead may intersect with a flow of washing solution to remove unwanted reagents and reaction products prior to detecting the detectable signal on the bead. This reaction and wash cycle can be repeated one or more times depending on the dimensions of the microfluidic chip. Thus, the entire microfluidic chip may allow for reaction and washing of components without interrupting the reaction.

[0014] In a preferred embodiment, a reaction between beads, capture molecule, preferably an antibody, and an analyte occur in the reactor to form a bead-capture molecule-analyte complex. The bead-capture molecule- analyte complex may enter the array of structures and may intersect in the array of structures with a mixture of the reagents. The reagents may include a label compound, which may include the label itself or a molecule with the label, such as a labeled antibody or labeled analyte. The label compound either may react with the analyte on the bead-capture molecule-analyte complex or may replace the analyte of that complex to form the labeled end construct attached to the bead. The beads with and without the labeled complex (the number of beads carrying a labeled complex may depend on the ratio between number of beads and concentration of analytes) then intersects with the washing solution in the array of structures. While flowing through the array of structures, the beads get washed from unwanted components before exiting the array of structures at the other end. In an alternative embodiment, a washing solution may be introduced to the array of structure so that the bead-capture molecule-analyte complex gets washed before mixing with the reagents.

[0015] In still another embodiment, a collection area and / or analysis area can be included in the microfluidic chip at the end of the array of structures. The collection and / or analysis area allows for the analysis and / or collection of the cleaned reaction components, preferably beads, after they travel through the array of pillars. The collection can happen with or without prior analysis. The flow of solution, reaction components can be achieved by various means, preferably by applying vacuum.

[0016] In embodiments, methods may include mixing a first plurality of beads with a sample to form a first mixture in a reactor of a microfluidic chip. The sample may include a plurality of analytes. Each bead of the first plurality of beads may be coupled to an affinity reagent. The affinity reagent may be configured to bind to the analyte. Methods may in addition include binding a first subset of the plurality of analytes to a plurality of affinity reagents coupled to the first plurality of beads in the reactor. Methods may further include flowing a first portion of the first mixture from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip. The first portion of the first mixture may include the first plurality of beads coupled to a first subset of the plurality of affinity reagents. Methods may further include coupling the first plurality of beads to a plurality of label compounds in the first fluidic path. Methods may in addition include forming a second mixture by flowing a solution in a second fluidic path. The second fluidic path intersects the first fluidic path. Methods may also include measuring an amount of the plurality of label compounds in the second mixture. Other embodiments of this aspect include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the actions of the methods.

[0017] In embodiments, methods may include mixing a first plurality of beads with a sample to form a mixture in a reactor of a microfluidic chip. The sample may include a plurality of analytes and a plurality of labels. Each bead of the first plurality of beads may be coupled to an affinity reagent. The affinity reagent may be configured to bind to the analyte. The plurality of labels may be configured to bind to the analyte. Methods may in addition include binding the plurality of analytes to a plurality of affinity reagents coupled to the first plurality of beads. Methods may also include coupling a first subset of the plurality of labels to the first plurality of beads in the reactor. Methods may further include flowing a first portion of the mixture from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip. The first portion of the mixture may include the first plurality of beads coupled to the first subset of the plurality of labels. Methods may inaddition include forming a second mixture by flowing a solution in a second fluidic path. The second fluidic path may intersect the first fluidic path. Methods may also include measuring an amount of the first subset of the plurality of labels in the second mixture. Other embodiments may include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the actions of the methods.

[0018] Systems and methods described herein also allow for efficient and accurate enrichment of analytes (e.g., nucleic acid molecules). A microfluidic chip with an array of structures for directing flow of beads is used for enrichment. A nano / microliter stream may be continuously drawn from a reservoir of beads and unbound nucleic acid molecules into the microfluidic chip. The bead may be attached to a capture agent (e.g., an oligonucleotide, an antibody), which is configured to bind to a target analyte (e.g., a nucleic acid molecule, an antigen, peptide biomarker, exosome, cell). A washing buffer removes non-specific interactions (e.g., unbound nucleic acid molecules, unbound antigens) from the bead surface after the beads enter the array of structures. The washing buffer and the array of structures are efficient at separating beads bound to target analytes (e.g., nucleic acid molecules) from unbound other (non-target) analytes. As a result, an efficient and effective enrichment of analytes can be achieved.

[0019] In embodiments, methods may include mixing a plurality of target analytes and a plurality of non-target analytes with a first plurality of beads. Each bead of the first plurality of beads is coupled to a respective affinity reagent. The affinity reagent may be configured to bind to the target analyte and to not bind to the non-target analyte. Methods may in addition include binding each target analyte of the plurality of target analytes to the respective capture agent coupled to a bead of the first plurality of beads to form a coupled first plurality of beads. Methods may also include transferring a mixture. The mixture may include the coupled first plurality of beads and the plurality of non-target analytes to a first reservoir of a microfluidic chip. Methods may further include flowing a first portion of the mixture from the reservoir through a first fluidic path defined by a first plurality of structures in the microfluidic chip, where the first portion of the mixture may include the coupled first plurality of beads and the plurality of non-target analytes. Methods may in addition include forming a second mixture by flowing a solution in a second fluidic path. The second fluidic path may intersect the first fluidic path. Methods may also include removing, using the solution, the plurality of non-target analytes from the coupled first plurality of beads in thefirst fluidic path. Methods may further include flowing the coupled first plurality of beads to a second reservoir at an end of the first fluidic path. Other embodiments of this aspect include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the actions of the methods.

[0020] In embodiments, systems may include a microfluidic chip. The microfluidic chip may include a reactor. The microfluidic chip may further include a plurality of structures defining a first fluidic path. The first fluidic path may be in fluid communication with the reactor, a reactant reservoir, a solution reservoir, and a manifold. The manifold may be configured to deliver a reactant from the reactant reservoir to intersect the first fluidic path. The manifold may also be configured to deliver a solution from the solution reservoir to intersect the first fluidic path. Systems may in addition include a plurality of beads disposed on the microfluidic chip. Each bead of the plurality of beads may have a diameter smaller than a width of the first fluidic path. Each bead of the plurality of beads may be bound to a first affinity reagent. Systems may also include an imaging detector.

[0021] A system of one or more computers can be configured to perform particular operations or actions by virtue of having software, firmware, hardware, or a combination of them installed on the system that in operation causes or cause the system to perform the actions. One or more computer programs can be configured to perform particular operations or actions by virtue of including instructions that, when executed by data processing apparatus, cause the apparatus to perform the actions.

[0022] A better understanding of the nature and advantages of embodiments of the present invention may be gained with reference to the following detailed description and the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows an example of an immunoassay.

[0024] FIG. 2 shows a graph of a kinetic profile of a binding reaction.

[0025] FIG. 3A shows an example kinetic reaction profile for a binding reaction according to embodiments of the present invention.

[0026] FIG. 3B shows a rate function established by the affinity constants of each binding entity according to embodiments of the present invention.

[0027] FIG. 4 illustrates mechanisms allowing for accurate determination of kinetic parameters according to embodiments of the present invention.

[0028] FIG. 5 shows steps in determining kinetic parameters according to embodiments of the present invention.

[0029] FIG. 6 shows a reactor and the mechanisms allowing for accurate determination of kinetic parameters according to embodiments of the present invention.

[0030] FIG. 7A illustrates a microfluidic chip for analyzing reaction kinetic parameters according to embodiments of the present invention.

[0031] FIG. 7B illustrates the mechanics of the washing of the beads according to embodiments of the present invention.

[0032] FIG. 8 depicts the relationship between sheer factor and displacement according to embodiments of the present invention.

[0033] FIGS. 9A, 9B, and 9C show possible orientations of structures according to embodiments of the present invention.

[0034] FIG. 10 illustrates an example of a two-step conjugation according to embodiments of the present invention.

[0035] FIG. 11 illustrates an example of how a deterministic lateral displacement (DLD) array can be used for the two-step conjugation reaction according to embodiments of the present invention.

[0036] FIG. 12 illustrates an embodiment of the two-step conjugation in a DLD array according to embodiments of the present invention.

[0037] FIG. 13 shows a graph of reaction kinetic profiles generated with a DLD array according to embodiments of the present invention.

[0038] FIG. 14A shows an example of multiplexing using different size beads according to embodiments of the present invention.

[0039] FIG. 14B shows the distribution of beads at different locations in the microfluidic chip according to embodiments of the present invention.

[0040] FIG. 15 is a graph of the bead intensity according to embodiments of the present invention.

[0041] FIG. 16 shows a graph of signal intensities over time according to embodiments of the present invention.

[0042] FIG. 17 shows a graph of reaction profiles with different ligand concentrations according to embodiments of the present invention.

[0043] FIG. 18 shows approximate solutions for experimentally relevant limiting cases according to embodiments of the present invention.

[0044] FIG. 19 shows graphs of the microsphere intensity versus the troponin concentration according to embodiments of the present invention.

[0045] FIG. 20 is a flowchart of an example process for analyzing reaction kinetics according to embodiments of the present invention.

[0046] FIG. 21 is a flowchart of an example process for analyzing reaction kinetics according to embodiments of the present invention.

[0047] FIG. 22 is a flowchart of an example process for determining a reaction kinetic parameter according to embodiments of the present invention.

[0048] FIG. 23A and FIG. 23B are a flowchart of an example process for analyzing a sample according to embodiments of the present invention.

[0049] FIG. 24 is a flowchart of an example process for performing a reaction with target analytes according to embodiments of the present invention.

[0050] FIG. 25 shows a system for analyzing reaction kinetics according to embodiments of the present invention.

[0051] FIG. 26 illustrates enrichment principles according to embodiments of the present invention.

[0052] FIG. 27 shows a reservoir and the mechanisms allowing for enrichment according to embodiments of the present invention.

[0053] FIG. 28 illustrates a microfluidic chip for enrichment according to embodiments of the present invention.

[0054] FIG. 29 is a flowchart of an example process for enriching or filtering target nucleic acid molecules according to embodiments of the present invention.

[0055] FIG. 30 is a flowchart of an example process for enriching or filtering target analytes according to embodiments of the present invention.

[0056] FIG. 31 shows a system for enriching target nucleic acid molecules according to embodiments of the present invention.

[0057] FIG. 32 shows a computer system according to embodiments of the present invention.DETAILED DESCRIPTION

[0058] Methods and systems involve a microfluidic chip with an array of pillars for directing flow of particles in order to analyze concentrations of analytes or reaction kinetics, to enrich for a target analyte, or to perform chemical or biological reactions through the array of pillars. The particles may be beads or cells. A bead is frequently used herein as an example for descriptive purposes, but any reference to a bead in this disclosure can also refer to a cell, unless context specifically indicates otherwise. Preferably the bead is made from a solid material like silica or polystyrene or any other suitable material. The analysis or target enrichment can involve an analyte, which may include an antigen, a nucleic acid molecule, a peptide biomarker, an exosome, or a cell. An antigen is frequently used herein as an example for descriptive purposes, but any reference to an antigen can include a general analyte or any analyte disclosed herein, unless context specifically indicates otherwise. Affinity reagents may bind to the analyte. Affinity reagents include an antibody, an oligonucleotide, an aptamer, peptide, polysaccharide, or any affinity reagent described herein. An antibody is frequently used herein as an example for descriptive purposes, but any reference to an antibody can include a general affinity reagent or any affinity reagent disclosed herein, unless context specifically indicates otherwise. The affinity reagent may bind to the particle.Another affinity reagent may be bound to a label. The label may be a fluorescent label, a quantum dot, fluorophore, chemiluminescent tag, electrochemical label, or other suitable label.I. ANALYZING CONCENTRATION OR KINETIC S OF REACTIONS

[0059] Current techniques of measuring concentration of analytes or kinetics of reactions (e.g., binding of an antigen to an affinity reagent) may involve interrupting a reaction to measure an amount at a specific time. Interrupting a reaction prevents real-time measurementof kinetics. Additionally, in order to obtain accurate and precise measurements, procedures may need to be followed rigorously. Errors from slight variations in procedure will result in inaccurate results, especially for fast reaction kinetics or measuring low concentrations of a reactant (e.g., an antigen).

[0060] Embodiments described herein include real-time measuring of the kinetics of reactions. Antigens, labels, affinity reagents, and beads may be mixed together in a batch reactor of a microfluidic chip. This time may be time to, the start of the reaction. A reaction may take place that may result in a bead being bound to a first affinity reagent, which is bound to an antigen, which is then bound to a second affinity reagent, which is bound to a label. A bead be attached to several first affinity reagents. Beads may be flowed out of the reactor continuously at a diagonal. A first bead may leave the reactor at time ti. A second bead may leave the reactor at time t2. A clean laminar flow may wash beads in a direction that intersects the diagonal. This flow may remove unbound labels, antigens, and / or affinity reagents from the beads. This clean laminar flow also may quench any reaction. The beads that remain in the diagonal flow may be bound to affinity reagents, antigens, and labels. The amount of labels of the beads may be measured in real-time at an output location. For example, the label may be a fluorescent label, and the intensity of the fluorescent signal may be measured.

[0061] The time of the signal measurement may be adjusted by the time the beads take to reach the output location from the batch reactor. Amounts of bound antigen can then be plotted versus time. Concentrations (e.g., absolute or relative concentrations) and / or kinetic rate constants can be determined using the measured amount of labels and the times of the measurement.

[0062] Embodiments described herein may allow for an accurate assessment of the reaction kinetic through mathematically fitting of the antigen concentration over time. The confidence in the concentration fitting may be continuously calculated with each new bead being measured. The measurement may be ended, when the satisfactory confidence level is achieved from the real-time mathematical fitting.

[0063] Several thousand microbeads may be incubated in the immunoassay reaction. Less than 1,000 may be needed to achieve a high confidence kinetic profile of the reaction and yield a high accuracy measurement. The mathematical modeling of the reaction kinetic profile may achieve a high confidence concentration fitting after less than 5 minutes reactionfor antigens in the nanomolar range, and less than 10 minutes in the picomolar range. Kinetics of binding reactions, dissociation reactions, and other reactions may be determined.

[0064] Suitable capture and / or measuring technologies may be used to form the surface attached labeled complex. The label to be measured can be formed by competing and noncompeting technologies. In the competing measuring technologies, the label is attached to the analyte and directly competes with unlabeled analytes already attached to the capture molecule on the bead surface. The complex to be measured may be formed by the capture molecule (e.g., capture antibody) and the labeled analyte. In non-competing measuring technologies, the complex to be measured is formed by the reaction of the labeled molecule.

[0065] Systems and methods are described in further detail in this disclosure.A. Reaction kinetics

[0066] FIG. 2 shows a graph of a kinetic reaction profile of a binding reaction with a given rate constant. The x-axis shows time. The y-axis shows signal intensity of a label indicating the completion of a binding reaction. The to at the bottom indicates the initial time. The teqindicates the time at which the binding reaction is at equilibrium. The different lines correspond to different initial concentrations of a reactant, with a higher line being a higher initial concentration. The initial phase of the binding reaction has a high slope. Monitoring the initial phase rather than the equilibrium phase may allow for precise kinetic fitting and a quicker result.

[0067] FIG. 3A shows an example kinetic reaction profile for a binding reaction. The x- axis shows the time in seconds. The y-axis shows the signal intensity of a label in arbitrary units. Line LI is the profile for a first ligand (e.g., analyte, antigen) concentration. Line L2 is the profile for a second ligand concentration. In endpoint detection techniques, measurements may be delayed until the reaction reaches equilibrium (e.g., at time teq) to determine the concentration. As shown in FIG. 3 A, the time may be around 10 minutes to reach equilibrium. Otherwise, measurements in the steep incline portion of the profiles should be rigorously carried out in order to be repeatable and comparable with other measurements.

[0068] FIG. 3B shows a rate function established by the affinity constants of each binding entity (i.e., affinity reagent). The equation includes the following variables: / is the signal intensity; / JS is the microbead concentration; A is the concentration of capture antibody (i.e., the concentration of the beads multiplied by the number of binding sites per bead); L is theconcentration of the ligand from the sample that is being measured; KD is the dissociation constant of the complex; konis the on rate constant of the complex; and t is the time of the reaction.

[0069] A transfer function converts a signal intensity value to a ligand / antigen concentration in the sample. With data of signal intensity as a function of time, the transfer function can use the rate function to determine the respective concentrations of each binding entity to the number of complexes being formed. However, when the reaction is interrupted at a set incubation time as with endpoint detection, the value of the ligand concentration is inferred from a calibration curve. The calibration curve would need to be determined from calibration samples and not from the test sample, and the calibration samples would need to be measured in the same manner as the test sample. Hence, using the rate function has advantages over endpoint detection in not needing a calibration performed.

[0070] With the kinetic reaction profile, the total ligand concentration can be calculated from fitting of the first few seconds of the reaction. By contrast, endpoint detection is typically performed at equilibrium, which may be minutes into the reaction.

[0071] Rate functions, such as in FIG. 3B, show that the ligand concentration can be calculated when the rate constants are known. Additionally, rate constants may be calculated when ligand concentrations are known. Both rate constants and ligand concentrations can be calculated from multiple experiments to generate multiple kinetic reaction profile curves.B. Kinetic immunoassay technique

[0072] Calculating kinetic parameters using rate functions can be performed in the first few seconds of the reaction if the signals for a bound reaction for a given reaction time can be determined accurately.1. Principles

[0073] FIG. 4 illustrates the mechanisms allowing for accurate determination of kinetic parameters. Stage 402 shows time to. At stage 402, a capture bead 404 is present. Capture beads may be bound to capture antibodies, including antibody 410. Antigens, including ligand 412, may be present in the same mixture as the capture beads. Additionally, detection antibody, such as detection antibody 416 may be present. Detection antibody 416 may be bound to label 420. One or more detection antibodies may be bound to a single label. Label420 may be a quantum dot, fluorophore, chemiluminescent tag, electrochemical label, or other suitable label.

[0074] Stage 424 shows the mixture after conjugation. At time tinc(“incubation time”), Ligands may bind to the detection antibody and / or the capture antibody. The expected binding reaction is a sandwich reaction, which forms a complex 428 of the capture bead bound to a ligand bound to a detection antibody with a label. Although FIG. 4 illustrates the capture bead with only seven capture antibodies, a capture bead may have 100 or more capture antibodies.

[0075] Stage 450 shows the mixture after washing the capture bead of ligands and detection antibodies bound to labels. Only the capture bead remains. The intensity of the labels therefore is related to the presence of bound ligands.

[0076] FIG. 5 shows steps in determining kinetic parameters using the components of FIG. 4. At stage 504, a reactor 508 may have an unknown ligand concentration. At stage 510, reagents are added at time to. Reagents include capture beads conjugated with capture antibodies and detection antibodies conjugated with labels.

[0077] Stage 520 shows continuous incubation. Reactions occur continuously in reactor 508 after time to.

[0078] Stage 530 shows a capture bead complex after removal of unbound residue 534, including ligands and detection antibodies conjugated with labels. The capture bead complex and unbound residue can be removed from reactor 508 at a distinct time (e.g., L, t2, ti). The separation of the capture bead complex from the unbound residue may be performed in a deterministic lateral displacement (DLD) microfluidics array 538.

[0079] As a result of a DLD array, a capture bead leaving the reservoir at time will reach a point first compared to a capture bead leaving at a later time t2. A capture bead leaving at a later time t2 will have a longer time for incubation and therefore more ligands and labels coupled to the bead. The intensity of beads at later times should be greater to reflect the greater number of ligands. Stage 540 shows that the initial unknown ligand concentration can be inferred from the intensities from capture beads over time.

[0080] FIG. 6 shows another illustration of a possible reactor and mechanisms allowing for accurate determination of kinetic parameters. Reactor 604 contains capture beads, including capture bead 608. Capture beads may be bound to capture antibodies, including antibody 610.Capture beads may bind with an antigen, including antigen 612. Antigens may bind with a labeled sandwich antibody, including labeled sandwich antibody 616. The labeled sandwich antibody 616 includes label 620. Label 620 may be a quantum dot, fluorophore, chemiluminescent tag, electrochemical label, or other suitable label. The expected binding reaction is a sandwich reaction 624, which forms a complex of the capture beads bound to an antigen bound to a labeled sandwich antibody. The contents in the reactor may be incubated. Although FIG. 6 illustrates each capture bead with only three capture antibodies, a capture bead may have 100 or more capture antibodies.

[0081] Arrow 628 represents a flow of material out of reactor 604. Area 632 shows possible materials in the output of the reactor. The materials illustrated include the product of a sandwich reaction, as well as an unbound antigen and an unbound labeled sandwich antibody. The beads may be drawn out from reactor 604 at a constant rate over a fixed amount of time.

[0082] Wash 636 represents a flow that removes the unbound antigen and unbound labeled sandwich antibody from the product of the sandwich reaction. The capture beads are not removed by the wash and instead proceed in a direction indicated by arrow 640 and arrow 644, which are different from the direction of the wash. The wash direction may be from top to bottom in this figure rather than from left to right.

[0083] Bead 648 represents a capture bead that has had time ti in the reactor. Bead 652 represent a capture bead that has had time L in the reactor, where L is a longer time than ti. Bead 648 is farther from reactor 604 than bead 652 because bead 648 left reactor 604 at an earlier time than bead 652. The same flow carries bead 648 and bead 652 so that bead 652 cannot pass bead 648. Bead 652 is depicted as a bead that has captured more than one antigen, each coupled to a labeled sandwich antibody. Bead 652 has spent longer in reactor 604 than bead 648 so bead 652 may capture more antigens.

[0084] The signal from the label or labels may be detected and quantified. The intensity of the signal may be related (e.g., proportional) to the amount of antigen on a bead. The signal intensity may be analyzed or plotted over time. A kinetic reaction profile can be determined from the signal intensity.

[0085] The error on each bead measurement may be controlled by the ensemble of molecules bound to the bead. The number of binding sites on each bead is known, and the number of ligands that should be bound to each bead may be mathematically known for anygiven time value. The error may be mathematically calibrated. For determinations of whether a concentration is above a threshold, a 99% confidence level may be achieved rapidly (e.g., within 5 minutes in the picomolar range). For example, a kinetic reaction profile may be used to determine whether the concentration of troponin I is above a threshold concentration that indicates a heart attack.

[0086] FIG. 7A illustrates a microfluidic chip for analyzing reaction kinetic parameters. One or more channels (e.g., channel 704) may lead to imaging input area 708. The channels may be from a reactor 710 (e.g., reactor 508 in FIG. 5 or reactor 604 in FIG. 6) or from a reservoir for a wash solution. Imaging input area 708 allows for brightfield images of beads with labels and unbound labels to be taken. Imaging input area 708 is optional. FIG. 7A is not shown to scale.

[0087] After going through imaging input area 708, the bead mixture goes through deterministic lateral displacement (DLD) array 712. DLD arrays are described in US 7,150,812, the entire contents of which are incorporated herein by reference for all purposes. DLD array 712 may include a plurality of structures. The structures may be pillars with a circular base or a rectangular (e.g., square) base. The structures may be in a regularly spaced array. The array may define straight paths through the structures. These straight paths may be offset from the path going from the channels through the imaging input area. The straight paths through DLD array 712 may not be parallel to the longitudinal axis of the chip. In FIG. 7 A, the longitudinal axis is illustrated in the vertical direction.

[0088] The solid lines indicate paths (e.g., path 716) that the beads follow through the DLD array. These paths are at a diagonal from imaging input area 708. The beads travel and are displaced laterally from their input. Oval 718 indicates that the capture antibody, detection antibody, and ligand are bound together. The beads travel to imaging output area 720. Imaging output area 720 is laterally displaced from imaging input area 708.

[0089] The dashed lines indicate paths (e.g., path 724) that a cleaning flow may follow. This cleaning flow may be delivered from one or more channels. The cleaning flow may be from buffer reservoir 730. The cleaning flow may be along the longitudinal axis. The cleaning flow directs unbound antigens and antibodies away from the beads, effectively washing the beads. With the longitudinal direction of the cleaning flow, the cleaning flow may go to imaging output area 720, which is aligned with imaging input area 708. Although only a few solid lines are illustrated, the cleaning flow may cover most (50%, 60%, 70%,80%, 90% or more) or the entirety of the DLD array, washing beads throughout their travel to imaging output area 720. Imaging output area 720 is for brightfield imaging and is optional.

[0090] At measurement area 736, the labels may be measured. Measurement area 736 may be a section of the structures. The beads follow consistent and predictable paths between structures, and signals from the labels of the beads can be reliably measured by an imaging device or other detector. For example, a high sensitivity quantitative CMOS camera may image microbeads and detect down to a single fluorescent or photoluminescent label. The cleaning flow may remove other labels to decrease or eliminate background signal. The cleaning flow may fully replace the liquid volume around each bead up to several hundred times.

[0091] The total immunoassay reaction may be as small as 1 pL, 5 pL, or 10 pL. The sampling of the reaction may be carried out on less than 5 microliters.

[0092] FIG. 7B illustrates the mechanics of the washing of the beads. Bead 750 has nonspecific interactions with labeled sandwich antibody 754 and other labeled sandwich antibodies. In this example, bead 750 is not bound to any antibodies and therefore is not bound to any antigens, which are therefore not bound to any labeled sandwich antibodies.

[0093] Arrows, including arrow 758, show laminar flow of a buffer solution. This flow cleans bead 750 of non-specific interactions. The flow of the buffer solution may be similar to path 724 in FIG. 7A. The flow may be in a direction parallel to the longitudinal axis of the microfluidic chip. The unbound labeled sandwich antibodies (e.g., labeled sandwich antibody 762) are washed away from bead 750 in the direction of the flow of the buffer solution.

[0094] Bead 750 is laterally displaced, which may be as explained with DLD array 712 in FIG. 7A. After lateral displacement, bead 766 may be clean with little or no labeled sandwich antibodies nearby (e.g., within a distance equal to the diameter of the bead).

[0095] FIG. 8 depicts the relationship between sheer factor and displacement. A laminar flow is in the direction (vertical) in the figure. Bead 804 is displaced in both they direction and x direction (horizontal) in traveling diagonally. Bead 804 is subjected to a Sheer factor, which is the force applied by the laminar flow on non-specifically bound entities. The Sheer factor is the difference between the vertical (y) velocity of bead 804 and the laminar flow. The x displacement determines how many times the liquid volume around the bead is fully renewed. In embodiments, a 6 pm bead that travels 5 mm in the x direction results in thebuffer around the bead (i.e., any liquid in contact with the bead surface) being exchanged 833 times.

[0096] FIGS. 9A-9C illustrate possible orientations of structures. The structures are offset from the longitudinal axis of the microfluidic chip to create lateral displacement in the flow of the beads. FIG. 9A shows the largest offset angle, and FIG. 9C shows the smallest offset angle. FIG. 9A shows an orientation of structures where starting from a given structure, three additional structures are passed to reach a structure that is shifted one over to the left from the starting structure. The shift is denoted as X, which is the distance from the center of one structure to the center of the adjacent structure. The angle of the offset can be calculated from tan'1(1 / 3). The orientation is denoted as N=3, where 3 is the number of structures for a shift to the right. FIG. 9B shows an orientation of N=5. FIG. 9C shows an orientation of N=10.

[0097] A lower N value will result in larger diameter beads being shifted laterally but may not move smaller diameter beads laterally. The relationship between the critical diameter and the N value can be determined from an empirical equation:where Dc is the critical diameter of the beads, G is the gap distance between adjacent structures, N is the value described in FIGS. 9A-9C, 0 is the angle of the rotational offset of a square lattice to arrive at the structures, and a and P are fitted parameters.

[0098] FIGS. 9A-9C show N as an integer, but N is not restricted to integer values. The structures may be a rotational offset of a square lattice by an angle 9. Angle 9 is continuous and not limited to values where N is an integer.

[0099] The gap spacing, G, and the height of the structures may be any value greater than the diameter of the beads. In some embodiments, the gap spacing and / or the height of the structures may be 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, 1.4 to 1.5, 1.5 to 2, 2 to 3, 3 to 5, 5 to 10, 10 to 20, or greater than 20 times larger than the diameter of the beads.2. Two-step conjugation

[0100] Some embodiments may include dividing the sandwich reaction into two steps. The first step may include binding the antigen to the capture bead via an antibody. The second step may include binding a label to the antigen-capture bead complex. The second step may be performed after the first step and in a different location than the first step.

[0101] A two-step conjugation may include several advantages over a single-step conjugation in certain embodiments. In some reaction schemes, a single-step conjugation may result in products other than a sandwich reaction complex of one label, one antigen, and one capture bead. As an example, an antigen may bind to a label without the antigen being bound to the capture bead. As another example, a single label may be configured to bind to multiple antigens. As a result, complexes may include one or more label compounds with multiple antigens, without a capture bead. Because these complexes are not immobilized on a capture bead, the antigens would not be detected. In some embodiments, labels may also bind to other proteins in a sample, which will reduce the number of labels and reduce the concentration of the desired sandwich reaction complex.

[0102] FIG. 10 illustrates an example of a two-step conjugation. The first row shows the first step of target capture. The second row shows the second step of labeling.

[0103] Stage 1004 shows time to. Ligands (e.g., ligand 1008) may be just introduced to the microspheres (e.g., bead 1012). The microspheres include several capture antibodies. Ligands may include antigens or any analytes described herein. Microspheres may include any capture bead described herein.

[0104] Stage 1020 shows a snapshot at time tinc, after some time where the ligands can conjugate to the microsphere. Microsphere-ligand complex 1024 includes a microsphere bound to several ligands. Unbound ligands (e.g., ligand 1028) are present at stage 1020.

[0105] Stage 1032 shows a snapshot after the microspheres are washed of unbound ligands. Microsphere-ligand complex 1036 is free of unbound ligands.

[0106] Target capture in stages 1004 and 1020 of the first step can be described by the equilibrium reaction: onl f + Ac_ ACL offiwhere L is the ligand concentration, Ac is the capture antibody concentration, and koniand koffi are rate constants.

[0107] Stage 1040 shows a snapshot at time ti after introducing labels (e.g., label 1044) to microsphere-ligand complex 1048. Labels include a detection antibody.

[0108] Stage 1060 shows a snapshot at time tiabei after labels are conjugated to the ligand. Sandwich complex 1064 includes labels bound to ligands.

[0109] Stage 1070 shows a snapshot after the sandwich complexes are washed of unbound labels. Sandwich complex 1074 is free of unbound labels.

[0110] The labeling in stages 1040 and 1060 can be described by the equilibrium reaction: on2ACL + ADACLADoff2 where L is the ligand concentration, Ac is the capture antibody concentration, AD is the detection antibody concentration, and kon2 and koff2 are rate constants.

[0111] FIG. 11 illustrates an example of how a DLD array can be used for the two-step conjugation reaction. The washing of unbound ligands and labels occur during the flow of the conjugated microspheres through a DLD array.

[0112] Stage 1104 is the sample with an unknown ligand concentration [L], The sample may include ligand 1108.

[0113] Stage 1112 shows a reactor after the addition of microspheres conjugated with a capture antibody (e.g., microsphere 1116). Stage 1112 is similar to stage 1020.

[0114] Stage 1120 shows the reactor after a certain duration of incubation. The reactor includes a microsphere-ligand complex 1124 and unbound ligands (e.g., ligand 1128).

[0115] The output of the reactor may be sent to a DLD array 1132. Additionally, detection antibody-conjugated labels may be added to DLD array 1132 from a reservoir 1136. The labels (e.g., label 1140) bind to ligands bound to the microsphere on sandwich complex 1144.

[0116] As a result flow dynamics through the DLD array, unbound residue 1148 is separated from microspheres, as described with FIGS. 4 to 7B.

[0117] Sandwich complexes including sandwich complex 1144 can be imaged at stage 1152. The concentration of the ligand can be inferred from a signal from the labels.

[0118] FIG. 12 illustrates an embodiment of the two-step conjugation in a DLD array. Reactor 1204 may include ligands (e.g., ligand 1208) and capture antibody -conjugated microspheres. The ligands may bind with the capture antibody-conjugated microspheres to form a ligand-microsphere complex (e.g., ligand-microsphere complex 1212). Reactor 1204 may also include unbound ligands (e.g., ligand 1208).

[0119] Flow with beads is drawn from reactor 1204 to DLD array 1216. Unbound ligands flow down path 1220, which may be parallel to the longitudinal axis of the microfluidic chip. The flow of unbound ligands may be aided by a buffer flow.

[0120] The ligand microsphere complexes may flow down a fluidic path 1224 defined by structures in DLD array 1216. The fluidic path may be offset from the longitudinal axis of the microfluidic chip. The fluidic path and any fluidic path described herein may be a path of the travel of solid beads or cells.

[0121] A reactant reservoir 1228 may include detection antibody-conjugated labels (e.g., label 1232). The detection antibody-conjugated labels may flow down path 1236, which may be parallel to the longitudinal axis of the microfluidic chip. The labels may interact with ligand-microsphere complexes in fluidic path 1224. Labels may bind to the ligand- microsphere complex, forming a sandwich complex of a microsphere, ligand, and label. Unbound labels may continue down path 1236. The flow of unbound labels may be aided by a buffer flow.

[0122] A reservoir 1240 may contain a buffer. The buffer may flow down the longitudinal axis of the microfluidic chip. The buffer flow may aid the washing of the unbound ligands and / or labels from the microspheres. The buffer flow may be across the microfluidic chip and may not be limited to only a path going through input imaging port 3 and output imaging port 3.

[0123] Oval 1242 indicates that the capture antibody, ligand, and detection antibody are bound together in a sandwich complex. Sandwich complexes (including sandwich complex 1244) may be imaged at output imaging ports 1248. The signal intensity of the microspheres may be correlated with the number of labels on a microsphere, which may be correlated to the number of ligands on the microsphere.C. Estimating reaction kinetic parameters

[0124] Systems and methods described herein can be used to analyze kinetics of different reactions.1. Dissociation rate constants

[0125] The dissociation rate constant (koff) was estimated with methods and systems described herein. In this experiment, the sandwich immunoassay was performed with human C reactive protein (hCRP). The immunoassay was incubated until equilibrium, and then the beads were run through the DLD array. The washed beads were trapped in holding traps designed in the chip, and the signal decay was monitored over time on a few beads to detect the signal loss function.

[0126] FIG. 13 shows a graph of reaction kinetic profiles generated with a DLD array and methods described herein. The x-axis shows time in minutes. The y-axis shows the signal intensity in arbitrary units. Each line corresponds to signal decay of a single microbead. All microbeads are from the same reaction, so are expected to have a similar koff.

[0127] The intensity of labels as a function of time can be represented by:Z(t) = Ioe~k°ffxtwhere Io is the initial intensity of the labels bound to microspheres, koffis the dissociation rate constant, and t is time.

[0128] The intensities at different times can be fit to the equation and & can be determined. The determined koff are shown in FIG. 13. The fit shows that we have a single exponential fit, and this demonstrates that only sandwich bound signal is being detected, and the rate at which it decays corresponds to the known L value for this antibody system. The R2values are provided, showing good fits to the data.2. Multiplexing

[0129] Different size beads and / or different types of labels can be used to test examine kinetics of different reactions with the same assay.

[0130] FIG. 14A shows an example with different size beads. Bead 1404 is a 10 pm diameter bead, bound to an antibody that captures C-reactive protein. Bead 1408 is an 8.2 pm diameter bead, bound to an antibody that captures IGF1. Bead 1412 is a 5.8 pm diameterbead, bound to an antibody that captures hepcidin. These beads, along with C-reactive protein, IGF1, hepcidin, and labeled sandwich antibodies are incubated in reactor 1416.

[0131] Flow with beads is drawn from reactor 1416 through a series of DLD arrays. Buffer from buffer reservoir 1420 is flowed to wash the beads to remove non-specific interactions as described herein. The series of DLD arrays separates the different size beads laterally. The DLD arrays are denoted as Nm, where m denotes the number of rows of additional pillars corresponding to a one-column shift in beads (indicated by N in FIGS. 9A-9C). The smallest beads (e.g., bead 1412) follows path 1424. The medium beads (e.g., bead 1408) follows path 1428. The largest beads (e.g., bead 1404) follows path 1432.

[0132] FIG. 14B shows the distribution of beads at different locations in the microfluidic chip. The smallest beads, bead 1412, are displaced the least amount laterally, ending in port 3 and target channels 13-19 (indicated by box 1436). Bead 1408 is displaced an intermediate amount laterally, ending mostly in port 5 and target channels 23-31 (indicated by box 1440). The largest beads, bead 1404, is displaced the largest amount laterally, ending mostly in port 7 and target channels 35-43 (indicated by box 1444).

[0133] The results show that different size beads can be used to analyze the kinetics or concentrations of different antigens at the same time.3. Signal intensity versus time

[0134] The signal intensity was tracked over time using methods and systems described herein. The beads are attached to streptavidin. Biotin is labeled with quantum dots.Streptavidin has a high affinity to bind with biotin.

[0135] Images of beads having reaction times of 1 min., 8 min., and 23 min. show brighter (i.e., higher signal intensity) beads with longer reaction times.

[0136] FIG. 15 is a graph of the bead intensity. The x-axis is the x-coordinate (e.g., left to right in FIG. 7A) of the pixel. The x-axis spans 450 pixels. The y-axis is the sum of the signal intensities from all pixels at a given x-coordinate. FIG. 15 shows that higher reaction times lead to higher signal intensities from the beads.

[0137] FIG. 16 shows a graph of signal intensities over time for 10 pm beads modified with streptavidin and biotin labeled with quantum dots. The x-axis is time in minutes. The y- axis is microsphere quantum dot occupancy, which represents the number of dots on thesurface of the bead and is a signal intensity. The reaction is run three times to generate the data points in the graph. The fitted line is for illustrative purposes. The graph shows a kinetic reaction profile. The profile has a steep increase then levels off as the reaction approaches equilibrium.4. Kinetic measurements

[0138] An experiment verified the capability of methods and systems described herein to analyze reaction kinetics in the picomolar concentration range.

[0139] FIG. 17 shows a graph of reaction profiles with different ligand concentrations in the picomolar range. The x-axis is time in minutes. The y-axis is microbead occupancy. The three lines are fitted lines for different concentrations of ligands: 1 pM, 3 pM, and 5 pM. The 5 pM ligand has the steepest increase and the highest occupancy near equilibrium. The 1 pM ligand has the least steep increase and the lowest occupancy near equilibrium.

[0140] Data in this graph can be analyzed to determine rate constants for the reaction. Additionally, data can be used for calibration for additional experiments. The concentration of a sample can be determined based on how closely the kinetic profile matches previous reaction data.5. Conjugation reaction model

[0141] A conjugation reaction model helps understand the reaction profiles in methods and systems described herein. The conjugation reaction model is determined using a Langmuir reaction model. There is no analytical solution to the general sandwich assay reaction model. The differential equation is solved numerically to find the relationship between the number of ligands and the number of sandwich complexes of ligand, capture antibody, and label antibody.

[0142] FIG. 18 shows approximate solutions for experimentally relevant limiting cases for single-step conjugation, where by is the primary (capture) antibody, L is the ligand, A2 is the secondary (label) antibody, and Nxis the number of x. The solutions are forwhich is the fraction of primary antibodies occupied with ligands that are also labeled with secondary4 antibodies as a function of time. For example, bead 1804 has an / «of-. Bead 1804 has seven primary antibody sites with five occupied with a ligand, and with four of the five ligands labeled with the secondary antibody.

[0143] Equation 1808 describes the fraction bound and labeled for conventional binding or non-depleting regime, where [Z] ~ [A2] » [^7].

[0144] Equation 1812 describes the fraction bound and labeled in titration or strongly- depleting regime, where [Aj] ~ [A2] » \L\.fbi(f) is proportional to [Z],

[0145] Equation 1816 describes a mode suitable for rare biomarker detection, where [A2] » [A 7] ~ [Z], The secondary antibody can be introduced in excess of A 7 and Z.

[0146] Section 1820 describes variables in the equations.6. Experiment results

[0147] The detection of troponin was performed using a DLD array. Anti-Troponin I antibodies clonel9C7mab (Capture), clone 16A1 Imab, and Troponin I were purchased from Hytest (Finland).

[0148] The beads were 10 pm amine modified silica beads purchased from NANOCS (NY), Mercaptoethylamine (Life Technology). Amine-to-sulfhydryl crosslinker and Zeba spin columns were purchased from Thermo Fisher Scientific. Phycoerythrin labeling kit (Abeam) was used to label clone 16A1 Imab according to manufacturer instructions.

[0149] A conjugation buffer was prepared from phosphate buffered saline (0.1 mM PBS, pH 7.2 EDTA) pH 6.5-7.5.

[0150] A 250 mM crosslinker stock solution of amine-to-sulfhydryl crosslinkers with soluble polyethylene glycol (PEG) spacer arms was prepared. Crosslinker (SM(PEG)4) in the amount of lOOmg (entire contents of vial, approximately 100 pL) was dissolved in 680 pL of dry DMSO.

[0151] Antibody reduction was performed. A volume of 125 pL antibody solution antibody solution 19C7cc with 125 pl conjugation buffer was obtained. Next 6 mg of monoethanolamine (MEA) was mixed in 100 pl conjugation buffer. A volume of 25 pl MEA solution was added to the antibody solution. The solutions were mixed, incubate at 37 °C for 90 minutes. The reduced antibody solution was purified with Zeba spin exclusion columns.

[0152] Microbeads were prepared by dissolving 36.8 mg silica beads in conjugation buffer 1 ml (estimated 0.3 nM amine groups).

[0153] The beads were spun down. The buffer was replaced. Crosslinker was added to dissolved microbead-NH2 at 1 mM final concentration by mixing 4 pL of the 250 mM crosslinker stock solution per milliliter of bead solution.

[0154] The reaction mixture was incubated for 30 minutes at room temperature or 2 hours at 4 °C. The microbeads were spun down washed with conjugation buffer. The spin down and wash were repeated two times overall.

[0155] The bead solution was combined with the linker and reduced antibody solution. The reaction mixture was incubated at room temperature for 30 minutes or 2 hours at 4 °C.

[0156] Spin down of the microbeads was performed. The microbeads were washed with conjugation buffer. The spin down and wash were repeated five to 10 times overall.

[0157] Labeling antibody conjugation was performed according to Phycoerythrin labeling kit instructions.

[0158] Fresh Troponin I aliquot was defrosted from -80 °C storage and diluted in pooled human plasma to make solutions of 0 pg / ml, 10 pg / ml, 100 pg / ml, 1 ng / ml, and 10 ng / ml (Troponin dilutions).

[0159] A volume of 60 pl of Troponin dilution was added to 20 pl of capture antibody conjugated silica bead solution, and incubated for 15 minutes (reaction media).

[0160] A volume of 10 pl of the reaction media was introduced in the channel 1 of the DLD array. A volume of 10 pl of phycoerythrin conjugated labeling antibody solution was added to channel 2 of the DLD array. A volume of 10 pl of IxPBS buffer was added to channel 3 of the DLD array.

[0161] The reaction media, the antibody solution, and the buffer were pulled through the DLD array by applying a negative pressure at the exit of the DLD array with a syringe pump.

[0162] As the microbeads get separated from the reaction media in channel 1, they traverse channel 2 where the labeling antibody solution is flowing, and arrive into channel 3, which contains clean PBS buffer. When the microbeads arrive at the bottom of channel 3, they are immobilized in microfluidic traps, where they are imaged. The fluorescence signal intensity of each microbead is measured against background. For each solution, a number of microbeads are measured to obtain robust statistics.

[0163] FIG. 19 shows graphs of the microsphere intensity versus the troponin concentration. The y-axis shows the microsphere intensity in arbitrary units on a log scale. The x-axis shows the troponin concentration in ng / ml. In the main graph, each sphere’s intensity is plotted. In the inset graph, the intensities of the different spheres at one concentration are averaged to a single point, and the range of intensities is shown with a line. This graph demonstrates that the intensity of a microsphere is proportional to the troponin concentration with methods using the DLD array.D. Example methods1. General conjugation

[0164] FIG. 20 is a flowchart of an example process 2000. In some implementations, one or more process blocks of FIG. 20 may be performed by a system, including system 1500 or any system described herein.

[0165] At block 2010, a first plurality of beads is mixed with a sample to form a mixture in a reactor of a microfluidic chip. The beads may include polystyrene, iron-oxide, silica, polymer, metal (e.g., gold, silver), or any other suitable material. The sample may include a plurality of analytes and a plurality of label compounds. Each bead of the first plurality of beads may be coupled to an affinity reagent. The analyte may be any analyte or ligand described herein. The affinity reagent may be configured to bind to the analyte. Each bead may be coupled to a plurality of affinity reagents. The plurality of label compounds may be configured to bind to the analyte. The label compounds may be a labeled sandwich antibody, as described herein, which may include a detectable label and an antibody configured to bind the analyte. The plurality of beads may be spherical. In some embodiments, cells may be used in the place of beads.

[0166] The sample may be prepared using a biological sample obtained from a subject. The biological sample may include blood, plasma, serum, urine, tissue, sweat, nasal excretions, or material from a mouth swab. In some embodiments, the biological sample may be from a subject that has a tumor. The sample may be prepared by extracting or concentrating analytes from the biological sample. In some embodiments, the sample may exclude extracting or concentrating analytes.

[0167] Analytes can be captured and cleaned for analysis. In an in vitro diagnostic method, the analytes may be biomarkers present in body fluids like blood, urine, saliva, orcerebrospinal fluid and can be peptides, proteins, lipids, nucleic acids, electrolytes, and other molecules and structures produced by the body. Dependent of type, concentration either alone or in relation to each other, the biomarkers can be used to detect, diagnose, guide, or monitor certain diseases and disease states or therapies. Embodiments described herein can also be used for life science research applications as a method to collect and discover new biomarkers to identify biomarkers indicative for certain diseases and disease states such as Alzheimer’s, Parkinsons, and Traumatic Brain Injury. In some embodiments, analytes may be released from the capture molecule and collected for external analysis after wash.

[0168] The analyte may be an antigen. The antigen may be associated with a disease or a disorder. The antigen may be a protein or a peptide. The antigen may have a concentration from 0 to 10 pM, 10 to 50 pM, 50 to 100 pM, 100 to 500 pM, 500 pM to 1 pM, 1 to 10 pM, 10 to 50 pM, 50 to 100 pM, or 100 to 500 pM. The antigen may be troponin I, C-reactive protein, IGF1, hepcidin, or any antigen described herein.

[0169] In some embodiments, the analyte may be a nucleic acid molecule. For example, the nucleic acid molecule may be DNA or RNA (e.g., mRNA). The nucleic acid molecule may be a biomarker for a disorder or disease. The nucleic acid molecule may be single-stranded.

[0170] The affinity reagent may be an antibody. The antibody may have an affinity to bind with a protein that is the antigen.

[0171] In some embodiments, the affinity reagent may be an oligonucleotide. The oligonucleotide may include a sequence of nucleotides complementary to a portion or all of the nucleic acid molecule analyte. The portion may be at least 3, 4, 5, 6, 7, 8, 9, 10, 10 to 15, 15 to 20, or 20 to 30 nucleotides. In some embodiments, the entire sequence of the nucleic acid molecule is complementary to a subsequence of nucleotides in the oligonucleotide.

[0172] Each label of the plurality of label compounds may include a fluorescent label, a quantum dot, a chemiluminescent label, or an electrochemical label.

[0173] In some embodiments, the label compound may include an oligonucleotide that includes a sequence of nucleotides complementary to a subsequence of the nucleic acid molecule analyte. For example, a first part of the nucleic acid molecule analyte may hybridize with the oligonucleotide attached to the bead, and a second part of the nucleic acid molecule analyte may hybridize with the oligonucleotide of the label compound.

[0174] In some embodiments, the label compound may include an enzyme. The enzyme may target double-stranded nucleic acid molecules. In this manner, the enzyme may recognize a nucleic acid molecule that has hybridized with an oligonucleotide attached to a bead.

[0175] In some embodiments, the label compound may include a reactant that undergoes a chemical reaction with the analyte. The reactant may bind with the analyte coupled on the bead. In some embodiments, the reactant and analyte may form a single product (e.g., A + B —> C). In some embodiments, the first product formed is an intermediate product, which may dissociate to form other products (e.g., A + B — > C — > D + E). The label compound may then become uncoupled to the bead. The label can promote a covalent reaction after binding. Such a reaction may be crosslinking that promotes the label staying attached to the ligand and preventing loss of the label. This can be achieved via peptide covalent binding reaction or via cross linking moieties that covalently bind the complex together.

[0176] At block 2020, the plurality of analytes is bound to a plurality of affinity agents coupled to the first plurality of beads in the reactor. Other analytes may be unbound in the reactor at that point in time. At later times, other analytes may bind to other affinity reagents of other beads in the reactor. In this disclosure, affinity reagents may be also termed affinity agents and vice versa.

[0177] At block 2030, a first subset of the plurality of label compounds is coupled to the first plurality of beads. Coupling may occur in the first fluidic path or in the reactor. Coupling may be by binding the plurality of label compounds to the plurality of affinity agents that are bound to the plurality of beads. Coupling the first subset of the plurality of label compounds to the plurality of analytes may occur before or after binding the plurality of analytes to the plurality of affinity reagents. For example, the plurality of label compounds may bind to the analytes before the analytes are bound to the plurality of affinity reagents. As another example, the plurality of label compounds may be flowed in a fluidic path to intersect the first fluidic path, and then the label compounds may couple to the beads, as illustrated with label 1232 and path 1236 in FIG. 12.

[0178] At block 2040, a first portion of the mixture is flowed from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip. The first portion of the mixture may include the first plurality of beads coupled to the first subset of the plurality of label compounds. The plurality of analytes and the plurality of affinity agents(and optionally the plurality of label compounds) may be in the reactor for a predetermined incubation time. After an incubation time, a flow out of the reactor may be started. In some embodiments, the predetermined incubation time may be 0 seconds, 0 to 10 seconds, 10 to 30 seconds, 30 seconds to 1 minute, or greater than 1 minute. The first portion of the mixture may not be identical to the remainder of the mixture. In some embodiments, the first portion of the mixture may be the first plurality of beads coupled to the first subset of the plurality of label compounds.

[0179] The plurality of structures may include a plurality of pillars. The plurality of pillars may be a portion of an array of pillars. The array of pillars may be a DLD array, including any described herein. The array of pillars may be characterized by a plurality of rows and a plurality of columns. The plurality of pillars may include pillars from at least five columns from the plurality of columns. In some embodiments, the plurality of pillars may include from 1 to 5 columns, from 5 to 10 columns, from 10 to 20 columns, from 20 to 30 columns, from 30 to 50 columns, or more than 50 columns from the plurality of columns. The structures in the plurality of structures may be identical.

[0180] The microfluidic chip may have a longitudinal axis. The first fluidic path and the longitudinal axis may form an angle in a range from 10 degrees to 60 degrees. For example, the angle may be in a range from 10 to 20 degrees, from 20 to 30 degrees, from 30 to 40 degrees, from 40 to 50 degrees, or from 50 to 60 degrees. The first fluidic path may be in a diagonal direction relative to the microfluidic chip. The first fluidic path may be determined by the average (e.g., mean, median, or mode) direction of the first plurality of beads. The angle formed may be an angle calculated with FIGS. 9A to 9C. For example, the angle may be equal to or within 5% or 10% of tan'1(1 / N), where N may be any number from 0.1 to 30.

[0181] At block 2050, a second mixture is formed by flowing a solution in a second fluidic path. The second fluidic path may intersect the first fluidic path. The first fluidic path and the second fluidic path may intersect at an angle in a range from 10 to 20 degrees, from 20 to 30 degrees, from 30 to 40 degrees, from 40 to 50 degrees, or from 50 to 60 degrees. The second fluidic path may be parallel to the longitudinal axis. The second fluidic path may be determined by the average (e.g., mean, median, or mode) direction of the solution. The initial direction of the second fluidic path may be in the same initial direction as the initial flow from the reactor to the plurality of structures. The solution may include phosphate buffer saline (PBS).

[0182] Process 2000 may further include removing a second subset of the plurality of label compounds from the first portion of the mixture to form a second mixture including the first plurality of beads. The removal may be performed using the solution. The second subset of the plurality of label compounds may not be coupled to the first plurality of beads. The solution may wash or clean the beads of non-specific interactions as described herein.

[0183] Process 2000 may include removing unbound analytes. The plurality of analytes may be a first plurality of analytes. The method may further include removing a second plurality of analytes, where the second plurality of analytes is not coupled to the first plurality of beads. The removal may be performed using the solution.

[0184] At block 2060, an amount of the first subset of the plurality of label compounds in the second mixture may be measured. Measuring the amount of label compounds may include measuring an intensity (e.g., a fluorescence intensity, pixel intensity, or electrical current). The intensity may be a normalized intensity. The location for measurement may be at a first distance from a longitudinal axis going through the outlet of the reactor. The first distance may be in a range from 0 to 1 mm, 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, 5 to 10 mm, or greater than 10 mm. The location for measurement may be at a second distance from the reactor. The second distance may be from 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, 5 to 10 mm, 10 to 15 mm, 15 to 20 mm, 20 to 30 mm, 30 to 50 mm, or greater than 50 mm.

[0185] In some embodiments, the amount in the second mixture may be measured along a plurality of locations along the first fluidic path. In this manner, a reaction progress may be monitored, including for when the reaction with the analyte forms an intermediate product that dissociates to form multiple products.

[0186] At block 2070, a reaction kinetic parameter is determined using the amount of the first subset of the plurality of label compounds. The reaction kinetic parameter may be any parameter used in a kinetic rate equation. The reaction kinetic parameter may be a concentration of the plurality of analytes. For example, the concentration may be an absolute or relative concentration in the reactor or in the sample. A relative concentration may be relative to the concentration of another analyte in the reactor or in the sample. The reaction kinetic parameter may be a rate constant characterizing a binding reaction of the affinity reagent to the analyte. The reaction kinetic parameter may be a rate constant characterizing a dissociation reaction of the affinity reagent to the analyte. In some embodiments, the rateconstants may be predetermined, and the concentration may be calculated using the predetermined rate constant. The reaction kinetic parameter may be determined using 10 to 20, 20 to 30, 30 to 40, 40 to 50, or 50 to 100 beads, or fewer than 200 beads.

[0187] In some embodiments, the reaction kinetic parameter may be the concentration of the plurality of analytes. The concentration may be compared to a threshold value. The threshold value may be a value of a concentration that indicates the presence of a disorder, or the threshold value may be a value of a concentration that is statistically different from the minimum concentration that indicates the presence of a disorder. For example, the threshold value may be 1, 2, or 3 standard deviations above the minimum concentration. When the analyte is troponin I, the threshold value may indicate the existence or onset of a heart attack.

[0188] If the subject from whom the sample is obtained is determined to have a disorder, the subject may be treated for the disorder. For example, an alert may be sent to a medical professional or an emergency or medical facility. If the subject is determined to have a heart attack, the subject may be treated for the heart attack, which may include administering aspirin, clot busters (thrombolytics or fibrinolytics), blood thinners (e.g., heparin), nitroglycerin, morphine, beta blockers, ACE inhibitors, and / or statins. In some embodiments, the subject may undergo surgery (e.g., coronary angioplasty and stenting, coronary artery bypass surgery).

[0189] Process 2000 may be multiplexed to analyze different analytes. The plurality of analytes may be a plurality of first analytes. The sample may include a plurality of second analytes. The first analyte may be different from the second analyte. The plurality of affinity reagents may be a plurality of first affinity reagents. Process 2000 may further include mixing a second plurality of beads with the sample to form the mixture. Each bead of the second plurality of beads may be coupled to a second affinity reagent. The second affinity reagent may be configured to bind to the second analyte. Process 2000 may include binding the plurality of second analytes to a plurality of second affinity reagents coupled to the second plurality of beads.

[0190] The first plurality of beads may be characterized by diameters in a first size range. The second plurality of beads may be characterized by diameters in a second size range. The first size range may not be the second size range. The first size range and the second size range may be non-overlapping. Beads in additional size ranges may be used. For example, beads having 3, 4, 5, 6, 7, 8, 9, or 10 size ranges may be used. Beads may be in a range from1 to 2 gm, 2 to 3 gm, 3 to 4 gm, 4 to 5 gm, 5 to 6 gm, 6 to 7 gm, 7 to 8 gm, 8 to 9 gm, 9 to 10 gm, 10 to 11 gm, 11 to 15 gm, 15 to 20 gm, 20 to 30 gm, or greater than 30 gm.

[0191] The plurality of structures may be a first plurality of structures. Process 2000 may further include flowing a second portion of the mixture from the reactor through a third fluidic path defined by a second plurality of structures in the microfluidic chip. The second plurality of structures may include different structures than the first plurality of structures.

[0192] Process 2000 further includes coupling a third subset of the plurality of label compounds to the second plurality of beads in the reactor. Process 2000 may include removing a fourth subset of the plurality of label compounds from the second portion of the mixture to form a fourth mixture including the second plurality of beads. The removal may be performed using the solution. Process 2000 may include measuring an amount of the third subset of the plurality of label compounds in the fourth mixture.

[0193] The label compounds used may be different to analyze different types of analytes. The plurality of label compounds may be a plurality of first label compounds. The sample may further include a plurality of second label compounds. The plurality of second label compounds may be configured to bind to the second analyte. The plurality of second label compounds may be different from the plurality of first label compounds. Process 2000 may further include coupling a first subset of the plurality of second label compounds to the second plurality of beads in the reactor. The first portion of the mixture may include the second plurality of beads coupled to the first subset of the plurality of second label compounds. Process 2000 may also include removing a second subset of the plurality of second label compounds from the first portion of the mixture to form the second mixture may include the second plurality of beads. The removal may be performed using the solution. Process 2000 may include measuring an amount of the first subset of the plurality of second label compounds coupled to the second plurality of beads in the second mixture.

[0194] Labels of the label compounds may have different colors. For example, 2, 3, 4, or 5 different colors of fluorophores can be used as labels. As another example, labels including quantum dots may have 2 to 5, 5 to 10, 10 to 15, or more than 15 different colors. Multiple colors can be used for each bead size.

[0195] In embodiments, process 2000 may include measuring an amount of label compounds on a first bead and then measuring an amount of label compounds on a second bead, where the second bead was released from the reactor after the first bead. The differencein the amount of labels and the time between measurements can be used to determine the reaction kinetic parameter.

[0196] Process 2000 may be repeated two or more times. Process 2000 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes described elsewhere herein.

[0197] In embodiments where cells are used in place of beads, the affinity reagent may also include surface markers and surface receptors. The analytes may also include viruses, bacteria, or other types of cells. Processes can be monitored in a cell, including the concentration or time needed to activate or trigger a cell.

[0198] Although FIG. 20 shows example blocks of process 2000, in some implementations, process 2000 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 20. Additionally, or alternatively, two or more of the blocks of process 2000 may be performed in parallel.2. Two-step conjugation

[0199] FIG. 21 is a flowchart of an example process 2100. Example process 2100 may be similar to example process 2000. Example process 2100 involves a two-step conjugation reaction. The reactor in process 2100 may not include label compounds. In some implementations, one or more process blocks of FIG. 21 may be performed by a system 2500 or any system described herein.

[0200] At block 2110, a first plurality of beads is mixed with a sample to form a first mixture in a reactor of a microfluidic chip. The sample may include a plurality of analytes. Each bead of the first plurality of beads may be coupled to an affinity reagent. The affinity reagent may be configured to bind to the analyte. Block 2110 may be performed in a similar manner as block 2010. However, in block 2110, the reactor may not include label compounds.

[0201] At block 2120, a first subset of the plurality of analytes is bound to a plurality of affinity reagents coupled to the first plurality of beads in the reactor. Block 2120 may be performed in a similar manner as block 2020.

[0202] At block 2130, a first portion of the first mixture is flowed from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip. The first portion of the first mixture may include the first plurality of beads coupled to a first subset of the plurality of affinity agents. Block 2130 may be performed in a similar manner as block 2040. The first mixture may not include label compounds when it is being flowed. The first fluidic path may be fluidic path 1224 in FIG. 12.

[0203] At block 2140, the first plurality of beads is coupled to a plurality of label compounds in the first fluidic path. Block 2140 may be performed in a similar manner as block 2030. The coupling in block 2140 may not occur in the reactor. Coupling may include binding the plurality of label compounds to the first subset of the plurality of analytes. The plurality of label compounds may be flowed from a reactant reservoir to the first fluidic path. The reactant reservoir may be reactant reservoir 1228 in FIG. 12. The amount of the plurality of label compounds may be in excess of the amount of the first subset of the plurality of analytes or the plurality of analytes. For example, the amount of the plurality of label compounds may be at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 50, or 50 to 100 times the amount of the first subset of the plurality of analytes or the plurality of analytes.

[0204] At block 2150, a second mixture is formed by flowing a solution in a second fluidic path. The solution may be flowed from a reservoir, including reservoir 1240 of FIG. 12. The second fluidic path may intersect the first fluidic path. Block 2150 may be performed in a similar manner as block 2050.

[0205] At block 2160, an amount of the plurality of label compounds in the second mixture is measured. Block 2160 may be performed in a similar manner as block 2060.

[0206] In some embodiments, process 2100 may further include determining a reaction kinetic parameter using the amount of the plurality of label compounds. Determining the reaction kinetic parameter may be performed in a similar manner as in block 2070.

[0207] Process 2100 may be multiplexed to analyze different analytes. The plurality of analytes may be a plurality of first analytes. The sample may include a plurality of second analytes. The first analyte is different from the second analyte. The plurality of affinity reagents may be a plurality of first affinity reagents. The process may further include mixing a second plurality of beads with the sample to form the first mixture. Each bead of the second plurality of beads may be coupled to a second affinity reagent. The second affinity reagent may be configured to bind to the second analyte and not the first analyte. The plurality ofsecond analytes may be bound to a plurality of second affinity reagents coupled to the second plurality of beads.

[0208] The first plurality of beads may be characterized by diameters in a first size range. The second plurality of beads is characterized by diameters in a second size range. The first size range is not the second size range. The first size range and the second size range may be any of the ranges described herein, including with process 2000.

[0209] The plurality of structures may be a first plurality of structures. Process 2100 may further include flowing a second portion of the first mixture from the reactor through a third fluidic path defined by a second plurality of structures in the microfluidic chip. The second plurality of structures may include different structures than the first plurality of structures.

[0210] Process 2100 may further include coupling a second subset of the plurality of label compounds to the second plurality of beads in the third fluidic path. Process 2100 may include measuring an amount of the second subset of the plurality of label compounds. Multiplexing in process 2100 may be similar to what is described in process 2000. Coupling the second subset of the plurality of label compounds to the second plurality of beads may not occur in the reactor.

[0211] Process 2100 may further include removing a fourth subset of the plurality of label compounds from the second portion of the first mixture to form a third mixture. The third mixture may include the second plurality of beads. Measuring the amount of the second subset of the plurality of label compounds may be in the third mixture.

[0212] The plurality of label compounds may be a plurality of first label compounds. The plurality of second label compounds may be configured to bind to the second analyte. The plurality of second label compounds may be different from the plurality of first label compounds. Process 2100 may further include coupling a first subset of the plurality of second label compounds to the second plurality of beads. The coupling may be outside the reactor and in a fluidic path (e.g., third fluidic path). The first portion of the first mixture may include the second plurality of beads. Process 2100 may include removing a second subset of the plurality of second label compounds from the first subset of the plurality of second label compounds. A third mixture may include the first subset of the plurality of second label compounds. Process 2100 may include measuring an amount of the first subset of the plurality of second label compounds coupled to the second plurality of beads in the third mixture.

[0213] In embodiments, process 2100 may include measuring an amount of label compounds on a first bead and then measuring an amount of label compounds on a second bead, where the second bead was released from the reactor after the first bead. The difference in the amount of labels and the time between measurements can be used to determine the reaction kinetic parameter.

[0214] Process 2100 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes described elsewhere herein.

[0215] Although FIG. 21 shows example blocks of process 2100, in some implementations, process 2100 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 21. Additionally, or alternatively, two or more of the blocks of process 2100 may be performed in parallel.3. Reaction kinetic parameter

[0216] FIG. 22 is a flowchart of an example process 2200. In some implementations, one or more process blocks of FIG. 22 may be performed by a system, including system 2500, computer system 10, or any system described herein. The blocks in process 2200 may be computer-implemented.

[0217] At block 2210, data including a reaction time and value of a signal reflecting an amount of a first subset of a plurality of label compounds may be received. The value of the signal may be obtained by all or part of process 2000, process 2100, or any method described herein.

[0218] The value of the signal may not be the absolute amount of the first subset of the plurality of label compounds. The value of the signal may be measured. The signal may be a fluorescence signal. The value may be an intensity.

[0219] At block 2220, a reaction kinetic parameter may be determined using the reaction time and the value of the signal. The reaction kinetic parameter may be an amount of the affinity reagent in the sample. Determining the reaction kinetic parameter may include inputting the time and the value of the signal into an equation relating time and value of the signal to the reaction kinetic parameter. The equation may be an analytical or simplified equation, including any equation described herein. The equation may be determinedempirically from one or more calibration samples having known values of the reaction kinetic parameter.

[0220] Determining the reaction kinetic parameter may include comparing the value of the signal at the time to a calibration value at the time. The calibration value may be determined from one or more calibration samples having known values of the reaction kinetic parameter at the time.

[0221] The reaction kinetic parameter may be any reaction kinetic parameter described herein. The reaction kinetic parameter may be used as described with process 2000, process 2100, or any method described herein. The reaction kinetic parameter is obtained using new types of data and new systems and techniques for acquiring the data.

[0222] Process 2200 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes described elsewhere herein.

[0223] Although FIG. 22 shows example blocks of process 2200, in some implementations, process 2200 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 22. Additionally, or alternatively, two or more of the blocks of process 2200 may be performed in parallel.4. Bead-by-bead measurements

[0224] FIG. 23A and FIG. 23B are a flowchart of an example process 2300 for analyzing a sample. Example process 2300 involves a method of measuring amounts of labels on two different beads. The process involves reading beads sequentially (e.g., one at a time).Example process 2300 is similar to example process 2000 and 2100. The measurement of labels of two different beads is implicit in example processes 2000 and 2100 and explained in more detail with process 2300. In some implementations, one or more process blocks of FIG. 23 may be performed by a system 2500.

[0225] At block 2305, a first plurality of beads is mixed with the sample to form a first mixture in a reactor of a microfluidic chip. The sample may include a plurality of analytes. Each bead of the first plurality of beads is coupled to a plurality of affinity reagents. Each affinity reagent of the plurality of affinity reagents is configured to bind to the analyte. Block 2305 may be performed in a similar manner as block 2110 or block 2010.

[0226] At block 2310, a first subset of the plurality of analytes is bound to a first subset of the plurality of affinity reagents coupled to a first bead of the first plurality of beads in the reactor. Block 2310 may be performed in a similar manner as block 2020 or block 2120.

[0227] At block 2315, a second subset of the plurality of analytes is bound to a second subset of the plurality of affinity reagents coupled to a second bead of the first plurality of beads in the reactor. Block 2315 may be performed in a similar manner as block 2020 or block 2120.

[0228] At block 2320, a first portion of the first mixture from the reactor is flowed through a first fluidic path defined by a plurality of structures in the microfluidic chip. The first portion of the first mixture may include the first bead coupled to the first subset of the plurality of affinity reagents. Block 2320 may be performed in a similar manner as block 2040 or block 2130, except for one bead instead of multiple beads. The first fluidic path may be fluidic path 1224 in FIG. 12.

[0229] At block 2325, the first bead of the first plurality of beads is coupled to a first subset of a plurality of label compounds in the first fluidic path. Block 2325 may be performed in a similar manner as block 2030 or block 2140 for a single bead instead of a plurality of beads.

[0230] At block 2330, a second portion of the first mixture is flowed from the reactor through the first fluidic path after flowing the first portion of the first mixture through the first fluidic path. The second portion of the first mixture may include the second bead coupled to the second subset of the plurality of affinity reagents. Block 2330 may be performed in a similar manner as block 2040 or block 2130 for one bead instead of multiple beads.

[0231] At block 2335, process 2300 may include coupling the second bead of the first plurality of beads to a second subset of the plurality of label compounds in the first fluidic path. Block 2335 may be performed in a similar manner as block 2030 or block 2140 for a single bead instead of a plurality of beads.

[0232] At block 2340, a solution is flowed in a second fluidic path. The second fluidic path intersects the first fluidic path. Block 2340 may be performed in a similar manner as block 2050 or block 2150.

[0233] At block 2345, the solution is mixed with the first portion of the first mixture. Block 2345 may be performed in a similar manner as block 2050 or block 2150.

[0234] At block 2350, a first amount of the first subset of the plurality of label compounds is measured after mixing the solution with the first portion of the first mixture. Block 2350 may be measured in a measurement area, including measurement area 2532 of FIG. 25. Block 2350 may be performed in a similar manner as block 2060 or block 2160 for one bead instead of multiple beads.

[0235] At block 2355, the solution is mixed with the second portion of the first mixture after mixing the solution with the first portion of the first mixture. Block 2344 may be performed in a similar manner as block 2050 or block 2150.

[0236] At block 2360, a second amount of the second subset of the plurality of label compounds is measured after measuring the first amount. Block 2360 may be measured in a measurement area, including measurement area 2532 of FIG. 25. Block 2360 may be performed in a similar manner as block 2060 or block 2160 for one bead instead of multiple beads.

[0237] Process 2300 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes (e.g., process 2000 or process 2100) described elsewhere herein.

[0238] Although FIG. 23 shows example blocks of process 2300, in some implementations, process 2300 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 23. Additionally, or alternatively, two or more of the blocks of process 2300 may be performed in parallel.5. Reactions with analytes

[0239] FIG. 24 is a flowchart of an example process 2400 for performing a reaction with target analytes. Example process 2400 may be similar to example processes 2000 and 2100, but label compounds are optional because the focus may be to react a reactant with an analyte efficiently. In some implementations, one or more process blocks of FIG. 24 may be performed by a system 2500.

[0240] At block 2410, a plurality of target analytes is mixed with a plurality of beads. Each bead of the plurality of beads is coupled to a respective affinity reagent. The affinity reagent is configured to bind to the target analyte. Block 2410 may be performed in a similar manner as block 2305, block 2110, or block 2010.

[0241] At block 2420, each target analyte of the plurality of target analytes is bound to the respective capture agent coupled to a bead of the plurality of beads to form a coupled plurality of beads. Block 2420 may be performed in a similar manner as block 2310, block 2020, or block 2120.

[0242] At block 2430, a mixture including the coupled plurality of beads is transferred to a first reservoir of a microfluidic chip. Block 2430 may be performed in a similar manner as block 2630. Block 2430 is optional. In some embodiments, the mixing of and the coupling of the plurality of target analytes with the plurality of beads may occur in the first reservoir.

[0243] At block 2440, a first portion of the mixture is flowed from the reservoir through a first fluidic path defined by a first plurality of structures in the microfluidic chip. The first portion of the mixture may include the coupled plurality of beads. Flowing the first portion of the mixture may include flowing the coupled plurality of beads sequentially (e.g., one-by- one) through the first fluidic path. Block 2440 may be performed in a similar manner as block 2320, block 2330, block 2030, or block 2140.

[0244] At block 2450, a plurality of reactants is flowed in a second fluidic path, where the second fluidic path intersects the first fluidic path. Block 2450 may be performed in a similar manner as block 2030, block 2140, block 2325, or block 2335.

[0245] At block 2460, the plurality of reactants is reacted with the plurality of target analytes of the coupled plurality of beads to form a plurality of reacted beads. The reaction may be a reaction that couples the reactant with the analyte. In some embodiments, the reaction may be any chemical reaction described herein.

[0246] At block 2470, the plurality of reacted beads is flowed to a second reservoir at an end of the first fluidic path. Block 2470 may be performed in a similar manner as block 2970 of FIG. 29.

[0247] Process 2400 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes (e.g., process 2000 or process 2100) described elsewhere herein.

[0248] In some embodiments, the plurality of reactants is a first plurality of reactants. Flowing the first plurality of reactants may include flowing a second plurality of reactants.Process 2400 may further include removing the second plurality of reactants from the plurality of reacted beads in the first fluidic path.

[0249] Process 2400 may further include flowing a solution in a third fluidic path, where the third fluidic path intersects the first fluidic path. The solution flowing through the first fluidic path may remove the second plurality of reactants from the plurality of reacted beads in the first fluidic path.

[0250] In some embodiments, process 2400 may further include coupling a plurality of label compounds to the plurality of reactants. An amount of the plurality of label compounds coupled to the plurality of reactants may be measured. Measuring may be measuring a fluorescence intensity or by any method described herein.

[0251] In some embodiments, measuring the amount of the plurality of label compounds coupled to the plurality of reactants may include measuring the amount at a plurality of locations along the first fluidic path. In this manner, the progress of the chemical reaction may be monitored over time.

[0252] In some embodiments, multiple reactants may be used. The reactant may be a first reactant, and the plurality of reacted beads may be a plurality of first reacted beads. The process may further include flowing a plurality of second reactants in a third fluidic path. The third fluidic path may intersect the first fluidic path. The plurality of second reactants may be reacted with the plurality of target analytes of the coupled plurality of beads to form a plurality of second reacted beads. The plurality of second reactants may have a label compound different from the label compound of the plurality of first reactants. In some embodiments, the second fluidic path is the same as the third fluidic path.

[0253] Although FIG. 24 shows example blocks of process 2400, in some implementations, process 2400 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 24. Additionally, or alternatively, two or more of the blocks of process 2400 may be performed in parallel.

[0254] Some embodiments may include methods of performing a reaction in a microfluidic chip. Reactions may include chemical reactions or physical reactions (e.g., adsorption). The method may include releasing a plurality of beads from a first reservoir to an array of structures in the microfluidic chip. Releasing may be the beads flowing through an outlet of the first reservoir. In some embodiments, the outlet of the first reservoir may be opened torelease the beads. The first reservoir may be the first reservoir in process 2400 or any reactor described herein.

[0255] The method may include displacing the plurality of beads laterally from a first longitudinal axis of the microfluidic chip in the array of structures. Displacing the plurality of beads may be similar to flowing beads in a first fluidic path defined by a plurality of structures, similar to block 2440, 2320, block 2330, block 2030, or block 2140.

[0256] The method may include flowing a reactant from a second reservoir to intersect with the plurality of beads displaced laterally from the first longitudinal axis. Flowing the reactant may be similar to flowing the plurality of reactants in a second fluidic path, as in block 2450, block 2030, block 2140, block 2325, or block 2335.

[0257] The method may include reacting the reactant with the plurality of beads or with a component attached to each bead of the plurality of beads. The reacting may be similar to block 2460.

[0258] The method may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes (e.g., process 2400) described elsewhere herein.

[0259] In a first implementation, the array of structures may include a plurality of parallel rows of structures and a plurality of parallel columns of structures. The array of structures is characterized by a second longitudinal axis, and the second longitudinal axis is offset from the first longitudinal axis. The array of structures may be any array of structures or pillars described herein.

[0260] In a second implementation, alone or in combination with the first implementation, releasing a plurality of beads from a first reservoir may include releasing the plurality of beads in a direction parallel to the first longitudinal axis.

[0261] In a third implementation, alone or in combination with the first and second implementation, flowing the reactant from the second reservoir may include flowing the reactant from the second reservoir in a direction parallel to the first longitudinal axis.

[0262] In a fourth implementation, alone or in combination with one or more of the first through third implementations, the first reservoir may include a plurality of analytes. Each bead of the plurality of beads is coupled to a plurality of affinity reagents in the firstreservoir. The affinity reagent is configured to bind to the analyte. The reactant may include a plurality of label compounds. The method further includes binding a first subset of the plurality of analytes to a first subset of the plurality of affinity reagents coupled to a plurality of beads in the first reservoir, where reacting the reactant (e.g., label compound) is with the component attached to each bead of the plurality of beads, and where the component is the analyte.

[0263] In a fifth implementation, alone or in combination with one or more of the first through fourth implementations, the analyte is an antigen, the affinity reagent is an antibody, and the label compound may include a fluorescent label.

[0264] A sixth implementation, alone or in combination with one or more of the first through fifth implementations, the method further includes flowing the plurality of beads out of the array of structures and detecting the plurality of label compounds on the plurality of beads outside of the array of structures.

[0265] A seventh implementation, alone or in combination with one or more of the first through sixth implementations, the method may include measuring an amount of label compounds on each bead of the plurality of beads.

[0266] In an eighth implementation, alone or in combination with one or more of the first through seventh implementations, where detecting the plurality of label compounds may include detecting each bead of the plurality of beads sequentially after the respective bead flows out of the array of structures.

[0267] In a ninth implementation, alone or in combination with one or more of the first through eighth implementations, the plurality of analytes is a first plurality of analytes, the first reservoir may include a second plurality of analytes, and displacing the plurality of beads laterally from the first longitudinal axis may include separating the second plurality of analytes from the plurality of beads.

[0268] A tenth implementation, alone or in combination with one or more of the first through ninth implementations, the method further includes flowing a solution from a third reservoir to the array of structures along the first longitudinal axis to separate the second plurality of analytes from the plurality of beads.

[0269] In an eleventh implementation, alone or in combination with one or more of the first through tenth implementations, the reactant is a first reactant. The method further includesmixing a plurality of second reactants with the plurality of beads. Each bead of the plurality of beads is coupled to a respective affinity reagent, and the affinity reagent is configured to bind to the first reactant. The method further includes binding each second reactant of the plurality of second reactants to the respective affinity reagent coupled to a bead of the plurality of beads. In some embodiments, the method may include transferring the plurality of beads to the first reservoir after the binding (e.g., the binding may occur outside of the chip). Reacting the first reactant may include reacting the first reactant with the second reactant attached to each bead of the plurality of beads.

[0270] In a twelfth implementation, alone or in combination with one or more of the first through eleventh implementations, the plurality of second reactants is a first plurality of second reactants, the first reservoir may include a second plurality of second reactants, and displacing the plurality of beads laterally from the first longitudinal axis may include separating the second plurality of second from the plurality of beads.

[0271] A thirteenth implementation, alone or in combination with one or more of the first through twelfth implementations, the method further includes flowing a solution from a third reservoir to the array of structures along the first longitudinal axis to separate the second plurality of second reactants from the plurality of beads.

[0272] In a fourteenth implementation, alone or in combination with one or more of the first through thirteenth implementations, the first reactant may include a label compound, and the method further may include detecting the label compound after the plurality of beads exits the array of structures.

[0273] In a fifteenth implementation, alone or in combination with one or more of the first through fourteenth implementations, the chemical reaction is used in the analysis of analytes in a sample bead.

[0274] A sixteenth implementation, alone or in combination with one or more of the first through fifteenth implementations, the method may include detecting each bead of the plurality of beads after exiting.E. Example systems

[0275] FIG. 25 shows a system 2500 for analyzing reaction kinetics. System 2500 may perform all or part of process 2000, process 2100, process 2200, process 2300, or process 2400. In embodiments, system 2500 may include a microfluidic chip 2504. Microfluidic chip2504 may include a reactor 2508. Reactor 2508 may have a volume from 1 to 5 pL, 5 to 10 pL, 10 to 15 pL, 15 to 20 pL, 20 to 30 pL, or greater than 30 pL. Reactor 2508 may have a volume of 20 pL or less. Reactor 2508 may be reactor 604, reactor 710, reactor 1204, or any reactor described herein. Reactor 2508 may be a reservoir.

[0276] Reactor 2508 may include an agitator or a heater. The agitator may include a stirrer or a sonicator. The agitation may be created by vibration or magnetic beads, which may be larger than the beads for coupling to the analytes. The agitator may create turbulence within the reactor.

[0277] Reactor 2508 may include a reactor outlet. In some embodiments, the outlet is an opening of reactor 2508 that leads to a manifold or a DLD array. In some embodiments, the outlet may be configured to be in an open or closed position. For example, the outlet may include a valve, a gate, or a seal (e.g., a liquid-impermeable membrane). Reactor 2508 may include a liquid that is inert and does not react with other components in system 2500.

[0278] Microfluidic chip 2504 may include a structure array 2512. The structure array may be a DLD array (e.g., DLD array 712). The structure array may include a plurality of parallel rows of structures and a plurality of parallel columns of structures. Microfluidic chip 2504 may have a first longitudinal axis 2540. The array of structures may have a second longitudinal axis 2544. Second longitudinal axis 2544 may be offset from first longitudinal axis 2540. The axes may not be parallel. Second longitudinal axis 2544 and first longitudinal axis 2540 may form an angle from 10 to 20 degrees, 20 to 30 degrees, 30 to 40 degrees, 40 to 50 degrees, 50 to 60 degrees, 60 to 70 degrees, or 70 to 80 degrees. The angle formed may be an angle calculated with FIGS. 9A to 9C. For example, the angle may be equal to or within 5% or 10% of tan'1(1 / N), where N is an integer from 1 to 30.

[0279] Each structure of the plurality of structures may be characterized by a diameter in a range from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm. The structures may have a height from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm. A structure may be separate from the closest structure by a distance in a range from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm. The height of the structures may be equal or about equal (e.g., within 5%,10%, or 20%) of the distance separating a structure from the closest structure. The diameter of the pillars may be equal or about equal to the height of the structures. The diameter of the beads may be calculated by the equationas explained above. The diameter of the beads may be a function of the distance between adjacent structures and the number of structures for a shift on structure to the right. As described throughout, structure array 2512 allows for lateral displacement of beads in a direction offset from first longitudinal axis 2540.

[0280] The structures may not be or include electrodes connected to a power source or magnets. In embodiments, microfluidic chip 2504 may also not include electrodes or magnets. In embodiments, a liquid may be disposed in the plurality of structures.

[0281] Structure array 2512 may include a plurality of structures defining a first fluidic path, which may be parallel or substantially parallel to second longitudinal axis 2544. The first fluidic path may be path 716 in FIG. 7 A. The first fluidic path may be in fluid communication with reactor 2508, a reactant reservoir 2510, and a solution reservoir 2516. As used herein, fluid communication may mean that a fluid can be disposed to be in continuous contact with the various units. In some embodiments, the first fluidic path may be in fluid communication with the reactor outlet of reactor 2508. For example, a membrane may separate the main reactor volume from the plurality of structures, but the reactor outlet itself may be in communication with the plurality of structures. Reactant reservoir 2510 may be reactant reservoir 1228 or any reactant reservoir described herein. A manifold 2520 may be configured to deliver a reactant from the reactant reservoir to intersect the first fluidic path. The manifold may deliver the solution in a direction parallel to first longitudinal axis 2540. For example, the path of the reactant may be path 2548. A plurality of label compounds may be disposed in reactant reservoir 2510. The reactant may be the label compound.

[0282] Manifold 2520 may be configured to deliver a solution from solution reservoir 2516 to intersect the first fluidic path. The solution may be a buffer solution to clean the beads. Solution reservoir 2516 may be buffer reservoir 1420. The manifold may deliver the solution in a direction parallel to first longitudinal axis 2540. For example, the path of the solution may be path 2552 or path 724 in FIG. 7A. Manifold 2520 is illustrated as having two input lanes into structure array 2512. However, manifold 2520 is not limited to two input lanes. Manifold 2520 may be configured to deliver a component in addition to the reactant and the solution. The component may be another reactant from another reactant reservoir or another solution from another solution reservoir.

[0283] The flow from reactor 2508, reactant reservoir 2510, and / or solution reservoir 2516 may be driven by fluidic pumps. System 2500 may include one or more fluidic pumps. In some embodiments, capillary force may replace pump-driven flow. Structure array 2512 may be separated from reactor 2508, reactant reservoir 2510, and / or solution reservoir 2516 by one or more membranes (e.g., liquid-impermeable membranes). The one or more membranes may be configured to be removable such that the removal initiates flow (e.g., capillary force- driven or pump driven) of liquids from reactor 2508, reactant reservoir 2510, and / or the solution reservoir 2516 to the first fluidic path. Reactant reservoir 2510 and / or solution reservoir 2516 may each have an outlet, which may be in fluid communication with the first fluidic path, similar to the reactor outlet being in fluid communication with the first fluidic path. The one or more membranes may seal reactor 2508, reactant reservoir 2510, and / or solution reservoir 2516 until they are removed (e.g., physically removed or punctured). The membranes may prevent mixing of the contents of any combination of reactor 2508, reactant reservoir 2510, and solution reservoir 2516. System 2500 may exclude moving parts (e.g., pumps, motors, mechanical stirrers), including moving parts for driving flow. Capillary force-driven flow is described in Aghajanloo et al., “Pumpless deterministic lateral displacement separation using a paper capillary wick,” Lab on a Chip (2023), the entire contents of which are incorporated herein by reference for all purposes.

[0284] In some embodiments, the plurality of structures may include different sections of structures. In embodiments, each section of the structures may have a different longitudinal axis than an adjacent section. In some embodiments, each section of the structures may have different gap distances, diameters, and / or heights than an adjacent section.

[0285] System 2500 may in addition include a plurality of beads or cells disposed on microfluidic chip 2504. The plurality of beads may be disposed in supply 2524. Supply 2524 may be a holding area for beads, analytes, and / or label compounds before they are introduced into reactor 2508 or reactant reservoir 2510. Each bead of the plurality of beads may have a diameter smaller than a width of the first fluidic path. Each bead of the plurality of beads may be bound to a first affinity reagent. The beads may be any beads described herein, including bead 608, bead 750, bead 766, bead 804, bead 1012, microsphere 1116, bead of ligandmicrosphere complex 1212, bead 1404, bead 1408, or bead 1412. The first affinity reagent may be an antibody or an oligonucleotide. For example, the first affinity reagent may be antibody 610 or any antibody described herein.

[0286] System 2500 may include a plurality of label compounds. The label compounds may not be coupled to the plurality of beads. The label compounds may be disposed in reactor 2508 and / or reactant reservoir 2510. Each label compound may include a second affinity reagent. Each second affinity reagent may be bound to a label. The second affinity reagent may be the antibody portion of labeled sandwich antibody 616. The second affinity reagent may be any antibody described herein. The label may be a fluorophore, quantum dot, chemiluminescent tag, electrochemical tag, or any label described herein.

[0287] System 2500 may include a plurality of analytes. The analytes may be any antigen described herein, including a protein. The plurality of first affinity reagents may be configured to bid to the analytes. The plurality of second affinity reagents may be configured to bind to the plurality of analytes.

[0288] The analytes, affinity reagents, label compounds, and / or beads may be located in supply 2524 or reactor 2508.

[0289] System 2500 may also include a detector 2528. Detector 2528 may be an imaging detector (e.g., a camera). Detector 2528 may be positioned to detect and / or measure a signal in measurement area 2532. Measurement area 2532 may be a portion of structure array 2512. Detector 2528 may be configured to detect the label. Detector 2528 may include one or more lenses and / or one or more lasers. In some embodiments, detector 2528 may be part of or contacting microfluidic chip 2504. In some embodiments, detector 2528 may be integrated into the microfluidic chip. Detector 2528 may include an application-specific integrated circuit (ASIC). Detector 2528 may be controlled by computer system 2536. Computer system 2536 may receive data from detector 2528. Data may indicate the intensity of the signal and / or a time when the signal was measured.

[0290] Measurement area 2532 may include a collection reservoir. The collection reservoir may be after the location where the labels are detected. The intersection of the reactant with the first fluidic path and / or the intersection of the solution with the first fluidic path may be between the reactor and the measurement area. A membrane may separate the main volume of the collection reservoir from the first fluidic path. The collection reservoir may include an inlet in communication with the first fluidic path even when the membrane is present. The removal of the membrane may initiate flow of liquids from the array of structures through the inlet to the collection reservoir. The flow may be driven by any force described herein.

[0291] System 2500 may be divided into separate interconnectable parts. For example, system 2500 may include a removable cartridge, which may be disposable, and a reusable device. The cartridge may include structure array 2512, reactant reservoir 2510, and / or solution reservoir 2516. The cartridge may also include a manifold configured to deliver a reactant from reactant reservoir 2510 to intersect the first fluidic path and / or a solution from solution reservoir 2516 to intersect the first fluidic path. The cartridge may be configured to reversibly connect with the reusable device. In some embodiments, the cartridge may include measurement area 2532.

[0292] The reusable device may include the components of system 2500 not part of the cartridge. For example, the reusable device may include reactor 2508. Additionally, the reusable device may include supply 2524, detector 2528, and computer system 2536. In some embodiments, the reusable device may include measurement area 2532. When the cartridge is connected with the reusable device, the reactor is in fluid communication with the first fluidic path.

[0293] The cartridge may include a reactant in reactant reservoir 2510. The cartridge may include a solution in solution reservoir 2516. The reactant and / or solution may be specific for a particular assay (e.g., a label configured to bind to a certain analyte). In some embodiments, the cartridge may include a liquid in structure array 2512. Different cartridges can be included for different assays. A kit may include a plurality of cartridges for different assays.

[0294] In some embodiments, system 2500 may include a sample reservoir configured to receiving a blood sample. For example, the blood sample may be blood obtained from a subject. A filter may separate the sample reservoir from at least one of the reactor and the reactant reservoir. The filter may be configured to retain red blood cells. The filter may pass plasma from the blood sample to at least one of the reactor and the reactant reservoir. System 2500 may allow for a blood sample from a subject to be directly processed and analyzed without additional sample preparation. Filters may include membranes or any suitable separation technology (including another DLD array).II. TARGET ENRICHMENT

[0295] Current techniques for enriching nucleic acid molecules face several limitations. Techniques include size-based separation, PCR amplification, and hybridization capture. These techniques may lack the level of specificity required to selectively enrich particularnucleic acid sequences, leading to false positives. Techniques may be labor intensive and time consuming, such as multiple rounds of purification, DNA fragmentation, and extensive sample handling. These processes can be prone to human error and are not ideal for high- throughput applications. Certain techniques may use a large amount of starting material and therefore may be unsuitable when the available nucleic acid sample is limited. Current techniques for enriching other analytes (e.g., antigens, peptide biomarkers, exosomes, cells from samples) face similar limitations.

[0296] Embodiments described herein have advantages over current techniques. The separation of beads bound to nucleic acid molecules of interest from unbound nucleic acid molecules (or other analytes) is effective and efficient. The array of structures allows for beads to flow down a distinct path from unbound nucleic acid molecules (or analytes). A wash solution successfully removes unbound nucleic acid molecules (or analytes) from beads. The separation of bound and unbound nucleic acid molecules (or analytes) is more complete than other techniques.A. Enrichment Principles

[0297] FIG. 26 illustrates enrichment principles used in methods and systems described herein. At stage 2604, a bead 2608 is coupled to an oligonucleotide 2612. Oligonucleotide 2612 is attached to a cleavable linker 2616, which is attached to bead 2608. An oligonucleotide is an illustrative example of an affinity agent (also called capture agent).

[0298] At stage 2620, bead 2608 is contacted with nucleic acid molecules. The nucleic acid molecules include a target nucleic acid molecule 2624 (i.e., a molecule in a region of interest) and a non-target nucleic acid molecule 2628. The mixture may be formed by beads being added to a mixture of the nucleic acid molecules or nucleic acid molecules may be added to the beads. Nucleic acid molecules are an illustrative example of an analyte. FIG. 26 can be adapted for other analytes and affinity agents.

[0299] At stage 2632, one or more target nucleic acid molecules hybridize to one or more oligonucleotides coupled to bead 2608. For example, double-stranded nucleic acid molecule 2636 is coupled to bead 2608.

[0300] Stage 2638a and stage 2638b illustrate separating the bead from the unbound nucleic acid molecules (both target and non-target). The separation occurs using adeterministic lateral displacement (DLD) microfluidics array, which is described elsewhere in this disclosure.

[0301] Stage 2640 illustrates the collection of beads after separation from unbound nucleic acid molecules.

[0302] At stage 2644, the linkers between the oligonucleotides and bead have been cleaved. Double-stranded nucleic acid molecules 2648 are separated from the beads. These doublestranded nucleic acid molecules can be amplified and analyzed (e.g., sequenced).

[0303] FIG. 27 shows an illustration of a possible reservoir and mechanisms allowing for enrichment or filtering of target analytes. The target analyte may be a nucleic acid molecule or any analyte described herein. Reservoir 2704 contains capture beads, including capture bead 2708. Capture beads may be bound to an affinity agent, including affinity agent 2710. Affinity agent 2710 may be an oligonucleotide or any affinity agent described herein. Capture beads may bind with a target analyte, including target analyte 2712. The reservoir may include non-target analytes, including non-target analyte 2716. Although FIG. 27 illustrates each capture bead with only three affinity agents, a capture bead may have 100 or more affinity agents. FIG. 27 also shows different sizes and shapes for the target analytes and non- target analytes for illustrative purposes, not to represent the actual scale of the molecules.

[0304] Arrow 2728 represents a flow of material out of reservoir 2704. Area 2732 shows possible materials in the output of the reservoir. The materials illustrated include the bead bound to a target analyte, as well as an unbound target analyte and an unbound non-target analyte. The beads may be drawn out from reservoir 2704 at a constant rate over a fixed amount of time.

[0305] Wash 2736 represents a flow that removes the unbound target analyte and unbound non-target analyte from the capture beads. The capture beads are not removed by the wash and instead proceed in a direction indicated by arrow 2740 and arrow 2744, which are different from the direction of the wash. The wash direction may be from top to bottom in this figure rather than from left to right.

[0306] Bead 2752 represent a capture bead separated from unbound analytes. The bound target analytes may be separated from the bead itself and analyzed further.

[0307] FIG. 28 illustrates a microfluidic chip for enriching target analytes. One or more channels (e.g., channel 2804) may convey flow from a reservoir 2810 (e.g., reservoir 2704 inFIG. 27). Reservoir 2810 includes a mixture of beads and unbound analytes. The analytes and beads may be nay disclosed herein. The flow includes this bead mixture.

[0308] After channel 2804, the bead mixture goes through deterministic lateral displacement (DLD) array 2812. DLD arrays are described in US 7,150,812, the entire contents of which are incorporated herein by reference for all purposes. DLD array 2812 may include a plurality of structures. The structures may be pillars with a circular base, a rectangular (e.g., square) base, a triangular base, or any polygon base. The structures may be in a regularly spaced array. The array may define straight paths through the structures. These straight paths may be offset from the path going from the channels from reservoir 2810. The straight paths through DLD array 2812 may not be parallel to the longitudinal axis of the chip. In FIG. 28, the longitudinal axis is illustrated in the vertical direction.

[0309] Path 2816 indicates a path that the beads follow through the DLD array. These paths are at a diagonal from before entry in input area 2820 into DLD array 2812. The beads travel to DLD array output area 2836. DLD array output area 2836 is laterally displaced from initial entry into DLD array in input area 2820.

[0310] The dashed lines indicate paths (e.g., path 2824) that a cleaning flow may follow. This cleaning flow may be delivered from one or more channels. The cleaning flow may be from buffer reservoir 2830. The cleaning flow may be along the longitudinal axis. The cleaning flow directs unbound target analytes (AT) and unbound non-target analytes (Ao) away from the beads, effectively washing the beads. With the longitudinal direction of the cleaning flow, the cleaning flow may go to DLD array output area 2836. Although only a few solid lines are illustrated, the cleaning flow may cover most (50%, 60%, 70%, 80%, 90% or more) or the entirety of the DLD array, washing beads throughout their travel to DLD array output area 2836. The cleaning flow may fully replace the liquid volume around each bead up to several hundred times.

[0311] Microfluidic chip output area 2836 may include a port to collect bound AT. Unbound AT and unbound Ao may be through a separate port. Reservoirs may also be used to collect output instead of ports.

[0312] The total volume in reservoir 2810 may be as small as 1 pL, 5 pL, or 10 pL.

[0313] FIG. 7B can be viewed as illustrating the mechanics of the washing of the beads. The beads may be washed of non-specific interactions with analytes rather than labeledsandwich antibodies. The laminar flow washes away unbound analytes (e.g., nucleic acid molecules) instead of labeled sandwich antibodies in the direction of the flow of the buffer solution.

[0314] FIG. 8 showing the relationship between sheer factor and displacement is applicable to target enrichment methods. The bead is washed of unbound analytes. Similarly, FIGS. 9A-9C show possible arrangements of pillars in the DLD array.B. Example Methods

[0315] FIG. 29 is a flowchart of an example process 2900 for enriching or filtering target nucleic acid molecules. In some implementations, one or more process blocks of FIG. 29 may be performed by a system 3100. The target nucleic acid molecules may correspond to a particular genomic region (e.g., a biomarker for a disease or disorder, including a particular single nucleotide polymorphism [SNP]). The genomic region may be a particular chromosome or subchromosomal region. The target nucleic acid molecules may be DNA or RNA (e.g., mRNA).

[0316] The sample of nucleic acid molecules may be prepared using a biological sample obtained from a subject. The biological sample may include blood, plasma, serum, urine, tissue, sweat, nasal excretions, or material from a mouth swab. In some embodiments, the biological sample may be from a subject that has a tumor. The sample may be prepared by extracting nucleic acid molecules from the biological sample.

[0317] In some embodiments, process 2900 may include separating a plurality of doublestranded nucleic acid molecules to form the plurality of target nucleic acid molecules, which may be single-stranded nucleic acid molecules. Separating the double-stranded nucleic acid molecules may be called DNA melting or DNA denaturation.

[0318] At block 2910, process 2900 may include mixing a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules with a first plurality of beads. The target nucleic acid molecule and the non-target nucleic acid molecules may be obtained from a biological sample. Each target nucleic acid molecule of the plurality of target nucleic acid molecules may include a target sequence. Each bead of the first plurality of beads may be coupled to a respective oligonucleotide. The oligonucleotide may include a first sequence complementary to the target sequence. The entirety or a portion of the oligonucleotide may be the first sequence. The entirety of or a portion of the target nucleic acid molecule mayhave the target sequence. The first sequence may be 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or over 50 nucleotides long. The beads, oligonucleotides, target nucleic acid molecules, and non-target nucleic acid molecules may be any described herein.

[0319] The first plurality of beads may be characterized by diameters in a first size range. The second plurality of beads may be characterized by diameters in a second size range. The first size range may not be the second size range. The first size range and the second size range may be non-overlapping. Beads in additional size ranges may be used. For example, beads having 3, 4, 5, 6, 7, 8, 9, or 10 size ranges may be used. Beads may be in a range from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm.

[0320] At block 2920, process 2900 may include binding each target nucleic acid molecule of the plurality of target nucleic acid molecules to the respective oligonucleotide coupled to a bead of the first plurality of beads to form a coupled first plurality of beads.

[0321] At block 2930, process 2900 may include transferring a mixture including the coupled first plurality of beads and the plurality of non-target nucleic acid molecules to a first reservoir of a microfluidic chip. Transferring may be by moving the mixture from another container to the first reservoir. In some embodiments, transferring the mixture may include transferring from a different reservoir on the microfluidic chip.

[0322] At block 2940, process 2900 may include flowing a first portion of the mixture from the reservoir through a first fluidic path defined by a first plurality of structures in the microfluidic chip. The first portion of the mixture may include the coupled first plurality of beads and the plurality of non-target nucleic acid molecules. The microfluidic chip may have a longitudinal axis. The first fluidic path and the longitudinal axis may form an angle in a range from 10 degrees to 60 degrees. The first fluidic path may be any first fluidic path described herein, including path 2816.

[0323] The first plurality of structures may include a plurality of pillars. The plurality of pillars may be a portion of an array of pillars. The array of pillars may be characterized by a plurality of rows and a plurality of columns. The plurality of pillars may include pillars from at least five columns from the plurality of columns. The plurality of pillars may be any plurality of pillars described herein, including those a DLD array and those illustrated in FIGS. 6A to 6C. The plurality of pillars may include pillars from at least five columns from the plurality of columns. In some embodiments, the plurality of pillars may include from 1 to5 columns, from 5 to 10 columns, from 10 to 20 columns, from 20 to 30 columns, from 30 to 50 columns, or more than 50 columns from the plurality of columns. The structures in the plurality of structures may be identical.

[0324] At block 2950, process 2900 may include forming a second mixture by flowing a solution in a second fluidic path. The second fluidic path intersects the first fluidic path. The second fluidic path may be parallel to the longitudinal axis. The solution may be any buffer solution or wash solution described herein.

[0325] The first fluidic path and the second fluidic path may intersect at an angle in a range from 10 to 20 degrees, from 20 to 30 degrees, from 30 to 40 degrees, from 40 to 50 degrees, or from 50 to 60 degrees. The second fluidic path may be parallel to the longitudinal axis. The second fluidic path may be determined by the average (e.g., mean, median, or mode) direction of the solution. The initial direction of the second fluidic path may be in the same initial direction as the initial flow from the reactor to the plurality of structures. The solution may include phosphate buffer saline (PBS).

[0326] At block 2960, process 2900 may include removing, using the solution, the plurality of non-target nucleic acid molecules from the coupled first plurality of beads in the first fluidic path. Removing the plurality of non-target nucleic acid molecules may include flowing the plurality of non-target nucleic acid molecules to a third reservoir on the microfluidic chip.

[0327] At block 2970, process 2900 may include flowing the coupled first plurality of beads to a second reservoir at an end of the first fluidic path. The second reservoir may be output area 2836 in FIG. 28.

[0328] In some embodiments, process 2900 further includes removing the coupled first plurality of beads from the second reservoir. For each bead of the coupled first plurality of beads, the respective oligonucleotide and the respective target nucleic acid molecule may be separated from the bead. Each bead of the first plurality of beads may be coupled to the respective oligonucleotide by a cleavable linker (e.g., a uracil). Separating the respective oligonucleotide and the respective target nucleic acid molecule from each bead of the coupled first plurality of beads may include using an enzyme to cleave at the linker. In some embodiments, the separation may be photoactivated.

[0329] The plurality of target nucleic acid molecules may be amplified after the separating to form an amplified plurality of target nucleic acid molecules. The amplified plurality of target nucleic acid molecules may be sequenced.

[0330] Process 2900 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes described elsewhere herein.

[0331] In some embodiments, different types of target nucleic acid molecules may be enriched. The plurality of target nucleic acid molecules may be a plurality of first target nucleic acid molecules. The oligonucleotide may be a first oligonucleotide. The plurality of non-target nucleic acid molecules may be a first plurality of non-target nucleic acid molecules. The process may further include mixing a plurality of second target nucleic acid molecules and the plurality of non-target nucleic acid molecules with a second plurality of beads. Each second target nucleic acid molecule of the plurality of second target nucleic acid molecules may include a second target sequence. Each bead of the second plurality of beads may be coupled to a respective second oligonucleotide. The second oligonucleotide may include a second sequence complementary to the second target sequence. The process may further include binding each second target nucleic acid molecule of the plurality of second target nucleic acid molecules to the respective second oligonucleotide coupled to a bead of the second plurality of beads to form a coupled second plurality of beads. Process 2900 may further include flowing a second portion of the mixture from the reservoir through a third fluidic path defined by a second plurality of structures in the microfluidic chip. The second plurality of structures may include different structures than the first plurality of structures. The mixture further may include the coupled second plurality of beads. The second portion of the mixture may include the coupled second plurality of beads and a second plurality of non- target nucleic acid molecules.

[0332] In embodiments, process 2900 may further include forming a third mixture by flowing the solution in a fourth fluidic path. The fourth fluidic path intersects the third fluidic path. Process 2900 may include removing, using the solution, the second plurality of non- target nucleic acid molecules from the coupled second plurality of beads in the third fluidic path. Process 2900 may include flowing the coupled second plurality of beads to a third reservoir at an end of the third fluidic path.

[0333] Although FIG. 29 shows example blocks of process 2900, in some implementations, process 2900 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 29. Additionally, or alternatively, two or more of the blocks of process 2900 may be performed in parallel.

[0334] FIG. 30 is a flowchart of an example process 3000 for enriching or filtering target analytes. Analytes may include a nucleic acid molecule, an antigen, a peptide biomarker, an exosome, a cell, or any analyte described herein. In some implementations, one or more process blocks of FIG. 30 may be performed by a system 3100.

[0335] At block 3010, process 3000 may include mixing a plurality of target analytes and a plurality of non-target analytes with a first plurality of beads. Each bead of the first plurality of beads may be coupled to a respective affinity agent. The affinity agent may be configured to bind to the target analyte and to not bind to the non-target analyte. The affinity agent may be an antibody, an oligonucleotide, an aptamer, peptide, polysaccharide, protein (e.g., DARPin), or any affinity agent described herein. The affinity agent may be anything configured to bind the analyte. Block 3010 may be similar to block 2910.

[0336] At block 3020, process 3000 may include binding each target analyte of the plurality of target analytes to the respective capture agent coupled to a bead of the first plurality of beads to form a coupled first plurality of beads. Block 3020 may be similar to block 2920.

[0337] At block 3030, process 3000 may include transferring a mixture may include the coupled first plurality of beads and the plurality of non-target analytes to a first reservoir of a microfluidic chip. Block 3030 may be similar to block 2930.

[0338] At block 3040, process 3000 may include flowing a first portion of the mixture from the reservoir through a first fluidic path defined by a first plurality of structures in the microfluidic chip. The first portion of the mixture may include the coupled first plurality of beads and the plurality of non-target analytes. Block 3040 may be similar to block 2940.

[0339] At block 3050, process 3000 may include forming a second mixture by flowing a solution in a second fluidic path. The second fluidic path intersects the first fluidic path. Block 3050 may be similar to block 2950.

[0340] At block 3060, process 3000 may include removing, using the solution, the plurality of non-target analytes from the coupled first plurality of beads in the first fluidic path. Block 3060 may be similar to block 2960.

[0341] At block 3070, process 3000 may include flowing the coupled first plurality of beads to a second reservoir at an end of the first fluidic path. Block 3070 may be similar to block 2970.

[0342] Although FIG. 30 shows example blocks of process 3000, in some implementations, process 3000 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 30. Additionally, or alternatively, two or more of the blocks of process 3000 may be performed in parallel. Process 3000 can be combined with parts of process 2900.C. Example systems

[0343] FIG. 31 shows a system 3100 for enriching target nucleic acid molecules. System 3100 may perform all or part of process 2900 or process 3000. In embodiments, system 3100 may include a microfluidic chip 3104. Microfluidic chip 3104 may include a reservoir 3108. Reservoir 3108 may have a volume from 1 to 5 pL, 5 to 10 pL, 10 to 15 pL, 15 to 20 pL, 20 to 30 pL, or greater than 30 pL. Reservoir 3108 may have a volume of 20 pL or less. Reservoir 3108 may be reservoir 2704, reservoir 2810, or any reactor described herein. Reservoir 3108 may include an agitator (e.g., a stirrer) or a heater. System 3100 may use any components from system 2500 as additional components.

[0344] Microfluidic chip 3104 may include a structure array 3112. The structure array may be a DLD array (e.g., DLD array 2812). The structure array may include a plurality of parallel rows of structures and a plurality of parallel columns of structures. Microfluidic chip 3104 may have a first longitudinal axis 3140. The array of structures may have a second longitudinal axis 3144. Second longitudinal axis 3144 may be offset from first longitudinal axis 3140. The axes may not be parallel. Second longitudinal axis 3144 and first longitudinal axis 3140 may form an angle from 10 to 20 degrees, 20 to 30 degrees, 30 to 40 degrees, 40 to 50 degrees, 50 to 60 degrees, 60 to 70 degrees, or 70 to 80 degrees. The angle formed may be an angle calculated with FIGS. 6A to 6C. For example, the angle may be equal to or within 5% or 10% of tan'1(1 / N), where N is an integer from 1 to 30.

[0345] Each structure of the plurality of structures may be characterized by a diameter in a range from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm. The structures may have a height from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm. A structure may be separate from the closest structure by a distance in a range from 1 to 2 pm, 2 to 3 pm, 3 to 4 pm, 4 to 5 pm, 5 to 6 pm, 6 to 7 pm, 7 to 8 pm, 8 to 9 pm, 9 to 10 pm, 10 to 11 pm, 11 to 15 pm, 15 to 20 pm, 20 to 30 pm, or greater than 30 pm. The height of the structures may be equal or about equal (e.g., within 5%, 10%, or 20%) of the distance separating a structure from the closest structure. The diameter of the pillars may be equal or about equal to the height of the structures. The diameter of the beads may be calculated by the equationas explained above. The diameter of the beads may be a function of the distance between adjacent structures and the number of structures for a shift on structure to the right.

[0346] The structures may not be or include electrodes connected to a power source or magnets. In embodiments, microfluidic chip 3104 may also not include electrodes or magnets.

[0347] Structure array 3112 may include a plurality of structures defining a first fluidic path, which may be parallel or substantially parallel to second longitudinal axis 3144. The first fluidic path may be path 2816 in FIG. 28. The first fluidic path may be in fluid communication with reservoir 3108 and a solution reservoir 3116. Reservoir 3108 may be at a first end of the first fluidic path. A manifold 3120 may be configured to deliver the solution in a direction parallel to first longitudinal axis 3140.

[0348] Manifold 3120 may be configured to deliver a solution from solution reservoir 916 to intersect the first fluidic path. The solution may be a buffer solution to clean the beads. Solution reservoir 3116 may be buffer reservoir 2830. The manifold may deliver the solution in a direction parallel to first longitudinal axis 3140. For example, the path of the solution may be path 3152 or path 2824 in FIG. 28. The path of the solution may be a second fluidic path, which may be defined by a plurality of structures that are different from the plurality of structures defining the first fluidic path. The solution reservoir may be at a first end of the second fluidic path.

[0349] In some embodiments, the plurality of structures may include different sections of structures. In embodiments, each section of the structures may have a different longitudinal axis than an adjacent section. In some embodiments, each section of the structures may have different gap distances, diameters, and / or heights than an adjacent section.

[0350] System 3100 may in addition include a plurality of beads disposed on microfluidic chip 3104. The plurality of beads may be disposed in supply 3124. Supply 3124 may be a holding area for beads and / or nucleic acid molecules before they are introduced into reservoir 3108. Binding (i.e., hybridization) of target nucleic acid molecules to oligonucleotides coupled on beads may occur in supply 3124. Each bead of the plurality of beads may have a diameter smaller than a width of the first fluidic path. Each bead of the plurality of beads may be bound to a first affinity reagent. The beads may be any beads described herein, including bead 2608, bead 2708, bead 750, or bead 1404. The nucleic acid molecules and / or beads may be located in supply 3124 or reservoir 3108. Supply 3124 may include reactants (e.g., primers) for hybridization.

[0351] Collection area 3132 is the end of the any fluidic paths for flow of beads or unbound nucleic acid molecules. Collection area 3132 may include a reservoir specifically for beads and / or a reservoir for unbound nucleic acid molecules. Collection area 3132 (e.g., reservoir for beads) may be at an opposite end of the first fluidic path as reservoir 3108. The reservoir for unbound nucleic acid molecules may be at an opposite end of the second fluidic path as solution reservoir 3116. Collection area 3132 may be DLD array output area 2836.

[0352] System 3100 may also include a handling system 3128. Handling system 3128 may transfer components from supply 3124 to reservoir 3108. Handling system 3128 may also extract beads or residue from collection area 3132. Handling system 3128 may transfer beads to a cleavage module. In addition, handling system 3128 may transfer nucleic acid molecules to an amplification module and / or a sequencing module. Handling system 3128 may be controlled by computer system 3136. Computer system 3136 may receive data from handling system 3128. Data may indicate the position and / or status of a handler (e.g., arm, end effector) of handling system 3128. Computer system 3136 may also control modules, including a sample extraction module, a hybridization module, a mixture collection module, a cleavage module, an amplification module, or a sequencing module. In some embodiments, supply 3124 may be the output of a sample extraction module, a hybridization module, and / or a mixture collection module. In some embodiments, collection area 3132 may be the input ofa cleavage module, an amplification module and / or a sequencing module. Microfluidic chip 3104 may have a direct input or output from another module. Process may run in a batch or continuous process through microfluidic chip 3104.III. COMPUTER SYSTEM

[0353] Any of the computer systems mentioned herein may utilize any suitable number of subsystems. Examples of such subsystems are shown in FIG. 32 in computer system 10. In some embodiments, a computer system includes a single computer apparatus, where the subsystems can be the components of the computer apparatus. In other embodiments, a computer system can include multiple computer apparatuses, each being a subsystem, with internal components. A computer system can include desktop and laptop computers, tablets, mobile phones and other mobile devices. Computer system 10 may be computer system 2236.

[0354] The subsystems shown in FIG. 32 are interconnected via a system bus 75. Additional subsystems such as a printer 74, keyboard 78, storage device(s) 79, monitor 76 (e.g., a display screen, such as an LED), which is coupled to display adapter 82, and others are shown. Peripherals and input / output (I / O) devices, which couple to I / O controller 71, can be connected to the computer system by any number of means known in the art such as input / output (I / O) port 77 (e.g., USB, Lightning). For example, I / O port 77 or external interface 81 (e.g. Ethernet, Wi-Fi, etc.) can be used to connect computer system 10 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via system bus 75 allows the central processor 73 to communicate with each subsystem and to control the execution of a plurality of instructions from system memory 72 or the storage device(s) 79 (e.g., a fixed disk, such as a hard drive, or optical disk), as well as the exchange of information between subsystems. The system memory 72 and / or the storage device(s) 79 may embody a computer readable medium. Another subsystem is a data collection device 85, such as a camera, microphone, accelerometer, and the like. Any of the data mentioned herein can be output from one component to another component and can be output to the user.

[0355] A computer system can include a plurality of the same components or subsystems, e.g., connected together by external interface 81, by an internal interface, or via removable storage devices that can be connected and removed from one component to another component. In some embodiments, computer systems, subsystem, or apparatuses can communicate over a network. In such instances, one computer can be considered a client andanother computer a server, where each can be part of a same computer system. A client and a server can each include multiple systems, subsystems, or components.

[0356] Aspects of embodiments can be implemented in the form of control logic using hardware circuitry (e.g., an application specific integrated circuit or field programmable gate array) and / or using computer software with a generally programmable processor in a modular or integrated manner. As used herein, a processor can include a single-core processor, multicore processor on a same integrated chip, or multiple processing units on a single circuit board or networked, as well as dedicated hardware. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will know and appreciate other ways and / or methods to implement embodiments of the present disclosure using hardware and a combination of hardware and software.

[0357] Any of the software components or functions described in this application may be implemented as software code to be executed by a processor using any suitable computer language such as, for example, Java, C, C++, C#, Objective-C, Swift, or scripting language such as Perl or Python using, for example, conventional or object-oriented techniques. The software code may be stored as a series of instructions or commands on a computer readable medium for storage and / or transmission. A suitable non-transitory computer readable medium can include random access memory (RAM), a read only memory (ROM), a magnetic medium such as a hard-drive, or an optical medium such as a compact disk (CD) or DVD (digital versatile disk) or Blu-ray disk, flash memory, and the like. The computer readable medium may be any combination of such storage or transmission devices.

[0358] Such programs may also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and / or wireless networks conforming to a variety of protocols, including the Internet. As such, a computer readable medium may be created using a data signal encoded with such programs. Computer readable media encoded with the program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer readable medium may reside on or within a single computer product (e.g., a hard drive, a CD, or an entire computer system), and may be present on or within different computer products within a system or network. A computer system may include a monitor, printer, or other suitable display for providing any of the results mentioned herein to a user.

[0359] Any of the methods described herein may be totally or partially performed with a computer system including one or more processors, which can be configured to perform the steps. Thus, embodiments can be directed to computer systems configured to perform the steps of any of the methods described herein, potentially with different components performing a respective step or a respective group of steps. Although presented as numbered steps, steps of methods herein can be performed at a same time or at different times or in a different order that is logically possible. Additionally, portions of these steps may be used with portions of other steps from other methods. Also, all or portions of a step may be optional. Additionally, any of the steps of any of the methods can be performed with modules, units, circuits, or other means of a system for performing these steps.IV. EMBODIMENTS

[0360] Embodiments may include the following. Methods may include detecting one bead than another bead.

[0361] Embodiment 1. A method for analyzing a sample, the method comprising: mixing a first plurality of beads with the sample to form a first mixture in a reactor of a microfluidic chip, wherein: the sample comprises a plurality of analytes, each bead of the first plurality of beads is coupled to a plurality of affinity reagents, and each affinity reagent of the plurality of affinity reagents is configured to bind to the analyte; binding a first subset of the plurality of analytes to a first subset of the plurality of affinity reagents coupled to a first bead of the first plurality of beads in the reactor; binding a second subset of the plurality of analytes to a second subset of the plurality of affinity reagents coupled to a second bead of the first plurality of beads in the reactor; flowing a first portion of the first mixture from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip, wherein: the first portion of the first mixture comprises the first bead coupled to the first subset of the plurality of affinity reagents; coupling the first bead of the first plurality of beads to a first subset of a plurality of label compounds in the first fluidic path; flowing a second portion of the first mixture from the reactor through the first fluidic path after flowing the first portion of the first mixture through the first fluidic path, wherein: the second portion of the first mixture comprises the second bead coupled to the second subset of the plurality of affinity reagents; coupling the second bead of the first plurality of beads to a second subset of the plurality of label compounds in the first fluidic path; flowing a solution in a second fluidic path, wherein the second fluidic path intersects the first fluidic path; mixing the solution withthe first portion of the first mixture; measuring a first amount of the first subset of the plurality of label compounds after mixing the solution with the first portion of the first mixture; mixing the solution with the second portion of the first mixture after mixing the solution with the first portion of the first mixture; and measuring a second amount of the second subset of the plurality of label compounds after measuring the first amount.

[0362] Embodiment 2. The method of Embodiment 1, further comprising: removing a third subset of the plurality of analytes from the first portion of the first mixture before coupling the first bead to the first subset of the plurality of label compounds in the first fluidic path.

[0363] Embodiment 3. The method of Embodiment 1, wherein the plurality of label compounds is a first plurality of label compounds, the method further comprising: flowing the first plurality of label compounds and a second plurality of label compounds in a third fluidic path, wherein the third fluidic path intersects the first fluidic path, and removing the second plurality of label compounds from the first fluidic path.

[0364] Embodiment 4. The method of Embodiment 1, wherein: the analyte is an antigen, the affinity reagent is an antibody, and each label of the plurality of label compounds comprises a fluorescent label.

[0365] Embodiment 5. The method of Embodiment 1, wherein: the plurality of structures comprises a plurality of pillars, the plurality of pillars is a portion of an array of pillars, the array of pillars is characterized by a plurality of rows and a plurality of columns, and the plurality of pillars comprises pillars from at least five columns from the plurality of columns.

[0366] Embodiment 6. The method of Embodiment 1, wherein: the microfluidic chip has a longitudinal axis, and the first fluidic path and the longitudinal axis form an angle in a range from 10 degrees to 60 degrees.

[0367] Embodiment 7. The method of Embodiment 1, wherein: the microfluidic chip has a longitudinal axis, and the second fluidic path is parallel to the longitudinal axis.

[0368] Embodiment 8. The method of Embodiment 1, wherein: the plurality of analytes is a first plurality of analytes, the method further comprising: removing, using the solution, a second plurality of analytes, wherein the second plurality of analytes is not coupled to the first plurality of beads.

[0369] Embodiment 9. The method of Embodiment 1, further comprising: determining a reaction kinetic parameter using the first amount and the second amount.

[0370] Embodiment 10. The method of Embodiment 9, wherein the reaction kinetic parameter is a concentration of the plurality of analytes.

[0371] Embodiment 11. The method of Embodiment 1, wherein the reaction kinetic parameter is a rate constant characterizing the binding reaction of the affinity reagent to the analyte.

[0372] Embodiment 12. The method of Embodiment 1, wherein: the plurality of analytes is a plurality of first analytes, the sample comprises a plurality of second analytes, the first analyte is different from the second analyte, and the plurality of affinity reagents is a plurality of first affinity reagents, the method further comprising: mixing a second plurality of beads with the sample to form the first mixture, wherein: each bead of the second plurality of beads is coupled to a second affinity reagent, and the second affinity reagent is configured to bind to the second analyte, and binding the plurality of second analytes to a plurality of second affinity reagents coupled to the second plurality of beads.

[0373] Embodiment 13. The method of Embodiment 12, wherein: the first plurality of beads is characterized by diameters in a first size range, the second plurality of beads is characterized by diameters in a second size range, and the first size range is not the second size range.

[0374] Embodiment 14. The method of Embodiment 13, wherein: the plurality of structures is a first plurality of structures, the method further comprising: flowing a third portion of the first mixture from the reactor through a third fluidic path defined by a second plurality of structures in the microfluidic chip, wherein the second plurality of structures comprises different structures than the first plurality of structures.

[0375] Embodiment 15. The method of Embodiment 14, further comprising: coupling a third subset of the plurality of label compounds to the second plurality of beads in the third fluidic path, and measuring a third amount of the third subset of the plurality of label compounds.

[0376] Embodiment 16. The method of Embodiment 15, further comprising: removing a fourth subset of the plurality of label compounds from the second portion of the first mixtureto form a second mixture comprising the second plurality of beads, wherein measuring the third amount of the third subset of the plurality of label compounds is in the second mixture.

[0377] Embodiment 17. The method of Embodiment 12, wherein: the plurality of label compounds is a plurality of first label compounds, a plurality of second label compounds is configured to bind to the second analyte, and the plurality of second label compounds is different from the plurality of first label compounds, the method further comprising: coupling a first subset of the plurality of second label compounds to the second plurality of beads, and measuring an amount of the first subset of the plurality of second label compounds coupled to the second plurality of beads.

[0378] Embodiment 18. The method of Embodiment 1, wherein: the analyte is a nucleic acid molecule, and the affinity reagent is an oligonucleotide comprising a sequence of nucleotides complementary to a portion of the nucleic acid molecule.

[0379] Embodiments may include methods with one-step or two-step conjugation.

[0380] Embodiment 19. A method for analyzing reaction kinetics of a binding reaction, the method comprising: mixing a first plurality of beads with a sample to form a mixture in a reactor of a microfluidic chip, wherein: the sample comprises a plurality of analytes and a plurality of label compounds, each bead of the first plurality of beads is coupled to an affinity reagent, the affinity reagent is configured to bind to the analyte, and the plurality of label compounds is configured to bind to the analyte; binding the plurality of analytes to a plurality of affinity reagents coupled to the first plurality of beads in the reactor; coupling a first subset of the plurality of label compounds to the first plurality of beads; flowing a first portion of the mixture from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip, wherein: the first portion of the mixture comprises the first plurality of beads coupled to the first subset of the plurality of label compounds; forming a second mixture by flowing a solution in a second fluidic path, wherein the second fluidic path intersects the first fluidic path; and measuring an amount of the first subset of the plurality of label compounds in the second mixture.

[0381] Embodiment 20. The method of Embodiment 19, wherein coupling the first subset of the plurality of label compounds to the first plurality of beads occurs in the first fluidic path.

[0382] Embodiment 21. The method of Embodiment 19, wherein coupling the first subset of the plurality of label compounds to the plurality of analytes occurs before binding the plurality of analytes to the plurality of affinity reagents.

[0383] Embodiments may include one-step conjugation.

[0384] Embodiment 22. A method for analyzing a sample in a microfluidic chip, comprising a reactant reservoir comprising beads, a first affinity agent capable of coupling to the bead surface and the sample, wherein at least one analyte in the sample is capable of coupling to the first affinity agent, further a second affinity agent coupled to a label wherein the second affinity agent is also able to couple to the at least first analyte and at least one solution reservoir wherein the microfluidic chip comprising a zone with an array of structures, the array of structures comprises a plurality of parallel rows of structures and a plurality of parallel columns of structures, the microfluidic chip has a first longitudinal axis, the array of structures has a second longitudinal axis, and the second longitudinal axis is offset from the first longitudinal axis and wherein the first reactant reservoir and the second reactant reservoir are in communication with the zone of array of structures through a communication structure (or port structure) and wherein the communication structure is configured to design and / or control the flow paths of liquids from the reservoirs; the method comprising: forming in the reactant reservoir at least one sandwich complex on at least one bead surface by coupling at least one bead, at least one first affinity agent, at least one first analyte, at least one second affinity agent with the label with each other, flowing the mixture of the first reactant reservoir through the communication structure into the array of structures, wherein the beads gets displaced laterally and intersect with the solution flowing into the structure of arrays through the communication structure of the solution reservoir and detecting the label on the bead when they leave the structure of arrays.

[0385] Embodiments may include determining reaction kinetic parameter with the reaction time and the value of a signal.

[0386] Embodiment 23. A method for analyzing reaction kinetics of a binding reaction, the method comprising: receiving data including a reaction time and value of a signal reflecting an amount of a first subset of a plurality of label compounds, wherein the value of the signal is obtained by: mixing a first plurality of beads with a sample to form a mixture in a reactor of a microfluidic chip, wherein: the sample comprises a plurality of analytes and the plurality of label compounds, each bead of the first plurality of beads is coupled to an affinity reagent,the affinity reagent is configured to bind to the analyte, and the plurality of label compounds is configured to bind to the analyte; binding the plurality of analytes to a plurality of affinity reagents coupled to the first plurality of beads in the reactor; coupling the first subset of the plurality of label compounds to the first plurality of beads; flowing a first portion of the mixture from the reactor through a first fluidic path defined by a plurality of structures in the microfluidic chip, wherein: the first portion of the mixture comprises the first plurality of beads coupled to the first subset of the plurality of label compounds; forming a second mixture by flowing a solution in a second fluidic path, wherein the second fluidic path intersects the first fluidic path; and measuring the value of the signal of the first subset of the plurality of label compounds in the second mixture; and determining a reaction kinetic parameter using the reaction time and the value of the signal.

[0387] Embodiment 24. The method of Embodiment 23, wherein determining the reaction kinetic parameter comprises: inputting the reaction time and the value of the signal into an equation relating reaction time and value of the signal to the reaction kinetic parameter.

[0388] Embodiment 25. The method of Embodiment 24, wherein the reaction kinetic parameter is an amount of the affinity reagent in the sample.

[0389] Embodiment 26. The method of Embodiment 23, wherein determining the reaction kinetic parameter comprises: comparing the value of the signal at the reaction time to a calibration value at the reaction time, wherein the calibration value is determined from one or more calibration samples having known values of the reaction kinetic parameter.

[0390] Embodiments may include enriching or filtering for certain molecules.

[0391] Embodiment 27. A method for enriching target nucleic acid molecules, the method comprising: mixing a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules with a first plurality of beads, wherein: each target nucleic acid molecule of the plurality of target nucleic acid molecules comprises a target sequence, each bead of the first plurality of beads is coupled to a respective oligonucleotide, and the oligonucleotide comprises a first sequence complementary to the target sequence, binding each target nucleic acid molecule of the plurality of target nucleic acid molecules to the respective oligonucleotide coupled to a bead of the first plurality of beads to form a coupled first plurality of beads; transferring a mixture comprising the coupled first plurality of beads and the plurality of non-target nucleic acid molecules to a first reservoir of a microfluidic chip; flowing a first portion of the mixture from the reservoir through a first fluidic pathdefined by a first plurality of structures in the microfluidic chip, wherein the first portion of the mixture comprises the coupled first plurality of beads and the plurality of non-target nucleic acid molecules; forming a second mixture by flowing a solution in a second fluidic path, wherein the second fluidic path intersects the first fluidic path; removing, using the solution, the plurality of non-target nucleic acid molecules from the coupled first plurality of beads in the first fluidic path; and flowing the coupled first plurality of beads to a second reservoir at an end of the first fluidic path.

[0392] Embodiment 28. The method of Embodiment 27, further comprising separating a plurality of double-stranded nucleic acid molecules to form the plurality of target nucleic acid molecules.

[0393] Embodiment 29. The method of Embodiment 27, further comprising: removing the coupled first plurality of beads from the second reservoir, and for each bead of the coupled first plurality of beads, separating the respective oligonucleotide and the respective target nucleic acid molecule from the bead.

[0394] Embodiment 30. The method of Embodiment 29, further comprising amplifying the plurality of target nucleic acid molecules to form an amplified plurality of target nucleic acid molecules after the separating.

[0395] Embodiment 31. The method of Embodiment 29, wherein: each bead of the first plurality of beads is coupled to the respective oligonucleotide by a uracil, separating the respective oligonucleotide and the respective target nucleic acid molecule from each bead of the coupled first plurality of beads comprises using an enzyme to cleave at the uracil.

[0396] Embodiment 32. The method of Embodiment 27, wherein removing the plurality of non-target nucleic acid molecules comprises flowing the plurality of non-target nucleic acid molecules to a third reservoir on the microfluidic chip.

[0397] Embodiment 33. The method of Embodiment 27, wherein: the plurality of target nucleic acid molecules is a plurality of first target nucleic acid molecules, the oligonucleotide is a first oligonucleotide, and the plurality of non-target nucleic acid molecules is a first plurality of non-target nucleic acid molecules, the method further comprising: mixing a plurality of second target nucleic acid molecules and the plurality of non-target nucleic acid molecules with a second plurality of beads, wherein: each second target nucleic acid molecule of the plurality of second target nucleic acid molecules comprises a second targetsequence, each bead of the second plurality of beads is coupled to a respective second oligonucleotide, and the second oligonucleotide comprises a second sequence complementary to the second target sequence, binding each second target nucleic acid molecule of the plurality of second target nucleic acid molecules to the respective second oligonucleotide coupled to a bead of the second plurality of beads to form a coupled second plurality of beads, flowing a second portion of the mixture from the reservoir through a third fluidic path defined by a second plurality of structures in the microfluidic chip, wherein the second plurality of structures comprises different structures than the first plurality of structures, wherein: the mixture further comprises the coupled second plurality of beads, and the second portion of the mixture comprises the coupled second plurality of beads and a second plurality of non-target nucleic acid molecules.

[0398] Embodiment 34. The method of Embodiment 33, further comprising: forming a third mixture by flowing the solution in a fourth fluidic path, wherein the fourth fluidic path intersects the third fluidic path, removing, using the solution, the second plurality of nontarget nucleic acid molecules from the coupled second plurality of beads in the third fluidic path, and flowing the coupled second plurality of beads to a third reservoir at an end of the third fluidic path.

[0399] Embodiment 35. The method of Embodiment 27, wherein: the first plurality of structures comprises a plurality of pillars, the plurality of pillars is a portion of an array of pillars, the array of pillars is characterized by a plurality of rows and a plurality of columns, and the plurality of pillars comprises pillars from at least five columns from the plurality of columns.

[0400] Embodiment 36. The method of Embodiment 27, wherein: the microfluidic chip has a longitudinal axis, and the second fluidic path is parallel to the longitudinal axis.

[0401] Embodiment 37. A method for enriching target analytes, the method comprising: mixing a plurality of target analytes and a plurality of non-target analytes with a first plurality of beads, wherein: each bead of the first plurality of beads is coupled to a respective affinity reagent, and the affinity reagent is configured to bind to the target analyte and to not bind to the non-target analyte, binding each target analyte of the plurality of target analytes to the respective affinity reagent coupled to a bead of the first plurality of beads to form a coupled first plurality of beads; transferring a mixture comprising the coupled first plurality of beads and the plurality of non-target analytes to a first reservoir of a microfluidic chip; flowing afirst portion of the mixture from the reservoir through a first fluidic path defined by a first plurality of structures in the microfluidic chip, wherein the first portion of the mixture comprises the coupled first plurality of beads and the plurality of non-target analytes; forming a second mixture by flowing a solution in a second fluidic path, wherein the second fluidic path intersects the first fluidic path; removing, using the solution, the plurality of non-target analytes from the coupled first plurality of beads in the first fluidic path; and flowing the coupled first plurality of beads to a second reservoir at an end of the first fluidic path.

[0402] Embodiments may include performing a reaction. Methods may be similar to other methods except the label compound may be optional.

[0403] Embodiment 38. A method for performing a reaction with target analytes, the method comprising: mixing a plurality of target analytes with a plurality of beads, wherein: each bead of the plurality of beads is coupled to a respective affinity reagent, and the affinity reagent is configured to bind to the target analyte, binding each target analyte of the plurality of target analytes to the respective capture agent coupled to a bead of the plurality of beads to form a coupled plurality of beads; transferring a mixture comprising the coupled plurality of beads to a first reservoir of a microfluidic chip; flowing a first portion of the mixture from the reservoir through a first fluidic path defined by a first plurality of structures in the microfluidic chip, wherein the first portion of the mixture comprises the coupled plurality of beads, wherein flowing the first portion of the mixture comprises flowing the coupled plurality of beads sequentially through the first fluidic path; flowing a plurality of reactants in a second fluidic path, wherein the second fluidic path intersects the first fluidic path; reacting the plurality of reactants with the plurality of target analytes of the coupled plurality of beads to form a plurality of reacted beads; and flowing the plurality of reacted beads to a second reservoir at an end of the first fluidic path.

[0404] Embodiment 39. The method of Embodiment 38, wherein: the plurality of reactants is a first plurality of reactants, and flowing the first plurality of reactants comprises flowing a second plurality of reactants, further comprising: removing the second plurality of reactants from the plurality of reacted beads in the first fluidic path.

[0405] Embodiment 40. The method of Embodiment 39, further comprising: flowing a solution in a third fluidic path, wherein the third fluidic path intersects the first fluidic path,wherein flowing the solution through the first fluidic path removes the second plurality of reactants from the plurality of reacted beads in the first fluidic path.

[0406] Embodiment 41. The method of Embodiment 38, further comprising: coupling a plurality of label compounds to the plurality of reactants, and measuring an amount of the plurality of label compounds coupled to the plurality of reactants.

[0407] Embodiment 42. The method of Embodiment 41, wherein measuring the amount of the plurality of label compounds coupled to the plurality of reactants comprises measuring the amount at a plurality of locations along the first fluidic path.

[0408] Embodiment 43. The method of Embodiment 38, wherein: the reactant is a first reactant, and the plurality of reacted beads is a plurality of first reacted beads, the method further comprising: flowing a plurality of second reactants in a third fluidic path, wherein the third fluidic path intersects the first fluidic path, and reacting the plurality of second reactants with the plurality of target analytes of the coupled plurality of beads to form a plurality of second reacted beads.

[0409] Embodiment 44. The method of Embodiment 43, wherein the second fluidic path is the same as the third fluidic path.

[0410] Embodiment 45. A method for performing a chemical reaction in a microfluidic chip comprising: at least a first reactant reservoir comprising a plurality of beads in a liquid and at least a second reactant reservoir comprising at least one reactant in a liquid wherein the reactant is capable of reacting with the bead surface or components attached to the bead surface, wherein the microfluidic chip comprises a zone with an array of structures, the array of structures comprises a plurality of parallel rows of structures and a plurality of parallel columns of structures, the microfluidic chip has a first longitudinal axis, the array of structures has a second longitudinal axis, and the second longitudinal axis is offset from the first longitudinal axis and wherein the first reactant reservoir and the second reactant reservoir are in communication with the zone of array of structures through a communication structure (or port structure) and wherein the communication structure is configured to design and / or control the flow paths of liquids from the reservoirs; wherein the beads gets displaced laterally and intersect with the reactant flowing vertically along the first longitudinal axis of the microfluidic chip and wherein the beads react with the reactant while the beads intersect with the vertical flow path of the reactant solution.

[0411] Embodiment 46. The method of Embodiment 45, wherein the chemical reaction is used in the analysis of analytes in a sample bead.

[0412] Embodiment 47. The method of Embodiment 45, further comprising detecting the bead after exiting the array of structures.

[0413] Embodiment 48. A cartridge comprising: a plurality of structures defining a first fluidic path, a reactant reservoir, a solution reservoir, and a manifold configured to deliver a reactant from the reactant reservoir to intersect the first fluidic path and a solution from the solution reservoir to intersect the first fluidic path, wherein: the cartridge is configured to reversibly connect with a reusable device to form a connected microfluidic chip system, the reusable device comprises a reactor, and the reactor is in fluid communication with the first fluidic path in the connected microfluidic chip system.

[0414] Embodiment 49. The cartridge of Embodiment 48, further comprising: a reactant disposed in the reactant reservoir, and a solution disposed in the solution reservoir.

[0415] Embodiment 50. A kit comprising the cartridge of Embodiment 48 and the reusable device comprising the detector and connectivity means / ports to external devices.

[0416] Embodiment 51. The kit of Embodiment 50, wherein the detector is part of the cartridge.

[0417] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0418] The above description of example embodiments of the present disclosure has been presented for the purposes of illustration and description and are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure. It is not intended to be exhaustive or to limit the disclosure to the precise form described nor are they intended to represent that the experiments are all or the only experiments performed. Although the disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of theteachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0419] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the disclosure being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

[0420] A recitation of “a”, “an” or “the” is intended to mean “one or more” unless specifically indicated to the contrary. The use of “or” is intended to mean an “inclusive or,” and not an “exclusive or” unless specifically indicated to the contrary. Reference to a “first” component does not necessarily require that a second component be provided. Moreover, reference to a “first” or a “second” component does not limit the referenced component to a particular location unless expressly stated. The term “based on” is intended to mean “based at least in part on.”

[0421] The claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only”, and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0422] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within embodiments of the presentdisclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0423] All patents, patent applications, publications, and descriptions mentioned herein are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. None is admitted to be prior art.

Claims

WHAT IS CLAIMED IS:

1. A system comprising: a microfluidic chip, wherein the microfluidic chip comprises: a reactor with a reactor outlet, an array of structures comprising a plurality of structures defining a first path, wherein: the microfluidic chip has a first longitudinal axis, the array of structures has a second longitudinal axis, the second longitudinal axis is offset from the first longitudinal axis, and the first path is in communication with the reactor outlet, a reactant reservoir, a solution reservoir, a manifold configured to deliver a reactant from the reactant reservoir to intersect the first path and to deliver a solution from the solution reservoir to intersect the first path; and a collection reservoir with a collection reservoir inlet in communication with the first path.

2. The system of claim 1, further comprising a detector configured to detect a label compound traveling to the collection reservoir.

3. The system of claim 1, wherein: the array of structures comprises a plurality of parallel rows of structures and a plurality of parallel columns of structures.

4. The system of claim 1, further comprising: a plurality of beads disposed on the microfluidic chip, wherein: each bead of the plurality of beads has a diameter smaller than a width of the first path, and each bead of the plurality of beads is bound to a first affinity reagent.

5. The system of claim 4, further comprising: an imaging detector, and a plurality of label compounds disposed in the reactant reservoir, wherein:the label compounds are not coupled to the plurality of beads, each label compound comprises a second affinity reagent, the imaging detector is configured to detect the label compound traveling to the collection reservoir, and the reactant is the label compound.

6. The system of claim 5, further comprising: a plurality of analytes disposed in the reactor, a plurality of first affinity reagents configured to bind to the plurality of analytes, and a plurality of second affinity reagents is configured to bind to the plurality of analytes.

7. The system of claim 6, wherein the plurality of second affinity reagents is disposed in the reactant reservoir.

8. The system of claim 1, wherein the reactor comprises an agitator.

9. The system of claim 2, wherein the detector is integrated into the microfluidic chip, and the detector comprises an application-specific integrated circuit.

10. The system of claim 2, wherein the detector comprises a lens and a laser.

11. The system of claim 1, wherein the manifold is further configured to deliver a component in addition to the reactant and the solution to intersect the first path.

12. The system of claim 1, further comprising: one or more first membranes separating the plurality of structures from the reactor, the reactant reservoir, and the solution reservoir, wherein the one or more first membranes are configured to be removable such that removal initiates flow of liquids from the reactor, the reactant reservoir, and the solution reservoir to the first path.

13. The system of claim 12, further comprising a pump, wherein the flow is driven by a pump.

14. The system of claim 12, further comprising: a second membrane separating the plurality of structures from the collection reservoir.

15. The system of claim 14, wherein the second membrane is configured to be removable such that removal initiates capillary force-driven flow of liquids from the array of structures to the collection reservoir.

16. The system of claim 1, further comprising a pump configured to drive a flow from the reactor to the first path.

17. The system of claim 1, further comprising an inert liquid disposed in the reactor.

18. The system of claim 1, further comprising: a sample reservoir configured to receive a blood sample, and a filter separating the sample reservoir from at least one of the reactor and the reactant reservoir, wherein the filter is configured to retain red blood cells and pass plasma from the blood sample to at least one of the reactor and the reactant reservoir.

19. The system of claim 1, wherein: the reactor outlet is configured to be in an open position or closed position, and the first path is in communication with the reactor when the reactor outlet is in the open position.

20. A method for performing a reaction in a microfluidic chip, the method comprising: releasing a plurality of beads from a first reservoir to an array of structures in the microfluidic chip; displacing the plurality of beads laterally from a first longitudinal axis of the microfluidic chip in the array of structures; flowing a reactant from a second reservoir to intersect with the plurality of beads displaced laterally from the first longitudinal axis; andreacting the reactant with the plurality of beads or with a component attached to each bead of the plurality of beads.

21. The method of claim 20, wherein: the array of structures comprises a plurality of parallel rows of structures and a plurality of parallel columns of structures, the array of structures is characterized by a second longitudinal axis, and the second longitudinal axis is offset from the first longitudinal axis.

22. The method of claim 20, wherein releasing the plurality of beads from the first reservoir comprises releasing the plurality of beads in a direction parallel to the first longitudinal axis.

23. The method of claim 20, wherein flowing the reactant from the second reservoir comprises flowing the reactant from the second reservoir in a direction parallel to the first longitudinal axis.

24. The method of claim 20, wherein: the first reservoir comprises a plurality of analytes, each bead of the plurality of beads is coupled to a plurality of affinity reagents in the first reservoir, the affinity reagent is configured to bind to the analyte, and the reactant comprises a plurality of label compounds, the method further comprising: binding a first subset of the plurality of analytes to a first subset of the plurality of affinity reagents coupled to the plurality of beads in the first reservoir, wherein reacting the reactant is with the component attached to each bead of the plurality of beads, and wherein the component is the analyte.

25. The method of claim 24, wherein: the analyte is an antigen, the affinity reagent is an antibody, and the label compound comprises a fluorescent label.

26. The method of claim 24, further comprising:flowing the plurality of beads out of the array of structures, and detecting the plurality of label compounds on the plurality of beads outside of the array of structures.

27. The method of claim 26, further comprising measuring an amount of label compounds on each bead of the plurality of beads.

28. The method of claim 26, where detecting the plurality of label compounds comprises detecting each bead of the plurality of beads sequentially after the respective bead flows out of the array of structures.

29. The method of claim 24, wherein: the plurality of analytes is a first plurality of analytes, the first reservoir comprises a second plurality of analytes, and displacing the plurality of beads laterally from the first longitudinal axis comprises separating the second plurality of analytes from the plurality of beads.

30. The method of claim 29, further comprising: flowing a solution from a third reservoir to the array of structures along the first longitudinal axis to intersect with the plurality of beads.

31. The method of claim 20, wherein: the reactant is a first reactant, the method further comprising: mixing a plurality of second reactants with the plurality of beads, wherein: each bead of the plurality of beads is coupled to a respective affinity reagent, and the affinity reagent is configured to bind to the first reactant, and binding each second reactant of the plurality of second reactants to the respective affinity reagent coupled to a bead of the plurality of beads, wherein reacting the first reactant comprises reacting the first reactant with the second reactant attached to each bead of the plurality of beads.

32. The method of claim 31, wherein:the plurality of second reactants is a first plurality of second reactants, the first reservoir comprises a second plurality of second reactants, and displacing the plurality of beads laterally from the first longitudinal axis comprises separating the second plurality of second reactants from the plurality of beads.

33. The method of claim 32, further comprising: flowing a solution from a third reservoir to the array of structures along the first longitudinal axis to separate the second plurality of second reactants from the plurality of beads.

34. The method of claim 31, wherein the first reactant comprises a label compound, and the method further comprises detecting the label compound after the plurality of beads exits the array of structures.

35. The method of claim 20, further comprising detecting each bead of the plurality of beads after exiting the array of structures.

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