Full-automatic biochemical measuring instrument system

Abstract

The invention discloses an instrument for a full-automatic table type NULISA platform and a related method. The instrument comprises an X-Y-Z portal frame, a microtiter plate objective table, an incubator, a quantitative qPCR module, a microtiter plate decontamination cleaner, a microtiter plate sealing device, a magnetic probe, a comb-shaped assembly, a reagent and consumable storage unit and a controller, wherein the magnetic probe and the comb-shaped assembly are used for sample mixing and magnetic bead extraction.

Description

Cross-referencing related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 501,355, filed May 10, 2023, which is incorporated herein by reference in its entirety.

[0002] In addition, all publications and patent applications mentioned in this specification are incorporated herein by reference. Background Technology

[0003] Biochemical assays use a set of reagents and follow a predetermined procedure to detect one or more target analytes in a biological sample. There is a strong demand for automated assay runs using instrument systems. Most such instruments on the market are designed to run a specific assay. Alternatively, users may need to purchase and install multiple off-the-shelf instruments, each performing a specific assay procedure (such as liquid transfer, plate washing, etc.), and integrate them into a "work unit" using a robotic arm. Such systems are typically custom-designed and integrated by the end-user, often resulting in bulky, unreliable systems that require significant maintenance effort.

[0004] To improve operational efficiency, multiple samples are typically measured in parallel on a support plate with multiple containers. Standard microtiter plates with 24 wells, 96 wells, and 384 wells are the most widely used measurement plates. For complex measurements involving multiple steps, a set of measurement plates is usually used in a single measurement run.

[0005] This invention discloses a compact instrument system designed for performing assays based on NULISA technology (WO2021113290). Because it includes functional modules capable of performing most key procedures for biochemical assays, it can be reconfigured to run a wide range of assays on microtiter plates in a fully automated manner.

[0006] Immunoassays are bioanalytical assays that follow a predetermined procedure to detect one or more target analytes in a biological sample. For example, enzyme-linked immunosorbent assay (ELISA) is a technique used to detect and quantify the presence of specific analytes, such as proteins, peptides, antibodies, and antigens. However, current immunoassays have limited performance in detecting bioanalytes such as proteins, and their limited sensitivity remains a technical bottleneck in many applications.

[0007] In traditional ELISA, plates or wells coated with capture antibodies are used to immobilize the target antigen in the sample. In magnetic bead-based ELISA, the capture antibody can be attached to the magnetic beads.

[0008] Using magnetic beads can improve sensitivity because they offer higher binding capacity. Magnetic beads also save end-user time, as bead-based ELISA typically requires shorter incubation times. Furthermore, magnetic beads are easily automated, making assays using them ideal for high-throughput applications.

[0009] Another way to improve the sensitivity of immunoassays is to use antibody sandwich pairs. However, other immunoassay techniques (such as immunoPCR, proximity linkage assay (PLA), proximity extension assay (PEA), single-molecule array (SIMOA), and single-molecule counting (SMC)) are still insufficient for analyzing low-abundance fractions of the proteome due to their limited sensitivity.

[0010] Nucleic acid ligation-mediated immune sandwich assay (NULISA) improves upon existing immunoassay techniques by employing various background inhibition mechanisms, such as the sequential capture / release mechanism based on paramagnetic beads described in U.S. Patent Application No. 17 / 330,331, the entire contents of which are incorporated herein by reference.

[0011] Furthermore, immunoassays typically involve multiple manual steps, which are time-consuming and can introduce costly errors (such as contamination or human error). Automating immunoassay procedures through instruments can improve operational efficiency and minimize contamination, impurities, and errors. To meet the growing testing demands, there is a need to promote the semi-automation or full automation of immunoassay procedures.

[0012] Semi-automation of multi-step immunoassays combines manual operation with automated instrumentation. It may require significant use of consumables (such as plates, combs, pipette tips, and reagents) to minimize contamination and can achieve high sensitivity for low concentrations of analytes. For example, ThermoFisher's KingFisher Flex system is an automated extraction instrument for the extraction and purification of nucleic acids and proteins after manual sample and plate preparation. While semi-automation eliminates some time-consuming manual steps, critical manual steps remain (e.g., manual plate transfer or manual start-up of the next automated instrument), which may ultimately limit the efficiency of the procedure.

[0013] Alternatively, automated workstations can further improve operational efficiency and sensitivity by almost completely reducing manual operations. In some cases, it has been explored to build automated immunoassay workstations using multiple dedicated instruments, each capable of performing specific procedures (such as liquid transfer, plate washing, etc.), and integrating them into a workstation using a robotic arm. For example, Beckman Coulter's Biomek workstation is a single-module workstation that supports tool interchangeability. These automated workstations are typically custom-configured and integrated by end-users, resulting in large sizes and high maintenance requirements. Operating and implementing specific procedures on these custom-designed workstations is another significant drawback for end-users.

[0014] The large size of these automated workstations also presents challenges for laboratory space layout. For example, the SIMOA HD-1 analyzer is a floor-standing instrument with a large footprint, measuring 55.7 × 35.3 × 63.5 inches. In contrast, benchtop workstations are compact, portable, and designed to be placed on laboratory workbenches, making them suitable for both small and large laboratory settings.

[0015] This invention discloses a compact instrument system designed to perform a multi-step, highly sensitive immunoassay based on NULISA technology (US Application No. 17 / 330,331) involving a sequential capture / release mechanism. Summary of the Invention

[0016] Implementation Scheme 1. A compact, fully automated instrument for performing dual capture and release multiplex immunoassays, comprising: A) Desktop chassis, including: 1) Controller; 2) A mechanical gantry crane capable of moving in three degrees of freedom; 3) A storage rack that accommodates multiple container support plates, accessible to both the user and the mechanical gantry; 4) Provides a controllable stage with multiple multi-container support plate positions, wherein the stage can move along the Y-axis (from front to back within the instrument housing). 5) A mixer, which serves as both a magnetic bead processor and a mixer, comprising multiple multi-container carrier plate platforms stacked vertically to each other; 6) A washer for cleaning multi-container support plates; and 7) Reader.

[0017] Implementation Scheme 2. A compact instrument designed to perform a multi-step, highly sensitive immunoassay involving a sequential capture / release process, comprising: A) Desktop chassis with a touchscreen display, including: 1) Controller; 2) A mechanical gantry crane capable of moving in three degrees of freedom; 3) A storage rack for accommodating multi-container carrier plates that can be accessed by both the user and the mechanical gantry; 4) A controllable stage that provides multiple positions for multi-container carrier plates, where the stage can move along the Y-axis (from front to back within the instrument housing); 5) A mixer that functions as a magnetic bead processor and a mixer, including multiple multi-container carrier plate platforms stacked vertically with respect to each other; 6) A washer for cleaning multi-container carrier plates; 7) An incubation and sealing device for incubating and sealing at least one multi-container carrier plate; and 8) A reader.

[0018] Embodiment 3. A fully automatic high-throughput precision proteomics instrument for performing ultra-high sensitivity analysis at a series of multiplex assay levels to support a wide range of biomarker profiling and the translation of validated biomarkers, including: A) A desktop housing with a touchscreen display, a user-accessible large-capacity reagent chamber, and a set of user-accessible compartment chambers, the desktop housing including: 1) A controller; 2) A controllable mechanical gantry equipped with an end effector, the end effector including a pipette, a multi-container carrier plate clamp, a laser position sensor, and a barcode scanner, the mechanical gantry enabling the end effector to move in three degrees of freedom along the X, Y, and Z axes; 3) A storage rack including a set of multi-container carrier plate compartments for receiving and accommodating at least one universal reagent kit and a consumable carrier device; 4) A controllable stage that provides multiple positions for multi-container carrier plates, where the stage can move along the Y-axis (from front to back within the instrument housing); 5) A controllable mixer that functions as a magnetic bead processor and a mixer, including multiple multi-container carrier plate platforms stacked vertically with respect to each other; 6) A controllable washer for cleaning multi-container carrier plates; 7) An incubation and sealing device for incubating and sealing at least one multi-container carrier plate; 8) A large-capacity fluid station including multiple containers; and 9) A controllable reader.

[0019] Embodiment 4. A desktop instrument for automatically performing multiple multiplex oligonucleotide-conjugated antibody proximity ligation assays in parallel on multiple biological samples, including: A) A desktop housing with a touchscreen display, a user-accessible large-capacity reagent chamber, and a set of user-accessible compartment chambers, the desktop housing including: 1) A controller; 2) A mechanical gantry equipped with an end effector, which includes a pipette, a multi-container support plate clamp, a laser position sensor, and a barcode scanner. The mechanical gantry enables the end effector to move in three degrees of freedom: X, Y, and Z. 3) Storage rack, comprising a set of multi-container carrier plate compartments for receiving and accommodating at least one general-purpose reagent kit and consumable carrier device; 4) Provides a stage with multiple multi-container support plate positions, wherein the stage can move along the Y-axis (from front to back within the instrument housing); 5) A mixer, which serves as both a magnetic bead processor and a mixer, comprising multiple multi-container carrier plate platforms stacked vertically to each other; 6) A washer for cleaning multi-container support plates; 7) Reader.

[0020] Implementation Scheme 5. In some implementation schemes, such as those including Schemes 1-4 and any other embodiments herein, at least one multi-container support plate includes a set of pre-selected paired binding portions for binding a particular analyte, wherein the paired binding portions include: (i) A first portion comprising a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker comprising a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and (ii) The second part includes a second antibody or second antibody fragment pre-selected for binding to a specific analyte that is the same as the first antibody or antibody fragment of the first part of the paired binding portion, a second nucleic acid target marker containing a second identifier that is specific to the specific analyte, and a second nucleic acid tag.

[0021] Implementation Scheme 6. A benchtop automated instrument for performing biochemical assays on biological samples containing multiple analytes, comprising: a) Controller; b) Controllable gantry crane; c) Controllable platform; d) A set of 5 multi-container support plates; e) Controlled quantitative PCR unit; f) Controllable plate washer; g) Controllable plate sealing device; h) Controlled-plate incubator; i) A controllable mixer, including a paramagnetic bead extractor containing magnetic poles and comb-like components; j) Storage racks accessible from the gantry, including: 1) Multiple first parts, comprising a first antibody or capture antibody fragment conjugated with a first nucleic acid tag, the first nucleic acid tag hybridizing with a first nucleic acid target marker and not covalently attached to the first antibody or first antibody fragment; 2) Multiple second parts, comprising a second antibody or second antibody fragment conjugated with a second nucleic acid target marker, wherein the second nucleic acid target marker hybridizes with a second nucleic acid tag and is not covalently attached to the second antibody or second antibody fragment; 3) First group, first base; 4) Second group, second basement; 5) A set of buffers; 6) Multiple double-stranded nucleic acid sample markers, including 5' and 3' protrusions; 7) Frames containing sealing membranes; and k) A controller, including a processor and a non-transitory machine-readable storage medium containing instructions executable by the processor to provide controlled operation of unit operations within the instrument, the operations controlled by the controller including: 1) A first solution is formed in the first multi-container support plate by mixing the following substances: i) at least a portion of a sample, ii) a portion of multiple first parts, and iii) One of multiple parts of Part II; 2) Incubate the first solution to form an immune complex comprising one of a plurality of first portions, one of a plurality of second portions, and at least one analyte from the sample; 3) By introducing the following substances, a sequential capture and release mechanism is implemented for immune complexes to remove contaminants and improve sensitivity: i) A portion of the first group of first substrates is used to capture at least a portion of immune complexes from a first solution in a first multi-container support plate, wherein the first solution in the first multi-container support plate is incubated, and the first group of paramagnetic beads is transferred to a second multi-container support plate by a mixer to form a second solution, thereby releasing at least a portion of the immune complexes, and the first group of first substrates is removed by a mixer; ii) A portion of the second set of second substrates to capture at least a portion of the immune complex from the second solution in the second multi-container plate, wherein the second solution is incubated, and the second set of second substrates is removed by a mixer to form a third solution in the third multi-container plate; 4) Introducing at least a portion of a plurality of double-stranded nucleic acid sample markers into a third solution to form at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule comprising a first nucleic acid target marker associated with a first antibody in at least one immune complex and at least one second nucleic acid target marker conjugated with a second antibody in at least one immune complex, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of a first antibody in the immune complex through one protrusion and with the second nucleic acid target marker conjugated with at least a portion of a second antibody in the immune complex through another protrusion, to complete the proximity connection between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one multiplex analyte-specific reporter molecule conjugated to a second antibody or a second antibody fragment and not covalently attached to a first antibody or a first antibody fragment; 5) At least one multiplex analyte-specific reporter molecule is collected in the fourth multi-container support plate, and then the multiplex analyte-specific reporter molecules are combined to form a pooled sample for the detection of analytes by NGS at atmolar level sensitivity.

[0022] Implementation Scheme 7. A benchtop automated instrument for performing biochemical assays on biological samples containing multiple analytes, comprising: a) Controller; b) Controllable gantry crane; c) Controllable platform; d) A set of 5 multi-container support plates; e) Controlled quantitative PCR unit; f) Controllable plate washer; g) Controllable plate sealing device; h) Controlled-plate incubator; i) A controllable mixer, including a paramagnetic bead extractor containing magnetic poles and comb-like components; j) Storage racks accessible from the gantry, including: 1) Multiple first parts, comprising a first antibody or capture antibody fragment conjugated with a first nucleic acid tag, the first nucleic acid tag hybridizing with a first nucleic acid target marker and not covalently attached to the first antibody or first antibody fragment; 2) Multiple second parts, comprising a second antibody or second antibody fragment conjugated with a second nucleic acid target marker, wherein the second nucleic acid target marker hybridizes with a second nucleic acid tag and is not covalently attached to the second antibody or second antibody fragment; 3) First group, first base; 4) Second group, second basement; 5) A set of buffers; 6) Multiple double-stranded nucleic acid sample markers, including 5' and 3' protrusions; 7) Frames containing sealing membranes; and k) A controller for controlling unit operations of an instrument, the controller including a non-transitory computer-readable storage medium having an executable file executable by the instrument controller to cause unit operations of the instrument to perform operations, the instrument operations including: 1) A first solution is formed in the first multi-container support plate by mixing the following substances: i) at least a portion of a sample, ii) a portion of multiple first parts, and iii) One of multiple parts of Part II; 2) Incubate the first solution to form an immune complex comprising one of a plurality of first portions, one of a plurality of second portions, and at least one analyte from the sample; 3) By introducing the following substances, a sequential capture and release mechanism is implemented for immune complexes to remove contaminants and improve sensitivity: i) A portion of the first group of first substrates is used to capture at least a portion of immune complexes from a first solution in a first multi-container support plate, wherein the first solution in the first multi-container support plate is incubated, and the first group of paramagnetic beads is transferred to a second multi-container support plate by a mixer to form a second solution, thereby releasing at least a portion of the immune complexes, and the first group of first substrates is removed by a mixer; ii) A portion of the second set of second substrates to capture at least a portion of the immune complex from the second solution in the second multi-container support plate, wherein the second solution is incubated, and the second set of second substrates is removed by a mixer to form a third solution in the third multi-container support plate; 4) Introducing at least a portion of a plurality of double-stranded nucleic acid sample markers into a third solution to form at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule comprising a first nucleic acid target marker associated with a first antibody in at least one immune complex and at least one second nucleic acid target marker conjugated with a second antibody in at least one immune complex, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of a first antibody in the immune complex through one protrusion and with the second nucleic acid target marker conjugated with at least a portion of a second antibody in the immune complex through another protrusion, to complete the proximity connection between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one multiplex analyte-specific reporter molecule conjugated to a second antibody or a second antibody fragment and not covalently attached to a first antibody or a first antibody fragment; 5) At least one multiplex analyte-specific reporter molecule is collected in the fourth multi-container support plate, and then the multiplex analyte-specific reporter molecules are combined to form a pooled sample for detection of the analyte by NGS at a molar sensitivity.

[0023] Implementation Scheme 8. A benchtop automated instrument for performing biochemical assays to detect analytes in at least a first biological sample and a second biological sample, comprising: a) Gantry frame; b) Platform; c) Multiple multi-container support plates, including a first plate, a second plate, a third plate, and a fourth plate; d) Quantitative PCR unit; e) Plate washer; f) Plate sealing device; g) Plate incubator; h) A mixer, including a paramagnetic bead extractor containing magnetic poles and comb-like components; i) Storage racks, including: 1) Multiple first parts, comprising a first antibody or capture antibody fragment conjugated with a first nucleic acid tag, the first nucleic acid tag hybridizing with a first nucleic acid target marker and not covalently attached to the first antibody or first antibody fragment; 2) Multiple second parts, comprising a second antibody or second antibody fragment conjugated with a second nucleic acid target marker, wherein the second nucleic acid target marker hybridizes with a second nucleic acid tag and is not covalently attached to the second antibody or second antibody fragment; 3) First group, first base; 4) Second group, second basement; 5) A set of buffers; 6) Multiple double-stranded nucleic acid sample markers, including 5' and 3' protrusions; 7) Frames containing sealing membranes; and j) A controller, including a processor and a non-transitory machine-readable storage medium containing instructions executable by the processor to provide controlled operation of unit operations within the instrument, the operations controlled by the controller including: 1) For each of the first assay reaction containing the first biological sample and the second assay reaction containing the second biological sample, a first solution is formed in the first multi-container support plate by mixing the following substances: i) at least a portion of a sample, ii) a portion of multiple first parts, and iii) One of multiple parts of Part II; 2) Incubate the first solution of the first assay reaction and the second assay reaction to form immune complexes with the analyte; 3) To remove contaminants and improve sensitivity, a dual capture and release mechanism is implemented for the immune complexes in each of the first and second assays using the following methods: i) Introduce a portion of the first group of first substrates into each of the first assay reaction and the second assay reaction to capture at least a portion of the immune complex from the first solution in the first multi-container support plate, wherein the first solution in the first multi-container support plate is incubated, and the first group of paramagnetic beads is transferred to the second multi-container support plate by a mixer to form a second solution, thereby releasing at least a portion of the immune complex, and the first group of first substrates is removed by a mixer. ii) Introduce a portion of the second set of second substrates into each of the first assay reaction and the second assay reaction to capture at least a portion of the immune complex from the second solution in the second multi-container plate, wherein the second solution is incubated, and remove the second set of second substrates by a mixer to form a third solution in the third multi-container plate; 4) Introduce at least a portion of a plurality of double-stranded nucleic acid sample markers into a third solution of each of the first and second assay reactions to form at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule comprising a first nucleic acid target marker associated with a first antibody in at least one immune complex and at least one second nucleic acid target marker conjugated to a second antibody in at least one immune complex, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of the first antibody in the immune complex through one protrusion and with the second nucleic acid target marker conjugated to at least a portion of the second antibody in the immune complex through another protrusion to complete the proximity linkage between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one multiplex analyte-specific reporter molecule conjugated to a second antibody or a second antibody fragment and not covalently attached to a first antibody or a first antibody fragment; 5) At least one multiplex analyte-specific reporter molecule is collected in the fourth multi-container support plate, and then the multiplex analyte-specific reporter molecule of the first assay reaction is combined with the multiplex analyte-specific reporter molecule of the second assay reaction to form a pooled sample for detection of the analyte by NGS at an amolar sensitivity.

[0024] Implementation Scheme 9. A method for parallel and automated determination of proximity linkages of multiple multiple oligonucleotide conjugated antibodies on multiple biological samples, comprising: A) In a first plate, a portion of a plurality of multiple pairwise binding moieties is introduced in parallel into a plurality of biological samples to form a plurality of immune complex forming solutions, wherein the multiple pairwise binding moieties comprise different pre-selected pairwise binding moieties for binding different specific analytes, the pairwise binding moieties including: i) The first portion of the paired binding portion includes a first antibody or first antibody fragment pre-selected for binding a specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second part of the pair-binding portion includes a second antibody or second antibody fragment pre-selected for binding a specific analyte that is the same as the first antibody or antibody fragment of the first part of the pair-binding portion, a second nucleic acid target marker containing a second identifier that is specific to the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first part of the pair binding portion is preselected for binding to the same first substrate for all different pair binding portions in multiplex assays, and the second nucleic acid tag of the second part of the pair binding portion is preselected for binding to the same second substrate for all different pair binding portions in multiplex assays; B) Parallel incubation of multiple immune complex formation solutions to form multiple multiple immune complexes; C) For the first time, a portion of a first substrate solution containing multiple first substrates is combined with multiple immune complex-forming solutions; D) The first nucleic acid tag of the first portion of the paired binding region binds to a portion of the first substrate for the first time; E) Firstly, multiple multiple immune complexes are extracted in parallel from multiple immune complex forming solutions through a portion of multiple first substrates to form multiple first immune complex purification solutions in a second plate; F) Multiple multiple immune complexes are eluted in parallel from multiple first immune complex purification solutions; G) Multiple first substrates are removed in parallel from multiple first immune complex purification solutions; H) The second step involves combining a portion of the second substrate solution containing multiple second substrates with multiple first immune complex purification solutions in parallel. I) The second nucleic acid tag of the second part of the paired binding portion binds to a part of the second substrate; J) A second step involves extracting multiple multiple immune complexes in parallel from multiple first immune complex purification solutions through a portion of multiple second substrates to form multiple second immune complex purification solutions in a third plate; K) Multiple first nucleic acid target markers (directly or indirectly) of the first part of the pair-binding part are linked in parallel with second nucleic acid target markers of the second part of the pair-binding part to form multiple multiplex analyte-specific reporter molecules; L) A third step involves extracting multiple multiple immune complexes (or multiple multiple analyte-specific reporter molecules) in parallel from multiple second immune complex purification solutions through a portion of multiple second substrates to form multiple third immune complex purification solutions in the second plate; M) Multiple multiple analyte-specific reporter molecules were further eluted in parallel from multiple third immune complex purification solutions; N) Replication of multiple multiplex analyte-specific reporter molecules; and O) Detect multiple replicated multiplex analyte-specific reporter molecules to identify (and quantify) specific analytes in multiple biological samples.

[0025] Implementation Scheme 10. A method for parallel and automated determination of proximity linkages of multiple multiple oligonucleotide conjugated antibodies on multiple biological samples, comprising: A) In a first multi-container carrier plate, a portion of a plurality of multiple multiple paired binding portions is introduced in parallel into a plurality of biological samples to form a plurality of immune complex forming solutions; The multiple pairwise binding portions include pre-selected different pairwise binding portions for binding different specific analytes, and these pairwise binding portions include: i) The first portion of the paired binding portion includes a first antibody or first antibody fragment pre-selected for binding a specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second part of the pair-binding portion includes a second antibody or second antibody fragment pre-selected for binding a specific analyte that is the same as the first antibody or antibody fragment of the first part of the pair-binding portion, a second nucleic acid target marker containing a second identifier that is specific to the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first part of the pair binding portion is preselected for binding to the same first substrate for all different pair binding portions in multiplex assays, and the second nucleic acid tag of the second part of the pair binding portion is preselected for binding to the same second substrate for all different pair binding portions in multiplex assays; B) Parallel incubation of multiple immune complex formation solutions to form multiple multiple immune complexes in a first multi-container support plate; C) For the first time, a portion of a first substrate solution containing multiple first substrates is combined with multiple immune complexes in a first multi-container carrier plate to form a solution combination; D) For the first time, the first nucleic acid tag of the first portion of the paired binding portion binds to a portion of the first substrate in the first multi-container carrier plate; E) For the first time, multiple multiple immune complexes are extracted in parallel from a first multi-container plate through a portion of multiple first substrates and transferred to a second multi-container plate; F) Clean the multiple multiple immune complexes in the second multi-container carrier plate; G) A second extraction of multiple multiple immune complexes from the second multi-container plate and transfer to the third multi-container plate; H) Multiple first substrates are eluted in parallel from multiple multiple immune complexes in a third multi-container carrier plate; I) Remove multiple first substrates in parallel from the third multi-container support plate; J) The second step involves combining a portion of a second substrate solution containing multiple second substrates with multiple multiple immune complexes in parallel with a third multi-container carrier plate; K) The second nucleic acid tag of the second portion of the paired binding portion is then bound to a portion of the second substrate of the third multi-container carrier plate; L) A third time, multiple multiple immune complexes are extracted in parallel from a third multi-container plate through a portion of multiple second substrates and transferred to a second multi-container plate; M) In the second multi-container carrier plate, multiple first nucleic acid target labels (directly or indirectly) of the first part of the paired binding portion on multiple multiple immune complexes are connected in parallel with second nucleic acid target labels of the second part of the paired binding portion to form multiple multiple analyte-specific reporter molecules; N) In the fourth step, multiple multiple immune complexes carrying analyte-specific reporter molecules are extracted in parallel from a second multi-container plate through a portion of multiple second substrates and transferred to a third multi-container plate; O) Multiple second substrates were eluted in parallel from multiple multiple immune complexes containing analyte-specific reporter molecules in a third multi-container carrier plate; P) Replication of multiple multiplex analyte-specific reporter molecules; and Q) Detect multiple replicated multiplex analyte-specific reporter molecules to identify (and quantify) the presence of a specific analyte in multiple biological samples.

[0026] Implementation Scheme 11. A method for parallel and automated determination of proximity linkages of multiple multiple oligonucleotide conjugated antibodies on multiple biological samples, comprising: A) In a first multi-container carrier plate, a portion of a plurality of multiple multiple paired binding portions is introduced in parallel into a plurality of biological samples to form a plurality of immune complex forming solutions; The multiple pairwise binding portions include pre-selected different pairwise binding portions for binding different specific analytes, and these pairwise binding portions include: i) The first portion of the paired binding portion includes a first antibody or first antibody fragment pre-selected for binding a specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second part of the pair-binding portion includes a second antibody or second antibody fragment pre-selected for binding a specific analyte that is the same as the first antibody or antibody fragment of the first part of the pair-binding portion, a second nucleic acid target marker containing a second identifier that is specific to the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first part of the pair binding portion is preselected for binding to the same first substrate for all different pair binding portions in multiplex assays, and the second nucleic acid tag of the second part of the pair binding portion is preselected for binding to the same second substrate for all different pair binding portions in multiplex assays; B) Parallel incubation of multiple immune complex formation solutions to form multiple multiple immune complexes in a first multi-container support plate; C) For the first time, a portion of a first substrate solution containing multiple first substrates is combined with multiple immune complexes in a first multi-container carrier plate to form a solution combination; D) For the first time, the first nucleic acid tag of the first portion of the paired binding portion binds to a portion of the first substrate in the first multi-container carrier plate; E) For the first time, multiple multiple immune complexes are extracted in parallel from a first multi-container plate through a portion of multiple first substrates and transferred to a second multi-container plate; F) Clean the multiple multiple immune complexes in the second multi-container carrier plate; G) A second extraction of multiple multiple immune complexes from the second multi-container plate and transfer to the third multi-container plate; H) Multiple first substrates are eluted in parallel from multiple multiple immune complexes in a third multi-container carrier plate; I) Remove multiple first substrates in parallel from the third multi-container support plate; J) The second step involves combining a portion of a second substrate solution containing multiple second substrates with multiple multiple immune complexes in parallel with a third multi-container carrier plate; K) The second nucleic acid tag of the second portion of the paired binding portion is then bound to a portion of the second substrate of the third multi-container carrier plate; L) A third time, multiple multiple immune complexes are extracted in parallel from a portion of a third multi-container plate through multiple second substrates and transferred to a fourth multi-container plate; M) In the fourth multi-container carrier plate, multiple first nucleic acid target labels (directly or indirectly) of the first part of the paired binding portion on multiple multiple immune complexes are linked in parallel with second nucleic acid target labels of the second part of the paired binding portion to form multiple multiple analyte-specific reporter molecules; N) The fourth time, multiple multiple immune complexes carrying analyte-specific reporter molecules are extracted in parallel from a portion of multiple second substrates and transferred to a third multi-container plate. O) Multiple second substrates were eluted in parallel from multiple multiple immune complexes containing analyte-specific reporter molecules in a third multi-container carrier plate; P) Replication of multiple multiplex analyte-specific reporter molecules; and Q) Detect multiple replicated multiplex analyte-specific reporter molecules to identify (and quantify) the presence of a specific analyte in multiple biological samples.

[0027] Implementation Scheme 12. A method for performing biochemical assays on biological samples to detect at least one analyte on a benchtop automated system, comprising: 1) Transfer at least one sample from the sample multi-container support plate to the first multi-container support plate; 2) Dilute the sample in the first multi-container support plate; 3) A first solution is formed in the second multi-container support plate by mixing the following substances: i) at least a portion of a sample, ii) A portion of a plurality of first parts, comprising a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker comprising a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag, and iii) A portion of a plurality of second portions, comprising a second antibody or second antibody fragment pre-selected for binding a specific analyte to a first antibody or antibody fragment identical to the first portion of the paired binding portion, a second nucleic acid target marker comprising a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; 4) Incubate the first solution to form an immune complex comprising one of a plurality of first portions, one of a plurality of second portions, and at least one analyte from the sample; 5) Implement a dual capture and release mechanism for immune complexes to remove contaminants and improve sensitivity through the following methods: i) Add a portion of the first substrate to capture at least a portion of the immune complex from the first solution in the second multi-container carrier plate; ii) Incubate the first solution in the second multi-container carrier plate to immobilize the immune complex on the surface of the first substrate; iii) The first substrate is transferred to a third multi-container support plate via a mixer to form a second solution with the elution buffer; iv) Clean the first substrate to remove unbonded impurities; v) Incubate the second solution in the third multi-container plate to release at least a portion of the immune complex; vi) Remove the first substrate using a mixer; vii) Add a portion of the second substrate to the second solution in the third multi-container support plate; viii) Incubate the second solution in the second multi-container carrier plate to fix the immune complex on the surface of the second substrate; ix) The second substrate is transferred to a third multi-container support plate via a mixer to form a third solution; x) Clean the second substrate to remove unbound impurities; 6) Add the bonding reagent to the third solution in the fourth multi-container support plate; 7) Introduce at least a portion of a plurality of double-stranded nucleic acid sample markers into a third solution in a fourth multi-container carrier plate to form at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule comprising a first nucleic acid target marker associated with a first portion of at least one immune complex and at least one second nucleic acid target marker conjugated to a second portion of at least one immune complex, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with a first portion of at least one portion of the immune complex by one protrusion and with the second nucleic acid target marker conjugated to a second portion of the immune complex by another protrusion, to complete proximity linkage between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one multiplex analyte-specific reporter molecule conjugated to a second antibody or a second antibody fragment and not covalently attached to a first antibody or a first antibody fragment; 8) Collect at least one multiplex analyte-specific reporter molecule in the first multi-container support plate and combine the solutions of the multiplex analyte-specific reporter molecules in the first multi-container support plate; and 9) Transfer the combined multiplex analyte-specific reporter molecule solutions from the first plate to the qPCR plate and detect the analytes by NGS with atmolar sensitivity.

[0028] Implementation Scheme 13. A rapid, compact instrument for automating the assay of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: a) Target kit, comprising: Multiple pre-selected paired binding moieties for binding a specific analyte, the paired binding moieties including: i) The first part includes a first antibody or first antibody fragment pre-selected for binding a specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; ii) The second part comprises a second antibody or second antibody fragment pre-selected for binding to a specific analyte identical to the first antibody or antibody fragment of the first part of the paired binding portion, a second nucleic acid target marker containing a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; and b) A gantry equipped with an end effector with pipettes and plate clamps, an incubator, and a substrate extractor / mixer for performing dual capture and release of immune complex purification using a portion of multiple paired binding parts.

[0029] Implementation Scheme 14. A rapid, compact instrument for automating the assay of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: a) Target kit, comprising: Multiple pre-selected paired binding moieties for binding a specific analyte, the paired binding moieties including: i) The first part includes a first antibody or first antibody fragment pre-selected for binding a specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; ii) The second part comprises a second antibody or second antibody fragment pre-selected for binding to a specific analyte identical to the first antibody or antibody fragment of the first part of the paired binding portion, a second nucleic acid target marker containing a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; and b) A gantry equipped with an end effector with pipettes and plate clamps, an incubator, and a substrate extractor / mixer for performing dual capture and release of immune complex purification using a portion of multiple paired binding parts.

[0030] Implementation Scheme 15. A rapid, compact instrument for automating the assay of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: a) Target kit, comprising: Multiple pre-selected paired binding moieties for binding a specific analyte, the paired binding moieties including: i) The first part includes a first antibody or first antibody fragment pre-selected for binding a specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; ii) The second part includes a second antibody or second antibody fragment pre-selected for binding to a specific analyte that is the same as the first antibody or antibody fragment of the first part of the paired binding portion, a second nucleic acid target marker containing a second identifier that is specific to the specific analyte, and a second nucleic acid tag; b) Test kit, containing: i) A first substrate solution comprising multiple first substrates, and ii) A second substrate solution comprising multiple second substrates, and iii) Ligation reagents, and optional nucleic acid sample-specific labels; c) Microtiter plate containing at least 96 wells; d) A gantry frame equipped with an end effector with pipettes and plate clamps, an incubator, and a substrate extractor / mixer for performing dual capture and release of immune complex purification using a portion of multiple paired binding moieties and a first substrate solution and a second substrate solution, and additionally using ligation reagents to ligate a portion (directly or indirectly) of a first nucleic acid target label of multiple paired binding moieties to a portion of a second nucleic acid target label of multiple paired binding moieties to form multiple analyte-specific reporter molecules.

[0031] Implementation Scheme 16. A benchtop instrument for automatically performing parallel assays of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: 1) A multi-container support plate containing multiple biological samples; 2) Storage rack, including: a) Target kit, comprising: Multiple multiple pairwise binding portions, each comprising pre-selected different pairwise binding portions for binding different specific analytes, the pairwise binding portions including: i) A first portion of the paired binding portion, the first portion comprising a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker comprising a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion, the second portion comprising a second antibody or second antibody fragment pre-selected for binding a specific analyte identical to the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker comprising a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first part of the pair binding portion is preselected for binding to the same first substrate for all different pair binding portions in multiplex assays, and the second nucleic acid tag of the second part of the pair binding portion is preselected for binding to the same second substrate for all different pair binding portions in multiplex assays; b) Test kit, containing: i) a first substrate solution comprising a plurality of first substrates; and ii) A second substrate solution comprising multiple second substrates; 3) Gantry crane, equipped with end effector with pipette and plate clamp; 4) Incubator; 5) Substrate extractor / mixer; 6) qPCR; 7) A first plate containing multiple first holes; 8) A second plate containing multiple second holes; 9) A third plate containing multiple third holes; and 10) Controller, wherein: a) The controller controls the gantry to introduce, in parallel, a portion of multiple multiple pairwise binding portions of the target kit and a portion of multiple biological samples into a portion of a plurality of first wells of the first plate to form multiple immune complex forming solutions in a portion of a plurality of first wells of the first plate; b) The controller controls the gantry and incubator to incubate multiple immune complex forming solutions in a portion of multiple first wells on the first plate in parallel to form multiple multiple immune complexes; c) The controller controls the gantry to combine a portion of a first substrate solution containing multiple first substrates in the test kit with a multiple immune complex forming solution in a portion of multiple first wells in the first plate; d) The controller controls the gantry and incubator to bind a first nucleic acid tag of the first portion of the multiple paired binding portion to a portion of the first substrate in a portion of a plurality of first wells of the first plate; e) The controller controls the substrate extractor to extract multiple multiple immune complexes in parallel from multiple immune complex formation solutions through a portion of a plurality of first substrates to form multiple first immune complex purification solutions in at least a portion of a plurality of second wells of a second plate, wherein a portion of the plurality of first substrates elutes from the multiple multiple immune complexes in the multiple first immune complex purification solutions. f) The controller controls the substrate extractor to extract portions of multiple first substrates in parallel from multiple first immune complex purification solutions; g) The controller controls the gantry to combine a portion of a second substrate solution containing multiple second substrates in the test kit with a portion of a multiple first immune complex purification solution in a portion of multiple second wells in the second plate; h) The controller controls the gantry and incubator to bind a portion of the second nucleic acid tag of the second portion of the paired binding portion to a portion of the second substrate of a portion of a portion of the first immune complex purification solution in a portion of a portion of a plurality of second wells in the second plate; i) The controller controls the substrate extractor to extract a portion of the second substrate in parallel in a portion of a plurality of first immune complex purification solutions in a portion of a plurality of second substrates in a plurality of second wells of a second plate, to form a plurality of multiplex analyte-specific reporter molecules in a portion of a plurality of third wells of a third plate by parallel linking a plurality of first nucleic acid target tags (directly or indirectly) of the first portion of the pair-binding portion with a plurality of second nucleic acid target tags of the second portion of the pair-binding portion. j) The controller controls the substrate extractor to extract multiple multiple analyte-specific reporter molecules in multiple third wells of a third plate back into multiple second wells of a second plate in parallel through a portion of multiple second substrates, to elute multiple second substrates in parallel from multiple multiple analyte-specific reporter molecules in multiple second wells of a second plate. k) The controller controls the gantry and thermal cycler to replicate multiple analyte-specific reporter molecules; and l) The controller controls qPCR to detect replicated multiple multiplex analyte-specific reporter molecules to identify (and quantify) specific analytes in multiple biological samples.

[0032] Implementation Scheme 17. A method for automating dual capture and release multiplex immunoassays on multiple biological samples, comprising: Operating instruments, which include: 1) Controller; 2) A mechanical gantry crane capable of moving in three degrees of freedom; 3) A storage rack that accommodates multiple container support plates, accessible to both the user and the mechanical gantry; 4) A mixer, which serves as both a bead processor and a mixer, comprising multiple multi-container carrier plate platforms; 5) Reader; and 6) The following components located within the storage rack: a) Multiple multi-container plates, including a first plate with multiple first wells, a second plate with multiple second wells, a third plate with multiple third wells, a fourth plate with multiple fourth wells, and a multi-container plate for measuring multiple biological samples; and b) Multi-container target delivery kit, comprising: Multiple multiple pairwise binding portions, each comprising pre-selected different pairwise binding portions for binding different specific analytes, the pairwise binding portions including: i) A first portion of the paired binding portion, the first portion comprising a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker comprising a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion, the second portion comprising a second antibody or second antibody fragment pre-selected for binding a specific analyte identical to the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker comprising a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first part of the pair binding portion is preselected for binding to the same first substrate for all different pair binding portions in multiplex assays, and the second nucleic acid tag of the second part of the pair binding portion is preselected for binding to the same second substrate for all different pair binding portions in multiplex assays; c) Multi-container test kit, comprising: i) a first substrate solution comprising a plurality of first substrates; and ii) A second substrate solution comprising multiple second substrates; Operate the instrument to control: a) A mechanical gantry for parallel introduction of a portion of multiple multiple pairwise binding portions of a target kit and a portion of multiple biological samples in a portion of multiple first wells of a first plate to form multiple immune complex forming solutions in a portion of multiple first wells of a first plate; b) A gantry and substrate mixer are used to incubate multiple immune complex forming solutions in a portion of multiple first wells of a first plate in parallel to form multiple multiple immune complexes; c) A gantry for combining a portion of a first substrate solution containing multiple first substrates in a test kit with multiple multiple immune complexes in a portion of multiple first wells in a first plate; d) A gantry and mixer, wherein a portion of a first nucleic acid tag of a first portion of a multiple pair-binding portion binds to a portion of a first substrate in a portion of a plurality of first wells of a first plate; e) A mixer for extracting, in parallel, multiple multiple immune complexes bound to multiple first substrates from a portion of a plurality of first wells of a first plate through a portion of a plurality of first substrates, and transferring them to a portion of a plurality of second wells of a second plate, and eluting the portions of multiple first substrates from the multiple multiple immune complexes in the portions of the plurality of second wells of the second plate; f) A mixer for parallel extraction of a portion of a plurality of first substrates from at least a portion of a plurality of multiple immune complexes in a plurality of second wells of a second plate; g) A gantry for combining a portion of a second substrate solution containing multiple second substrates with multiple multiple immune complexes in a portion of multiple second wells of a second plate; h) A gantry and mixer, which bind a portion of a second nucleic acid tag to a portion of a second substrate in a plurality of second wells of a second plate; i) A mixer for parallel extraction of portions of multiple multiplex immune complexes bound to portions of portions of the second substrates in portions of multiple second wells of a second plate, and transfer to portions of multiple third wells of a third plate, and for forming multiple multiplex analyte-specific reporter molecules by parallel linking multiple first nucleic acid target tags (directly or indirectly) of the first portion of the paired binding portions with multiple second nucleic acid target tags of the second portion of the paired binding portions; j) A mixer for parallel extraction of multiple multiple analyte-specific reporter molecules bound to a portion of a portion of a second substrate from a portion of a portion of a portion of a third plate through a portion of a plurality of second substrates, and for transfer back to a portion of a portion of a plurality of second wells of the second plate, and for parallel elution of multiple second substrates from a portion of a plurality of multiple analyte-specific reporter molecules in a portion of a plurality of second wells of the second plate; k) A gantry for transferring at least a portion of a plurality of multiplex analyte-specific reporter molecules from a portion of a plurality of second wells of a second plate to a portion of a plurality of fourth wells of a fourth plate; and l) Gantry frame to move the fourth plate to the reader.

[0033] Implementation Scheme 18. A compact, fully automated instrument for performing dual capture and release multiplex immunoassays, comprising: A) Desktop chassis, including: 1) A mechanical gantry crane capable of moving in three degrees of freedom; 2) A storage rack that accommodates multiple container support plates, accessible to both the user and the mechanical gantry; 3) Provides a controllable stage with multiple multi-container support plate positions, wherein the stage is movable along the Y-axis (from front to back within the instrument housing); and 4) Mixer, which serves as a magnetic bead processor and mixer, comprises multiple multi-container carrier plate platforms stacked vertically to each other.

[0034] Implementation Scheme 19. Perform the process of any one of the methods in Implementation Schemes 9-12 and 17 using the instrument according to any one of Implementation Schemes 1-5 and 18.

[0035] In some embodiments, such as embodiments 1-19 and any other embodiments herein, one of the first or second nucleic acid tags comprises a nucleotide-rich sequence. The first or second nucleic acid tag comprises one or more of the following nucleotide-rich sequences: poly-A, poly-T, poly-C, or poly-G sequences. The first nucleic acid tag comprises a poly-A or poly-T sequence.

[0036] In some embodiments, such as embodiments 1-19 and any other embodiments herein, one of the first or second nucleic acid tags comprises an immobilization reagent. The first or second nucleic acid tag comprises one or more of the following immobilization reagents: biotin, streptavidin, EDC, DCC, NHS ester, imine ester, maleimide, haloacetyl, pyridyl disulfide, hydrazide, alkoxyamine, aryl azide, diaziridine, or a chemically selective linker. The second nucleic acid tag is conjugated with biotin.

[0037] In some implementations, such as implementations 1-19 and any other implementations herein, the first nucleic acid tag is not covalently attached to the first antibody or antibody fragment.

[0038] In some implementations, such as implementations 1-19 and any other implementations herein, the first nucleic acid target marker is not covalently attached to the first antibody or the first antibody fragment.

[0039] In some implementations, such as implementations 1-19 and any other implementations herein, the second nucleic acid tag is not covalently attached to the second antibody or a second antibody fragment.

[0040] In some embodiments, such as embodiments 1-19 and any other embodiments herein, a first antibody or a first antibody fragment pairs with a second antibody or a second antibody fragment to bind to a non-overlapping epitope of the analyte and form an immune complex.

[0041] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the first antibody or first antibody fragment has a binding affinity of at least 10 for the analyte. -4 M, at least 10 -6 M, at least 10 -9 M or higher, and the second antibody or second antibody fragment has a binding affinity of at least 10 to the analyte. -4 M, at least 10 -6 M, at least 10 -9 M or higher.

[0042] In some implementations, such as implementations 1-19 and any other implementations herein, the first nucleic acid target marker includes a first identification barcode.

[0043] In some implementations, such as implementations 1-19 and any other implementations herein, the second nucleic acid target marker includes a second identification barcode.

[0044] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument further includes a target kit. The target kit comprises a plurality of wells of a multi-container plate, (i) wherein a first well of at least one of the plurality of wells in the target kit comprises a first portion; and (ii) a second well of at least one of the plurality of wells in the target kit comprises a second portion. The first and second wells are identical wells of the multi-container plate. Alternatively, the first and second wells may be different wells of the multi-container plate.

[0045] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the target kit further comprises a plurality of wells of a multi-container plate, (i) wherein a third well of at least one of the plurality of wells of the target kit contains a first nonfunctional binder comprising a third antibody or a third antibody fragment that specifically binds the analyte; and (ii) wherein a fourth well of at least one of the plurality of wells of the target kit contains a second nonfunctional binder comprising a fourth antibody or a fourth antibody fragment that specifically binds the analyte. The first nonfunctional binder binds the same epitope of the analyte in the same manner as in the first portion. The third antibody or third antibody fragment is identical to the first antibody or first antibody fragment of the first portion. The fourth antibody or fourth antibody fragment is identical to the second antibody or second antibody fragment of the second portion. The third and fourth wells may be identical wells of a multi-container plate. The third and fourth wells may be different wells of a multi-container plate. Furthermore, the first, second, third, and fourth wells may be identical wells of a multi-container plate. The first, second, third, and fourth wells may be different wells of a multi-container plate.

[0046] In some implementations, such as implementations 1-19 and any other implementations herein, the first nonfunctional binder is mixed with the first portion in a predetermined ratio.

[0047] In some implementations, such as implementations 1-19 and any other implementations herein, the second nonfunctional binder is mixed with the second portion in a predetermined ratio.

[0048] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument further includes a detection kit. The detection kit comprises multiple wells of a multi-container plate. (i) wherein at least one of the plurality of pores contains a first substrate solution containing a plurality of first substrates; (ii) wherein at least one of the plurality of pores contains a second substrate solution comprising a plurality of second substrates; and (iii) The third well in at least one of the plurality of wells contains a ligation reagent and an optional nucleic acid sample-specific label.

[0049] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the first substrate is a first paramagnetic bead. The first paramagnetic bead is coupled with a first binding sequence capable of binding to a first nucleic acid tag on a first antibody or a first antibody fragment. The first paramagnetic bead may be coated with a first binding sequence capable of binding to a first nucleic acid tag on a first antibody or a first antibody fragment.

[0050] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the second substrate is a second paramagnetic bead. The second paramagnetic bead is coupled with a second binding group capable of binding to a second nucleic acid tag associated with a second antibody or a second antibody fragment. The second paramagnetic bead may be coated with a second binding group capable of binding to a second nucleic acid tag associated with a second antibody or a second antibody fragment.

[0051] In some implementations, such as those 1-19 and any other implementations herein, the first binding sequence is preselected for hybridization with a first nucleic acid tag on a first antibody or a fragment of a first antibody.

[0052] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the first binding sequence coupled to the first paramagnetic bead is a poly-A or poly-T sequence. The poly-A sequence from the first nucleic acid tag hybridizes with the poly-T sequence from the first paramagnetic bead. Alternatively, the poly-T sequence from the first nucleic acid tag hybridizes with the poly-A sequence from the first paramagnetic bead.

[0053] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the second binding group coupled to the second paramagnetic bead is streptavidin or avidin.

[0054] In some embodiments, such as embodiments 1-19 and any other embodiments herein, biotin from the second nucleic acid tag binds to streptavidin from the second paramagnetic bead. Alternatively, biotin from the second nucleic acid tag binds to avidin from the second paramagnetic bead.

[0055] In some implementations, such as implementations 1-19 and any other implementations herein, the bond between the first nucleic acid tag and the binding sequence is an orthogonal bond.

[0056] In some implementations, such as those 1-19 and any other implementations herein, the bond between the second nucleic acid tag and the binding group is an orthogonal bond.

[0057] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the first, second, and third wells of the multi-container plate of the detection kit are located within the multi-container plate of the target kit. Alternatively, the first and second wells of the target kit are located within the detection kit.

[0058] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument further includes an incubation sealer for incubating and / or sealing at least one multi-container carrier plate.

[0059] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument also includes a large-capacity fluid station containing multiple containers.

[0060] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the mechanical gantry further includes an end effector comprising a pipette, a multi-container carrier clamp, a laser position sensor, and a barcode scanner, the mechanical gantry enabling the end effector to move in three degrees of freedom of the X, Y, and Z axes.

[0061] In some implementations, such as embodiments 1-19 and any other embodiments herein, the reader includes a qPCR unit for qPCR reading.

[0062] In some implementations, such as embodiments 1-19 and any other embodiments herein, the reader includes qPCR for preparing mixed libraries that can be used for next-generation sequencing (NGS).

[0063] In some implementations, such as those 1-19 and any other implementations herein, the reader is capable of identifying and / or quantifying nucleic acid reporter molecules.

[0064] In some implementations, such as embodiments 1-19 and any other embodiments herein, the housing also includes a touchscreen display.

[0065] In some implementations, such as embodiments 1-19 and any other embodiments herein, the storage rack includes a set of multi-container compartments for receiving and accommodating at least one universal reagent kit and consumable carrier.

[0066] In some implementations, such as embodiments 1-19 and any other embodiments herein, the storage rack further includes: a) Target reagent kit; b) Test kits; and c) At least one consumable carrying device, including: i) PCR multi-container plate, ii) Framed sealing film, iii) Multiple pipette tips, and iv) Multiple multi-container support plates.

[0067] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the framed sealing membrane has one or more perforated lines configured to allow easy and complete separation of the membrane from the frame by tearing. The framed sealing membrane seals the perforations when heat-welded to the edge of a multi-container support plate. The framed sealing membrane comprises a metal layer. The framed sealing membrane can be punctured by a pipette tip.

[0068] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the controller includes a processor and a non-transitory machine-readable storage medium containing processor-executable instructions to provide controlled operation of components within the instrument. Alternatively, the controller includes a processor and a non-transitory machine-readable storage medium containing processor-executable pre-programmed instructions to perform dual capture and release multiplex immunoassays.

[0069] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the controller includes a processor and a non-transitory machine-readable storage medium containing instructions executable by the processor to provide controlled operation of unit operations within the system, the operations controlled by the controller including: a) A first solution is formed in the first multi-container support plate by mixing the following substances: i) at least one part of a biological sample, ii) a portion of multiple first parts, and iii) One of multiple parts of Part II; b) Incubate the first solution in an incubator to form an immune complex comprising one of a plurality of first portions, one of a plurality of second portions, and at least one analyte from a biological sample; c) Implement a dual capture and release mechanism for immune complexes by introducing the following substances to remove unbound contaminants and improve sensitivity: i) A portion of the first paramagnetic bead is used to capture at least a portion of the immune complex from a first solution in a first multi-container support plate, wherein the first solution in the first multi-container support plate is incubated, and the first paramagnetic bead is transferred to a second multi-container support plate by an extractor / mixer to form a second solution, thereby releasing at least a portion of the immune complex, and the first paramagnetic bead is removed by an extractor / mixer. ii) A portion of the second paramagnetic beads to capture at least a portion of the immune complex from the second solution, wherein the second solution is incubated and the second set of paramagnetic beads is removed by an extractor / mixer to form a third solution in a third multi-container carrier plate.

[0070] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the contaminant may include an unbound first portion or an unbound second portion. The contaminant may include a non-target analyte. The contaminant may include an unbound target analyte.

[0071] In some implementations, such as implementations 1-19 and any other implementations herein, the operation controlled by the controller further includes: a) Clean the first solid surface of the first substrate to remove unbound contaminants; and b) Clean the second solid surface of the second substrate to remove unbound contaminants.

[0072] In some implementations, such as implementations 1-19 and any other implementations herein, the operation controlled by the system controller further includes: a) Introducing at least a portion of a plurality of nucleic acid sample markers into a third solution to form at least one nucleic acid reporter molecule, the nucleic acid reporter molecule comprising a first nucleic acid target marker associated with a first portion of at least one immune complex and at least one second nucleic acid target marker conjugated to a second portion of at least one immune complex, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of the first portion of the immune complex via one protrusion and with the second nucleic acid target marker conjugated to at least a portion of the second portion of the immune complex via another protrusion, to complete proximity linkages between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one nucleic acid reporter molecule conjugated to the first portion and not covalently attached to the second portion; and b) At least one nucleic acid reporter molecule is collected in a fourth multi-container plate and then the nucleic acid reporter molecules are combined to form a pooled sample for detection of the analyte by NGS with atmolar sensitivity and a wide dynamic range.

[0073] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, the operation controlled by the controller also includes cleaning the multi-container plate cleaner previously used to perform the capture-release mechanism and reintroducing the immune complex into the cleaned multi-container plate.

[0074] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument is pre-configured to perform dual capture and release multiplex immunoassays.

[0075] In some implementations, such as those 1-19 and any other implementations herein, the instrument is an all-in-one instrument with integrated qPCR capabilities, capable of processing sample assays from sample preparation to data or to merged NGS libraries.

[0076] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, the instrument is capable of atmolar-level sensitivity detection. Atmolar-level sensitivity detection can be at least 0.1, at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or higher atmolars.

[0077] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument is capable of amolar-level sensitivity detection and has a wide dynamic range of up to 12, 14, 16, 18, or 20 orders of magnitude, including the upper limit of detection and the lower limit of detection of a component in the target set.

[0078] In some implementations, such as those including implementations 1-19 and any other implementations herein, the instrument is capable of performing dual capture and release multiplex immunoassays on more than 10, 25, 35, 50, 75, or 100 samples in parallel.

[0079] In some implementations, such as those 1-19 and any other implementations herein, the instrument can perform dual capture and release multiple immunoassays of multiple samples, starting from the assay sample, without human intervention.

[0080] In some implementations, such as those including implementations 1-19 and any other implementations herein, the instrument can perform dual capture and release multiple immunoassays of multiple samples starting from the assay sample in less than 1 hour, less than 2 hours, less than 3 hours, or less than 4 hours without human intervention.

[0081] In some implementations, such as those including implementations 1-19 and any other implementations herein, the instrument can perform dual capture and release multiple immunoassays of multiple samples from assay samples to qPCR readings in less than 3 hours, less than 4 hours, less than 5 hours, or less than 6 hours without human intervention.

[0082] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, the instrument automatically performs the assay of multiple oligonucleotide conjugated antibodies for multiple biological samples, from sample preparation, purification via dual capture and release of immune complexes to the formation of multiple analyte-specific reporter molecules for multiple analytes in multiple biological samples, within 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours, with sensitivity as low as 10 atmol, 20 atmol, 30 atmol, 40 atmol, 50 atmol, 100 atmol, or 150 atmol.

[0083] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument completes the dual capture and release mechanism in less than 0.5 hours, less than 1 hour, less than 2 hours, or less than 3 hours.

[0084] In some implementations, such as those 1-19 and any other implementations herein, the instrument contains sufficient reagents and other consumables to run at least two, three, four, five, or six assay cycles without requiring the user to reload the reagents and other consumables.

[0085] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument is a benchtop instrument.

[0086] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument has a height of less than 50 inches, less than 48 inches, less than 46 inches, less than 44 inches, less than 42 inches, less than 40 inches, less than 38 inches, less than 36 inches, less than 34 inches, less than 32 inches, or less than 30 inches (excluding the stage).

[0087] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the base of the instrument has a depth of less than 36 inches, less than 32 inches, less than 30 inches, less than 28 inches, less than 26 inches, less than 24 inches, less than 22 inches, or less than 20 inches, and a width of less than 50 inches, less than 48 inches, less than 46 inches, less than 44 inches, less than 42 inches, less than 40 inches, less than 38 inches, less than 36 inches, less than 34 inches, less than 32 inches, or less than 30 inches.

[0088] In some implementations, such as implementations 1-19 and any other implementations herein, the instrument is capable of high-throughput measurements.

[0089] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the sample is a serum sample, a plasma sample, or a cerebrospinal fluid sample. The sample may be derived from a cell sample or a tissue sample.

[0090] In some implementation schemes, such as those including schemes 1-19 and any other implementation scheme herein, the sample has more than 10, more than 50, more than 100, more than 150, more than 200, more than 250, more than 300, more than 350, more than 400, more than 450, or more than 500 analytes.

[0091] In some implementation schemes, such as schemes 1-19 and any other schemes herein, the sample volume is less than 50 μL, less than 20 μL, less than 15 μL, less than 10 μL, less than 5 μL, or less than 1 μL.

[0092] In some implementations, such as embodiments 1-19 and any other embodiments herein, the analyte is a protein or peptide.

[0093] In some implementation schemes, such as those 1-19 and any other implementation scheme herein, multiplex assays are performed to detect at least 50 analytes, at least 100 analytes, at least 150 analytes, or at least 200 analytes.

[0094] In some implementations, such as implementations 1-19 and any other implementations herein, an automated instrument is used to perform a dual capture and release immunoassay procedure, wherein for a single assay run, the instrument consumes no more than 3, 4, 5, 6, or 7 multi-container carrier plates.

[0095] In some implementations, such as implementations 1-19 and any other implementations herein, an automated instrument is used to perform a dual capture and release immunoassay procedure, wherein for a single assay run, the instrument consumes no more than 1, 2, 3, or 4 boxes of pipette tips.

[0096] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes for capturing immune complexes on a substrate, and reuses the at least one plate to hold the solution containing immune complexes for eluting immune complexes from the substrate.

[0097] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes for capturing immune complexes on a substrate, washing the at least one plate, and reusing the at least one plate to hold the solution containing immune complexes for eluting immune complexes from the substrate.

[0098] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, and reuses the at least one plate to hold a solution containing immune complexes bound to a second substrate.

[0099] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing an immune complex bound to a first substrate for eluting the first substrate from the immune complex, and uses at least one plate to hold a solution containing an immune complex bound to a second substrate for eluting the second substrate from the immune complex.

[0100] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes for eluting a first substrate from the immune complexes, and reuses the at least one plate to hold a solution containing immune complexes for eluting a second substrate from the immune complexes.

[0101] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes for eluting a first substrate from the immune complexes, washing the at least one plate, and reusing the at least one plate to hold a solution containing immune complexes for eluting a second substrate from the immune complexes.

[0102] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold the solution containing the immune complex before capturing the immune complex on the second substrate, and reuses the at least one plate to hold the solution containing the immune complex for eluting the second substrate from the immune complex.

[0103] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold the solution containing the immune complex before capturing the immune complex on the second substrate, and after capturing the immune complex on the second substrate, the at least one plate is reused to hold the solution containing the immune complex.

[0104] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, and uses the at least one plate to hold the solution containing immune complexes for capturing immune complexes on a second substrate.

[0105] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, and reuses the at least one plate to hold the solution containing immune complexes for eluting the immune complexes from a second substrate.

[0106] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, and uses the at least one plate to hold a solution containing immune complexes for capturing immune complexes on a second substrate, and reuses the at least one plate to hold a solution containing immune complexes for eluting immune complexes from the second substrate.

[0107] In some embodiments, such as embodiments 1-19 and any other embodiments herein, during the first substrate elution, the instrument uses at least one plate to hold a solution containing immune complexes bound to the first substrate, and uses the at least one plate to hold a solution containing immune complexes for capturing immune complexes on a second substrate, and reuses the at least one plate to hold a solution containing immune complexes for eluting immune complexes from the second substrate.

[0108] In some embodiments, such as embodiments 1-19 and any other embodiments herein, before capturing immune complexes on the second substrate, the instrument uses at least one plate to hold the solution containing the immune complexes, washes the at least one plate, and after capturing immune complexes on the second substrate, reuses the at least one plate to hold the solution containing the immune complexes.

[0109] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, washes the at least one plate, and reuses the at least one plate to hold the solution containing immune complexes for capturing immune complexes on a second substrate.

[0110] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, washes the at least one plate, and reuses the at least one plate to hold the solution containing immune complexes for eluting the immune complexes from a second substrate.

[0111] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to hold a solution containing immune complexes bound to a first substrate, washes the at least one plate, and reuses the at least one plate to hold the solution containing immune complexes for capturing immune complexes on a second substrate, and reuses the at least one plate to hold the solution containing immune complexes for eluting immune complexes from the second substrate.

[0112] In some embodiments, such as embodiments 1-19 and any other embodiments herein, during the elution of the first substrate, the instrument uses at least one plate to hold a solution containing immune complexes bound to the first substrate, washes the at least one plate, and reuses the at least one plate to hold the solution containing immune complexes for capturing immune complexes on the second substrate, and reuses the at least one plate to hold the solution containing immune complexes for eluting immune complexes from the second substrate.

[0113] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to carry the immune complex solution for capturing the immune complex on the substrate, then washes the at least one plate, and reuses the at least one plate for eluting the immune complex solution from the substrate.

[0114] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain the immune complex solution during cleaning, and the at least one plate is reused to contain the immune complex solution during the formation of the reporter molecule.

[0115] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain a solution containing an immune complex bound to a first substrate, and reuses the at least one plate to contain a solution containing an immune complex bound to a second substrate.

[0116] In some embodiments, such as embodiments 1-19 and any other embodiments herein, during cleaning, the instrument uses at least one plate to contain a solution containing immune complexes bound to a first substrate, and during connection, the at least one plate is reused to contain a solution containing immune complexes bound to a second substrate.

[0117] In some embodiments, such as embodiments 1-19 and any other embodiments herein, prior to elution of the first substrate, the instrument uses at least one plate to contain a solution containing immune complexes bound to the first substrate, and the at least one plate is reused to contain a solution containing immune complexes bound to the second substrate prior to elution of the second substrate.

[0118] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain an immune complex formation solution for forming immune complexes, and uses the at least one plate to capture immune complexes on a substrate.

[0119] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain an immune complex formation solution for forming immune complexes, and uses the at least one plate to capture immune complexes on a first substrate.

[0120] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain an immune complex forming solution for forming immune complexes, and uses the at least one plate to capture immune complexes on a first substrate, and reuses the at least one plate to contain a solution containing an immune complex solution bound to the first substrate.

[0121] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain an immune complex forming solution for forming immune complexes, and uses the at least one plate to capture immune complexes on a first substrate, washes the at least one plate, and reuses the at least one plate to contain a solution containing an immune complex solution bound to the first substrate.

[0122] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain an immune complex forming solution for forming immune complexes, and uses the at least one plate to capture immune complexes on a first substrate, and reuses the at least one plate to contain a solution containing the immune complex solution bound to the first substrate for washing.

[0123] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain an immune complex forming solution for forming immune complexes, and uses the at least one plate to capture immune complexes on a first substrate, washes the at least one plate, and reuses the at least one plate to contain a solution containing the immune complex solution bound to the first substrate for washing.

[0124] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least the same plate for containing the immune complex solution, for capturing the immune complex on the substrate, and for eluting the immune complex from the substrate.

[0125] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least the same plate for containing an immune complex formation solution for forming immune complexes, and uses at least the same plate for capturing immune complexes on a first substrate.

[0126] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least the same plate for containing immune complexes, for eluting the first substrate, and for capturing immune complexes on the first substrate.

[0127] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least the same plate for containing the immune complex solution for eluting the first substrate and for capturing the immune complex on the second substrate.

[0128] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, the instrument uses at least the same plate to contain the first substrate solution and the second substrate solution.

[0129] In some implementations, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least the same plate to contain the first base solution, cleans the at least the same plate, and reuses the same plate to contain the second base solution.

[0130] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, the instrument reuses at least one plate for carrying a solution containing the target analyte from one step of the process to a subsequent step of the process.

[0131] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, the instrument reuses at least one plate for carrying a solution containing immune complexes from one step of the process to a subsequent step of the process.

[0132] In some embodiments, such as embodiments 1-19 and any other embodiments herein, the instrument uses at least one plate to contain a solution comprising at least one immune complex, which is a precursor to the target analyte, and the at least one plate is reused to carry the solution containing the immune complex from one step of the process to a subsequent step of the process.

[0133] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, from the formation of the immune complex through multiple process steps to the formation of the corresponding nucleic acid analyte reporter molecule, the instrument transfers the sample, i.e., a solution containing the target analyte or the analyte captured by the immune complex, without any pipette tip contacting the sample.

[0134] In some implementations, such as those including embodiments 1-19 and any other embodiments herein, from the formation of the immune complex through multiple process steps to the formation of the corresponding nucleic acid analyte reporter molecule, the instrument transfers the sample, i.e., a solution containing the target analyte or the analyte captured by the immune complex, without any pipette tip contact with the sample.

[0135] In some embodiments, an apparatus for performing an immunoassay is provided, comprising: a microtiter plate manipulator configured to be operatively coupled to a microtiter plate having a plurality of reaction wells (e.g., 20-500 wells, such as 96 or 384 wells, wherein the working volume of each well is at least 5 μL, such as 10-100 μL or 20-90 μL, and / or the total volume of each well is greater than 150 μL, such as 190-250 μL). μL), especially for receiving assay sample solutions; microtiter plate sealers for applying sealing components to the surface of microtiter plates to seal the solution in each well of the microtiter plate and reduce any contamination and / or loss of the solution in each well; magnetic bead extraction systems for mixing solutions (e.g., assay samples) and extracting magnetic beads from the solution and additives in the wells of microtiter plates; incubators for promoting the binding of antibodies to analytes in assay samples; washing systems for washing samples and / or contaminants in the wells (including decontamination and cleaning systems for decontamination of microtiter plates); polymerase chain reaction (PCR) systems; and more. The device includes, and optionally, an imaging system configured to acquire images of at least a portion of the solution in each well (the assay sample or the result of the assay sample after all or at least a portion of the NULISA procedure); an xyz gantry equipped with an end effector having a microtiter plate holder (plate manipulator), a pipetting subsystem, and at least one position sensor; and a computer-implemented control system configured to automatically operate, manage, and coordinate all or at least some of the systems within the device (including at least the plate manipulator, plate sealer, magnetic bead extractor, incubator, PCR system, and / or gantry), and / or receive image-related information from the imaging system.

[0136] In some embodiments, the device for sealing a microtiter plate is equipped with a membrane-containing frame, wherein the membrane may be self-adhesive, thermoformable, transparent, and / or well-sealing, and wherein the membrane-containing frame is operatively coupled to the microtiter plate. The frame is structured and positioned to apply a sealing assembly to the surface of the microtiter plate to seal all or at least some of the pores within the microtiter plate, wherein the contents of each pore (e.g., a solution or assay sample) are substantially isolated from the contents of each of the other plurality of sealed assay sites; and a controller configured to automatically operate the sealer to apply the sealing assembly to the plurality of assay sites.

[0137] In some embodiments, an apparatus is provided for inserting beads into measurement sites on a measurement consumable, comprising: a measurement consumable manipulator configured to be operatively coupled to a measurement consumable having a plurality of measurement sites on its surface; a bead loader configured to insert a single bead into a single measurement site such that each bead-containing measurement site will contain no more than one bead; and a controller configured to automatically operate the bead loader to insert a single bead into a single measurement site.

[0138] In some embodiments, an apparatus for performing a assay is provided, comprising: an assay consumable manipulator configured to operatively couple to an assay consumable having a plurality of assay sites on its surface; a sample loader configured to load an assay sample containing analyte molecules or particles of unknown concentration into at least a portion of the plurality of assay sites, such that the plurality of assay sites containing the assay sample are free of or contain a single analyte molecule or particle; a detector configured to probe at least a portion of the assay sites containing the assay sample and determine the proportion of analyte molecules or particles contained in the probed plurality of assay sites; and a computer-implemented system configured to receive information from the detector and determine from the information a measured value of the unknown concentration of analyte molecules or particles in the assay sample.

[0139] In some embodiments, an apparatus is provided for inserting beads into measurement sites on a measurement consumable, comprising: a measurement consumable manipulator configured to be operatively coupled to the measurement consumable, wherein the surface of the measurement consumable includes a plurality of measurement sites; a bead applicator configured to apply a plurality of magnetic beads to the surface of the measurement consumable or to place a plurality of magnetic beads at a position proximate to the surface; a bead loader including a magnetic field generator located near the measurement consumable and configured to generate relative motion between the magnetic beads and the measurement sites; and a controller configured to automatically operate the bead loader to generate relative motion between the magnetic beads and the measurement sites and to insert the beads into the measurement sites.

[0140] In some embodiments, an apparatus is provided for removing excess beads from a test consumable having a surface comprising a plurality of test sites, comprising: a test consumable manipulator operatively coupled to the test consumable, wherein the test consumable comprises a plurality of beads, wherein a first portion of the beads is contained within the test sites and a second portion of the beads is located on the surface of the test consumable but not contained within the test sites; a wiper configured to remove substantially all of the second portion of the beads from the surface; and a controller configured to automatically operate the wiper to remove the second portion of the beads.

[0141] In some embodiments, a measurement consumable is provided, comprising a surface having a plurality of measurement sites, each measurement site having a volume of about 10 liters to about 50 picoliters; and at least one channel formed on the surface, at least partially surrounding the plurality of measurement sites, the channel being positioned and structured to collect excess measurement sample liquid applied to the surface and overflowing from the measurement sites.

[0142] In some embodiments, an automated method is provided for forming a plurality of sealed test sites to perform a test, comprising: operatively associating a test consumable having a surface containing a plurality of test sites with a sealer device including a sealer and a controller; and applying a sealing assembly to the plurality of test sites using the sealer device such that a plurality of sealed test sites are formed, wherein the contents of each sealed test site are substantially isolated from the contents of each of the other plurality of sealed test sites.

[0143] In some embodiments, a method for inserting beads into a reaction vessel on a measurement consumable is provided, comprising: generating a magnetic field near a surface of the measurement consumable containing a plurality of reaction vessels, such that the magnetic field vector of the magnetic field points from the surface to the bottom of the reaction vessel and / or the periphery of the surface; delivering a plurality of magnetic beads near the surface; and generating relative motion between the magnetic beads and the reaction vessel.

[0144] In some embodiments, a method for forming a plurality of sealed reaction vessels to perform a assay is provided, comprising: applying a sealing assembly to a surface, thereby associating a assay consumable comprising a plurality of assay sites on the surface with the sealing assembly, wherein, once associated with the sealing assembly, the contents of each assay site are substantially isolated from the contents of each of the other plurality of assay sites without the need to maintain any pressure applied to the sealing assembly; and wherein the volume of each assay site is approximately 10 femtoliters to approximately 50 picoliters.

[0145] In some embodiments, a method for forming a plurality of sealed reaction vessels to perform a assay is provided, comprising: associating the assay consumable, which has a plurality of assay sites on its surface, with the sealing assembly by applying a sealing component to a surface of an assay consumable and applying pressure to the sealing component, wherein, after associating the sealing component with the assay consumable, the contents of each assay site are substantially isolated from the contents of each of the other plurality of assay sites; wherein the sealing component includes a pressure-sensitive adhesive such that, when pressure is applied to the sealing component, the pressure-sensitive adhesive is activated and the adhesive forms an adhesive bond between the sealing component and the surface of the assay consumable; wherein the volume of each assay site is about 10 femtoliters to about 50 picoliters.

[0146] In some embodiments, a method for forming a plurality of sealed measurement sites to perform a measurement is provided, comprising: providing a measurement consumable having a surface comprising a plurality of measurement sites, wherein each measurement site has a volume of about 10 femtoliters to about 50 picoliters; and applying a liquid substantially immiscible with the liquid contained within the plurality of measurement sites to the plurality of measurement sites, thereby forming a plurality of sealed measurement sites, wherein the contents of each sealed measurement site are substantially isolated from the contents of each of the other plurality of sealed measurement sites.

[0147] In some embodiments, an apparatus for removing beads from the surface of a measuring consumable is provided, comprising: a first magnet located near the surface of the measuring consumable and positioned opposite to a surface containing a plurality of measuring sites; a second magnet, a third magnet, and a metal object, wherein the second magnet and the third magnet are located near the surface containing the plurality of measuring sites, and such that opposite poles of the second magnet and the third magnet are positioned opposite each other; wherein the metal object is located between the second magnet and the third magnet.

[0148] In some implementations, the instrument partially achieves a compact design by rationally using and reusing sample plates (e.g., 96-well microtiter plates) to perform multiple complex steps in the assay. This presents several challenges, as the instrument must achieve extremely high levels of purity for the immune complex solution while avoiding contaminants typically associated with plate reuse, and still maintain minimal use of valuable resources (such as washing solutions and other reagents), minimal waste, and minimal process time. For example, in some implementations, the instrument may reuse the sample plate (e.g., a 96-well microtiter plate) in the main flow of the process, which involves taking the sample solution and forming an immune complex forming solution (where immune complexes are formed). The immune complex solution is then mixed with a first substrate to form a first immune complex capture solution, where immune complexes are captured on the first substrate. The first substrate with bound immune complexes is separated from the first immune complex capture solution and washed to remove contaminants (e.g., impurities, uncaptured immune complexes, incomplete immune complexes, antibodies, unbound analytes, blocked analytes, non-target analytes, process reagents, and fragments thereof). The first substrate bound with the immune complex is then mixed with an elution solution to elute the first substrate from the immune complex, forming a first purified immune complex solution. The first purified immune complex solution is then mixed with a second substrate to form a second immune complex capture solution, wherein immune complexes are captured on the second substrate. The second substrate bound with the immune complex is separated from and washed with the second immune complex capture solution to remove contaminants. The second substrate bound with the immune complex is then mixed with a linking solution to form an immune complex linking solution (in which an immune complex reporter molecule is formed). The second substrate bound with the immune complex (and the immune complex reporter molecule) is then separated from and washed with the immune complex linking solution to remove contaminants. The second substrate bound with the immune complex is then mixed with an elution solution to elute the second substrate from the immune complex, forming a second purified immune complex solution containing the immune complex reporter molecule.

[0149] In some embodiments, such as during the execution of a process, the instrument in some embodiments can clean and reuse sample plates (e.g., 96-well microtiter plates) used in the main flow of the analyte assay process. Specifically, the reuse of plates instead of using additional sterile plates is unique in processes where analytes are treated with immune complexes and the immune complexes are purified through a dual capture and release step to minimize the presence of contaminants that could cause noise in the final detection and analysis, and this part allows the process to be performed in a smaller space. This contributes to a compact instrument design. For example, during the execution of a process, the instrument in some embodiments can reuse plates in the main flow of the process, which requires taking a sample solution and forming an immune complex forming solution (where immune complexes are formed) in a first plate. The immune complex solution in the first plate is then mixed with a first substrate to form a first immune complex capture solution (also in the first plate), where immune complexes are captured on the first substrate. A first substrate bound with immune complexes is removed from a first plate and washed in a second plate to remove contaminants (e.g., impurities, uncaptured immune complexes, incomplete immune complexes, antibodies, unbound analytes, blocked analytes, non-target analytes, process reagents, and fragments thereof). Optionally, the first substrate bound with immune complexes is removed from the second plate and transferred to the first plate for additional washing. The first substrate bound with immune complexes is then transferred to a third plate and mixed with an elution solution to elute the first substrate from the immune complexes, forming a first purified immune complex solution in the third plate. The first purified immune complex solution is then mixed with a second substrate in the third plate to form a second immune complex capture solution, wherein immune complexes are captured on the second substrate in the third plate. The second substrate bound with immune complexes is removed from the third plate and washed in a fourth plate to remove contaminants. The second substrate bound with immune complexes is then mixed with a ligation solution in the (washed) second plate to form an immune complex ligation solution (wherein an immune complex reporter molecule is formed) in the second plate. The second substrate, bound to the immune complex (and the immune complex reporter molecule), is then separated from the second plate and washed in a (washed) fourth plate to remove contaminants. The second substrate bound to the immune complex is then removed from the fourth plate and mixed with an elution solution in a (washed) third plate to elute the second substrate from the immune complex, forming a second purified immune complex solution containing the immune complex reporter molecule in the third plate.

[0150] In some implementations, the instrument's compact design is achieved by incorporating a magnetic bead processor ("mixer") equipped with a multi-layer, multi-container carrier plate platform, a plate washer, and a controller programmed to efficiently control the components to perform NULISA multiplex analysis (e.g., see...). Figures 85-90Specifically, this compact instrument is capable of performing NULISA multiplex analysis on large numbers of samples (e.g., more than 48 samples in parallel) with fast processing times (e.g., less than 8 hours, 7 hours, or 6 hours), and processing times for the dual capture and release process section are at least 3 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hours. It achieves the high purity required for high sensitivity, for example, below 150 atmol, 100 atmol, or 50 atmol, by combining a mixer and a plate washer with paramagnetic beads to efficiently utilize (and reuse) consumables such as pipette tips and multi-container plates. The instrument section utilizes a mixer to transfer samples (i.e., solutions containing specific analytes or target analytes or analytes captured by immune complexes), minimizing the steps requiring pipette tip contact with the sample in the process, thus consuming fewer pipette tips. Similarly, the instrument utilizes a plate washer to clean multi-container plates in the process, allowing for plate reuse and reducing the number of plates required in the process.

[0151] In some implementation schemes, refer to Figures 85-86 This compact instrument efficiently executes processes from the initial step where a sample is placed into a multi-container plate (first plate) to form an immune complex solution, until a second purified capture immune complex containing a nucleic acid reporter molecule is mixed with the elution solution in a multi-container plate (third plate), without any pipette tip contact with the sample. For example, see reference... Figure 85 The procedure shown involves a compact instrument processing samples in a first plate (where immune complexes are formed and captured on a first set of paramagnetic beads). Samples are then transferred to a second plate via the first set of paramagnetic beads for washing, followed by a transfer back to a reusable first plate (washed with a plate washer) for an optional second wash. Samples are then transferred to a third plate via the first set of paramagnetic beads (where immune complexes are eluted from the first set of paramagnetic beads, the first paramagnetic beads are removed from the third plate, and the sample solution containing immune complexes to be captured on the second set of paramagnetic beads remains in the third plate). Samples are then transferred to a reusable second plate via the second set of paramagnetic beads and washed. Samples are then transferred to a fourth plate via the second set of paramagnetic beads (for ligation to form nucleic acid reporter molecules). Samples are transferred back to a reusable second plate (washed again with a plate washer) and washed. Samples are then transferred to a reusable third plate via the second set of paramagnetic beads to elute the second paramagnetic beads. Finally, samples are transferred to PCR plates using pipette tips and placed into the PCR unit within the instrument.

[0152] In some implementations, such as reference Figure 86The procedure shown involves a compact instrument processing samples in a first plate (where immune complexes are formed and captured on a first set of paramagnetic beads). Samples are then transferred to a second plate via the first set of paramagnetic beads for washing, and the samples are transferred back to a reusable first plate (washed with a plate washer) for an optional second wash. Samples are then transferred to a third plate via the first set of paramagnetic beads (where immune complexes are eluted from the first set of paramagnetic beads, the first set of paramagnetic beads are removed from the third plate, and the sample solution containing immune complexes to be captured on the second set of paramagnetic beads remains in the third plate). Samples are then transferred to a fourth plate via the second set of paramagnetic beads and washed. Samples are then transferred to a reusable second plate (washed with a plate washer) via the second set of paramagnetic beads for ligation to form a nucleic acid reporter molecule. Samples are then transferred to a reusable third plate (washed with a plate washer) via the second set of paramagnetic beads to elute the second set of paramagnetic beads. Finally, samples are transferred to a PCR plate using a pipette tip and placed into the PCR unit within the instrument. Attached Figure Description

[0153] Figure 1 The diagram illustrates the "cloud hub" assay ecosystem, which includes cloud-based software programs ("APP") that provide a user interface on any computing device, a set of assay-specific reagents and consumables, and automated assay instruments.

[0154] Figure 2 The diagram illustrates the system-level hardware architecture of the publicly disclosed instrument.

[0155] Figure 3A -C illustrates an exemplary design implementation of the disclosed system.

[0156] Figure 4A -B illustrates a specific design implementation of a storage rack that includes three structurally identical "compartments," each providing shelving space to hold a set of reagents and consumables for performing assay runs.

[0157] Figure 5 A specific implementation of the gantry design is shown, which allows the end effector to move in three degrees of freedom: X, Y, and Z axes.

[0158] Figure 6 A specific embodiment of the end effector is shown, which is equipped with two air displacement pipettes (ADP), a plate clamp, a laser distance sensor, and a barcode scanner.

[0159] Figure 7A -B describes a specific implementation where the relative position between a distance sensor and a specially designed target can be determined in three dimensions. More specifically, Figure 7A -B illustrates the use of an optical distance sensor fixed to its end effector to perform gantry position calibration. Figure 7AThe image shows a distance sensor approaching a target block, with a laser beam passing through the upper edge of the target block. Figure 7B This shows the change in the measured distance (y-axis) as the sensor scans over the top edge of the block.

[0160] Figure 8A- Figure B illustrates a specific embodiment of the stage, providing six plate positions. More specifically, Figure 8A shows a perspective view; and Figure 8B A top view is shown.

[0161] Figure 9 illustrates a specific implementation of the corner actuator mechanism, where each actuator is spring-loaded to the "closed" position. More specifically, Figure 9A- Figure B illustrates an exemplary embodiment of a push mechanism for aligning a plate to the edge of a recess in the platform. In Figure 9A, a cam 9004 below the platform forces a spring-loaded corner pusher 9002 to open, allowing the plate to be inserted into or removed from the recess at one end of its stroke. As the plate moves away from the end of its stroke, the cam 9004 releases the pusher 9002 to push the plate to the corner of the recess.

[0162] Figure 10A -C illustrates a specific implementation of the mechanism used to limit the upward movement of a plate placed in a groove. More specifically, Figure 10A The pusher is shown in the open position, at which point the plate can be inserted into or removed from the groove via the overhanging lugs on the edge. Figure 10B shows the pusher pushing the plate towards the corner of the groove. Figure 10C As shown in detail, the flange of the plate is restricted below the overhang of the lug and cannot move upward.

[0163] Figure 11A -C describes the concept and working principle of a bead processing device with multiple vertically stacked plate platforms. More specifically, Figure 11A A front view of the structure is depicted, showing a magnetic head 11002 with magnetic poles, a plastic comb 11004 with a sleeve, and a measuring plate placed on a vertical positioning platform 11006. Figure 11B The illustration shows a bead handling device when the bead touches the plate on the upper platform, wherein the magnetic pole is inserted into the sleeve 11008. Figure 11C A bead handling device is shown when the bead touches the plate on the lower platform.

[0164] Figure 12A -C illustrates a design concept where the upper measurement plate is placed on a "detachable" platform to further reduce the module's footprint. More specifically, Figure 12A -C describes the concept of a detachable platform. Figure 12A The detachable platform is shown when the board on the upper platform is touched; and Figure 12B -C indicates a detachable platform when the board on the lower platform is touched.

[0165] Figure 13A -B illustrates another concept for the disclosed bead processor, which has a fixed, vertically positioned platform for measuring the plate. An opening is located at the center of the upper platform, allowing magnetic poles and a sleeve to pass through the opening to access the plate on the lower platform. More specifically, Figure 13A and 13B The panels on the accessible upper and lower platforms are shown respectively.

[0166] Figure 14A- F shows Figure 13A -Exemplary design of the fixed plate platform concept illustrated in Figure B.

[0167] Figure 15A -C describes the "ear-like" alignment features on the cannula comb. Figure 15A ) and the mechanism on the disclosed bead processor for aligning the ferrule comb with the magnetic head ( Figure 15B , 15C More specifically, Figure 15A The image shows a comb-shaped sleeve with an "ear-like" structure on a substrate (as indicated by a circle). Figure 15B The diagram shows the engagement of the "finger-like" and "ear-like" structures to align the comb-like components with their carriers. Figure 15B The diagram shows an "ear-shaped" structure 15002 on a comb substrate, a comb-shaped support device 15004, a rotating component 15006, a sleeve 15008, and a "finger-shaped" component 15010 on the rotating component. Figure 15C The "baffle" 15012 is shown engaging with the underside of the comb to secure it to its support.

[0168] Figure 16 The diagram illustrates the basic conceptual structure of the disclosed plate washer, which includes one or more stripe-type dispensing heads 16004 and matrix-type suction heads 16002.

[0169] Figure 17 The illustration shows a matrix-type aspiration head, with partitioned manifolds at the top of the needle matrix, each manifold connecting to a portion of the needles in the entire matrix.

[0170] Figure 18 An exemplary system architecture for the disclosed cleaner is depicted, comprising two distribution strips, wherein distribution strip #1 is shared by buffers 1 & 2 (B1 & B2) and selected via a three-way valve, and distribution strip #2 is dedicated to buffer 3. The suction manifold is divided into two independent sections, each connected to a suction pump.

[0171] Figure 19A -B illustrates an innovative method for eliminating fluid residue trapped in dead zones of the suction manifold without increasing the power of the suction pump. More specifically, Figure 19A This shows that when liquid is drawn from one end or the center of the manifold, residue 19002 is produced in the "dead zone" section of the manifold. Figure 19B This shows “residue-free” aspiration of liquid from both ends of the manifold.

[0172] Figure 20A -B illustrates a specific implementation of the system-level piping and instrumentation diagram (PID) for the disclosed plate washer. More specifically, Figure 20A and 20B The dispensing side and the aspiration side are shown separately.

[0173] Figures 21A and 21B illustrate details of the disclosed design embodiment of the plate washer. More specifically, Figures 21A and 21B show perspective and side views of the plate washer.

[0174] Figure 22 illustrates the conceptual structure of the disclosed incubator & sealer module, which includes an upper and lower temperature control block and a heat-insulating shell.

[0175] Figure 23 illustrates the operation process of the disclosed incubation & sealing module for incubation.

[0176] Figure 24A -E illustrates the operational procedure of the disclosed incubation & sealing module for sealing test plates or sample plates.

[0177] Figure 25A- D illustrates an exemplary design of the disclosed incubator & sealer module.

[0178] Figure 26A- Figure E illustrates a specific implementation of a high-capacity station, including a high-capacity station door, a high-capacity nest, and a tubular manifold. More specifically, Figure 26A shows a high-capacity station (door closed) and a tubular manifold carrying tubing immersed in a high-capacity fluid bottle (the door panel has been removed for clarity). Figure 26B The tubular manifold is shown rising to open the large-capacity station door (plate removed). The tubing is lifted from the large-capacity fluid bottle, allowing the bottle to be removed or inserted into its respective nest. Figure 26C An overall view of the safety switch mechanism is shown. Figure 26D The safety switch mechanism is shown in the extended "safe" position, at which point the door position can be raised or lowered (switch "open"). Figure 26E The safety switch mechanism is shown in the compressed "stop" position, at which point the door is lowered to the barrier position (switch "closed").

[0179] Figure 27 The illustration shows a specific embodiment of the frame design in a disclosed framed sealing membrane (FSF). [The membrane is preferably mounted on the frame.] Figure 28 A specific implementation design of the perforated pattern on the disclosed FSF is shown.

[0180] Figure 29A-C illustrates a specific design implementation of a publicly disclosed generic reagent cartridge, including a base, cap, and several different types of reagent containers. More specifically, Figure 29A The components of the box are shown, including container 29002, TRIB 29004, MONO 29006, base 29008, and lid 29010. Figure 29B The base containing the container is depicted. Figure 29C The assembled box is depicted.

[0181] Figure 30A -C illustrates a specific embodiment of the disclosed consumable carrier. More specifically, Figure 30A The container with a lid and seven consumable items is shown. Figure 30B A locking cap with spring-loaded fingers is shown to accommodate a stack of consumables for transport. Figure 30C This shows the consumable carrier ready to be loaded into the instrument after the cover has been removed.

[0182] Figure 31 The illustration shows a large-capacity fluid bottle with mechanical features to prevent incorrect insertion into instruments and a device to reduce fluid splashing.

[0183] Figure 32 The illustration shows one implementation of the Alamar Biosciences ARGO HT instrument.

[0184] Figure 33 The illustration shows one implementation of the Alamar Biosciences ARGO HT instrument.

[0185] Figure 34 The diagram illustrates the single-weight measurement workflow in the ARGO HT instrument.

[0186] Figure 35 The illustration shows one implementation of the multiplex determination workflow in the ARGO HT instrument.

[0187] Figure 36 The illustration shows the buttons, icons, and symbols of the ARGO HT software.

[0188] Figure 37 The diagram illustrates the buttons, icons, and symbols of the Alamar NULISA Analysis (ANA) software.

[0189] Figure 38 The image shows a front view of the Alamar Biosciences ARGO HT instrument.

[0190] Figure 39 The illustration shows the compartmental components of the ARGO HT instrument.

[0191] Figure 40 The illustration shows the large-capacity fluid assembly of the ARGO HT instrument.

[0192] Figure 41 The illustration shows a rear view of the ARGO HT instrument.

[0193] Figure 42 The image shows the ARGO HT main screen after startup.

[0194] Figure 43 The diagram illustrates the single-weight measurement workflow in the ARGO HT instrument.

[0195] Figure 44 The image shows the home screen of the Alamar NULISA analysis (ANA) software.

[0196] Figure 45 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software.

[0197] Figure 46 The diagram shows the experimental interface (with selected experiments) of the Alamar NULISA analysis (ANA) software.

[0198] Figure 47 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying well assignment).

[0199] Figure 48 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (showing the function to create a new project).

[0200] Figure 49 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying the function to create a new experiment).

[0201] Figure 50 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying the experimental editing function).

[0202] Figure 51 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying the experimental scheduling function).

[0203] Figure 52 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying scheduled experiments).

[0204] Figure 53The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying a prompt to load the experiment into the first compartment).

[0205] Figure 54 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (indicating that the experiment has been loaded into the first compartment and is ready to run).

[0206] Figure 55 The image shows the experimental progress bar of the Alamar NULISA analysis (ANA) software.

[0207] Figure 56 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (showing the experimental setup and uninstallation).

[0208] Figure 57 The illustration shows an overview of the reagent loading in the compartments of the ARGO HT instrument.

[0209] Figure 58 The illustration shows the compartment of the ARGO HT instrument (which displays the loading of the suction head box).

[0210] Figure 59 The illustration shows the compartment of the ARGO HT instrument (which displays the loading of the consumables box).

[0211] Figure 60 The illustration shows the compartments of the ARGO HT instrument (which display the loading of samples and reagent kits).

[0212] Figure 61 The diagram shows the experimental interface of the Alamar NULISA analysis (ANA) software (displaying options for viewing experimental results).

[0213] Figure 62 The image shows the experimental results interface of the Alamar NULISA analysis (ANA) software (displaying a prompt indicating the loading of results).

[0214] Figure 63 The image shows the experimental results loading interface of the Alamar NULISA analysis (ANA) software.

[0215] Figure 64 The image shows the Alamar NULISA analysis (ANA) interface (with selected results).

[0216] Figure 65 The diagram shows the Alamar NULISA analysis (ANA) interface (with QC results).

[0217] Figure 66The Alamar NULISA analysis (ANA) interface is illustrated (with heatmap results).

[0218] Figure 67 The diagram shows the Alamar NULISA analysis (ANA) interface (with a results table).

[0219] Figure 68 The image shows the Alamar NULISA Analyzer (ANA) instrument interface (with selected instruments).

[0220] Figure 69 The image shows the Alamar NULISA Analyzer (ANA) instrument interface (with selected instruments).

[0221] Figure 70 The image shows the Alamar NULISA Analyzer (ANA) instrument interface (with selected experiments).

[0222] Figure 71 The diagram shows the Alamar NULISA analysis (ANA) settings interface (with user group tab selection).

[0223] Figure 72 The diagram shows the Alamar NULISA analysis (ANA) settings interface (with the option to add and remove group members).

[0224] Figure 73 The diagram shows the Alamar NULISA analysis (ANA) settings interface (with instrument tab selection).

[0225] Figure 74 The image shows the "Account Settings" drop-down menu.

[0226] Figure 75 The image shows the "Manage Your Account" interface.

[0227] Figure 76 The image shows the "User Management" interface.

[0228] Figure 77 The image shows the "Create New User" interface.

[0229] Figure 78 The image shows the "User Details" interface.

[0230] Figure 79 The image shows the "Upload User Files" dialog box.

[0231] Figure 80 The image shows a "template file" in Microsoft Excel.

[0232] Figure 81 The illustration shows a large-capacity fluid assembly.

[0233] Figure 82 The illustration shows the removal of the waste liquid bottle.

[0234] Figure 83 The illustration shows how to remove the consumable bottle.

[0235] Figure 84A The illustration shows a quick reference guide for running the experiment.

[0236] Figure 84B The diagrams provide a quick reference guide for analyzing data, turning instruments on and off, and maintaining them.

[0237] Figure 85 The diagram illustrates the steps involved in designing a compact, fully automated instrument to perform NULISA multiplex analysis.

[0238] Figure 86 The diagram illustrates the steps involved in designing a compact, fully automated instrument to perform NULISA multiplex analysis.

[0239] Figure 87 The diagram illustrates the steps involved in designing a compact, fully automated instrument to perform NULISA multiplex analysis.

[0240] Figure 88 The diagram illustrates the steps involved in designing a compact, fully automated instrument to perform NULISA multiplex analysis.

[0241] Figure 89 The diagram illustrates the steps of a compact, fully automated instrument designed to perform the NULISA dual capture and release multiple mechanisms.

[0242] Figure 90 The diagram illustrates the steps involved in designing a compact, fully automated instrument to perform NULISA multiplex analysis.

[0243] Figure 91A The figure shows the standard curve of IFNL1 generated on the ARGO HT instrument.

[0244] Figure 91B The figure shows the standard curve of CSF2 generated on the ARGO HT instrument. Detailed Implementation

[0245] definition Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms. Generally, the nomenclature and techniques used herein in molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art.

[0246] As used herein, the term “detection” or its grammatical equivalents are broadly used to include any means of determining the presence (i.e., whether) of an analyte or of any form of measurement of an analyte. Thus, “detection” can include determining, measuring, or evaluating the presence or absence, or quantity, or location of an analyte. This includes quantitative, semi-quantitative, and qualitative determinations, measurements, or evaluations. Such determinations, measurements, or evaluations can be relative (e.g., when detecting two or more different analytes in a sample) or absolute. Therefore, when used in the context of quantifying a target analyte in a sample, the term “quantification” can refer to absolute quantification or relative quantification. Absolute quantification can be achieved by adding one or more control analytes at known concentrations and / or by comparing the detection level of the target analyte with a known control analyte (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection levels or quantities of two or more different target analytes to provide a relative quantification (i.e., relative to each other) of each of the two or more different analytes.

[0247] As used herein, the term "analyte" can be any substance (e.g., a molecule) or entity to be detected by the assay methods provided herein. An analyte is the target of the assay methods provided herein. Therefore, an analyte can be any biomolecule or chemical compound to be detected, such as a peptide or protein, nucleic acid molecule, or small molecule (including organic and inorganic molecules). An analyte can be a cell or microorganism (including a virus), or a fragment or product thereof. An analyte can be any substance or entity for which specific binding agents can be developed and which can bind at least two "binding agents" simultaneously. In some embodiments, the analyte is a protein or polypeptide. Therefore, analytes of interest include protein molecules such as polypeptides, proteins, or prions, or any molecule or fragment thereof containing a protein or polypeptide component. In some embodiments, the analyte is a complete or partial protein molecule. An analyte can also be a single molecule or a complex comprising two or more molecular subunits that can be covalently or non-covalently bound to each other and can be the same or different. Therefore, analytes detectable by the assay methods described herein can be complex analytes (which can be protein complexes). Such complexes can be homopolymers or heteropolymers. Aggregates of molecules (e.g., proteins) can also be target analytes. Aggregate analytes can be aggregates of the same protein or different proteins. Analytes can also be complexes composed of proteins or peptides, or nucleic acid molecules (such as DNA or RNA). In some embodiments, the analyte is a complex composed of proteins and nucleic acids, for example, regulatory factors such as transcription factors.

[0248] As used herein, the term "sample" can be any biological and clinical sample, including, for example, any cell or tissue sample of an organism, or any bodily fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc. It also includes environmental samples, such as soil and water samples, or food samples. Samples can be freshly prepared or pretreated by any convenient means (e.g., for storage).

[0249] Representative samples therefore include any material containing biomolecules or other desired or target analytes, including, for example, food and related products, clinical and environmental samples. Samples can be biological samples, including viral or cellular material (including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts, and organelles). Such biological material includes all types of mammalian and non-mammalian animal cells, plant cells, algae (including cyanobacteria), fungi, bacteria, protozoa, etc. Representative samples also include whole blood and blood-derived products (such as plasma, serum, and erythrocyte sedimentation rate), blood cells, urine, feces, cerebrospinal fluid, or any other bodily fluid (e.g., respiratory secretions, saliva, breast milk, etc.), tissues, biopsy samples, cell cultures, cell suspensions, conditioned media, or other samples of cell culture components. Samples can be pretreated in any convenient or desired manner to prepare them for the methods disclosed herein. For example, samples can be treated by cell lysis or purification, analyte separation, etc.

[0250] As used herein, the term “binding” or its grammatical equivalent refers to an interaction between molecules (e.g., a binding agent and an analyte, or a presenting group and a receiving group) that forms a complex. Interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. As used herein, a “binding agent” in relation to an analyte is any molecule or entity capable of binding to an analyte. In some embodiments, the binding agent binds specifically to its target analyte, i.e., the binding affinity of the binding agent to the target analyte is higher than its binding affinity to other components in the sample. In some embodiments, the binding of the binding agent to the target analyte can be distinguished from non-target analytes because the binding agent either does not bind to non-target analytes, or the binding is negligible or undetectable, or any such non-specific binding (if it occurs) is at a relatively low level (distinguished). The binding between the target analyte and its binding agent is typically non-covalent. The binding agents used in the methods provided herein can be covalently conjugated to a presenting group (e.g., a nucleic acid tag) without substantially reducing the binding affinity of the binding agent to its target analyte.

[0251] A binding agent with a high binding affinity to the target analyte can be selected. In some embodiments, the binding affinity between the binding agent and the target analyte can be at least about 10. -4 M, at least about 10 -6M, or at least 10 -9 M or higher. The binder can be a variety of different types of molecules, as long as it exhibits the required binding affinity to the target analyte. In other embodiments, the binder can have moderate or even low affinity to its target analyte, for example, below about 10. -4 M.

[0252] The conjugate can be a macromolecule. In some embodiments, the conjugate is an antibody, or a binding fragment, derivative, or mimic thereof. When antibodies are the conjugate, they can be derived from polyclonal compositions (where heterogeneous antibody populations with different specificities are each conjugated to the same presenting group) or monoclonal compositions (where homologous homogeneous antibody populations with the same specificity for the target analyte are each conjugated to the same presenting group). Therefore, the conjugate can be a monoclonal antibody or a polyclonal antibody.

[0253] In some implementations, the binder is an antibody fragment, derivative, or mimic, wherein these fragments, derivatives, and mimics possess the required binding affinity to the target analyte. Such antibody fragments or derivatives typically contain at least V of the target antibody. H and V L To preserve the binding properties of the target antibody. In some embodiments, the binder is an antibody fragment that binds the analyte. As used herein, an antibody fragment refers to a molecule that, in addition to the complete antibody, contains a portion of the antibody and is typically an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, microantibodies, bivariate antibodies (DVD), monovariate antibodies (e.g., camel-derived antibodies, alpaca-derived antibodies), monovariate (VHH) of heavy chain antibodies, and multispecific antibodies formed from antibody fragments. In some embodiments, the binder is Fab. In some embodiments, the binder is scFv. In some embodiments, the binder is a monovariate antibody.

[0254] In some embodiments, the binder is an antibody mimic. An antibody mimic can be a molecule similar to an antibody, capable of specifically binding to an antigen, but structurally independent of the antibody. Antibody mimics are typically artificial peptides with a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes also considered antibody mimics. Antibody mimics known in the art include avidins, affilins, affimimers, affitin-like proteins, α-antibodies, anti-calmodulin, aptamers, affimimers, DARPin repeats, fynomers, Kunitz domain peptides, monomeric antibodies, and nanoclamps (nanoCLAMP).

[0255] In some implementations, polynucleotide aptamers are suitable as binding agents. Polynucleotide aptamers can be RNA oligonucleotides, which can selectively bind proteins in a manner very similar to receptors or antibodies (Conrad et al.). Methods Enzymol (1996), 267 (Combinatorial Chemistry), 336-367). The antibodies, fragments, derivatives and mimics described above are available from commercial sources and / or can be prepared using any convenient technique, wherein methods for producing polyclonal antibodies, monoclonal antibodies, fragments, derivatives and mimics (including recombinant derivatives thereof) are known to those skilled in the art (e.g., U.S. Patent Nos. 5,851,829 and 5,965,371).

[0256] In addition to antibody-based peptides / peptides or protein-based binding domains, the binding agent can also be a lectin, a soluble cell surface receptor or its derivative, an affinity compound, or any combination of derived proteins or peptides from phage display or ribosome display, or any type of combination peptide or protein library.

[0257] The binder can also be a ligand. Ligand binders can have different sizes. In some embodiments, the ligand binder has a size of about 50 to about 10,000 Daltons, about 50 to about 5,000 Daltons, or about 100 to about 1,000 Daltons. In some embodiments, the ligand binder has a size with a molecular weight of about 10,000 Daltons or greater.

[0258] In some embodiments, the binder is a small molecule capable of binding to the target analyte with the desired affinity. The small molecule can be an organic small molecule. The small molecule may contain one or more functional groups required for structural interactions with the target analyte, such as groups required for hydrophobic, hydrophilic, electrostatic, or even covalent interactions. When the target analyte is a protein, the small molecule binder may contain functional groups required for structural interactions with the protein, such as hydrogen bonds, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and typically contains at least an amino, amide, thiol, carbonyl, hydroxyl, or carboxyl group. In some embodiments, at least two of the above functional groups are contained. The small molecule binder may also contain regions that can be modified and / or participate in covalently linking to a presenting group (e.g., a nucleic acid tag) without substantially adversely affecting the ability of the small molecule to bind to its target analyte.

[0259] Small molecule binders may also comprise cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the aforementioned functional groups. Small molecule binders may also comprise structures found in biomolecules, including peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations thereof. Such compounds can be screened to identify compounds of interest. Various screening schemes are known in the art.

[0260] Small molecule binders can also be derived from naturally occurring or synthetic compounds, which can be obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, various methods exist for the random and directed synthesis of a wide range of organic compounds and biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are readily available or easily prepared. Furthermore, libraries and compounds generated naturally or synthetically can be easily modified using conventional chemical, physical, and biochemical methods and can be used to generate combinatorial libraries. Known small molecules can be chemically modified in a directed or random manner, such as by acylation, alkylation, esterification, and amidation, to produce structural analogs. Therefore, small molecule binders can be obtained from libraries of naturally occurring or synthetic molecules, including libraries of compounds generated through combinatorial processes (i.e., libraries of compound diversity). When obtaining small molecule binders from such libraries, convenient binding affinity assays are needed to screen for small molecule binders that exhibit some of the desired affinity for protein targets.

[0261] The assay methods provided herein use a first and a second binder that binds a non-interfering "epitope" of the analyte. As understood in the art, an epitope of an analyte is a site on the surface of the analyte where a binder binds. An epitope can be a localized region on the surface of the analyte. An epitope can consist of chemically active surface groups of a molecule, such as an amino acid or sugar side chain. An epitope can have specific three-dimensional structural features and specific charge features. An epitope can be a continuous fragment of an analyte molecule. An epitope can also be a molecule composed of more than one discontinuous fragment of an antigen linked together. If the analyte is a polypeptide or protein, its epitope can include continuous or discontinuous sequences along the primary sequence of the polypeptide chain. In some embodiments, the first and second binders used in the assay methods disclosed herein are molecules of the same type. For example, both the first and second binders can be monoclonal antibodies that bind a non-interfering epitope of the analyte. In some embodiments, the first and second binders can be different. For example, the first binder can be an antibody, and the second binder can be a small molecule.

[0262] The term "binding portion," when used to refer to an analyte, refers to a portion comprising one or more molecules such that the portion as a whole can specifically bind to the analyte. A binding portion may include one or more binding agents, one or more target markers, one or more sample markers, and / or one or more presenting groups. A binding portion may also include binding agents, target markers, sample markers, and / or presenting groups. Alternatively, a binding portion may include binding agents, target markers, and / or presenting groups. Molecules in a binding portion may be held together as a portion by a combination of covalent, non-covalent, or covalent and non-covalent molecular interactions. Alternatively, molecules in a binding portion may be held together by interactions with molecules of a portion of a non-binding portion (e.g., the analyte or one or more receiver groups). Furthermore, molecules in a binding portion may be held together as a portion by (i) interactions between molecules within the binding portion and (ii) interactions with molecules of a portion of a non-binding portion (e.g., the analyte or one or more receiver groups). In one embodiment, the binding portion comprises or is composed of a binding agent. In some embodiments, the binding portion comprises or is composed of a target marker. In some embodiments, the binding portion comprises or is composed of a presenting group. In other embodiments, the binding portion comprises or is composed of a sample marker. In one embodiment, the binding portion comprises a binder and a target marker or is composed of a binder and a target marker. In some embodiments, the binding portion comprises a binder and a presenting group or is composed of a binder and a presenting group. In some embodiments, the binding portion comprises a binder and a sample marker or is composed of a binder and a sample marker. In a further embodiment, the binding portion comprises a target marker and a presenting group or is composed of a target marker and a presenting group. In one embodiment, the binding portion comprises a target marker and a sample marker or is composed of a target marker and a sample marker. In other embodiments, the binding portion comprises a presenting group and a sample marker or is composed of a presenting group and a sample marker. In other embodiments, the binding portion comprises a binder, a target marker, and a presenting group or is composed of a binder, a target marker, and a presenting group. In some embodiments, the binding portion comprises a binder, a target marker, and a sample marker or is composed of a binder, a target marker, and a sample marker. In some embodiments, the binding portion comprises or consists of a binder, a presenting group, and a sample marker. In some embodiments, the binding portion comprises or consists of a target marker, a presenting group, and a sample marker. In other embodiments, the binding portion comprises or consists of a binder, a target marker, a presenting group, and a sample marker. In some embodiments, the binding portion comprises or consists of any one of a binder, a target marker, a presenting group, and a sample marker.In some embodiments, the binding portion comprises any two (in any combination or arrangement) of a binder, a target marker, a presenting group, and a sample marker, or is composed of any two (in any combination or arrangement) of a binder, a target marker, a presenting group, and a sample marker. In some embodiments, the binding portion comprises any three (in any combination or arrangement) of a binder, a target marker, a presenting group, and a sample marker, or is composed of any three (in any combination or arrangement) of a binder, a target marker, a presenting group, and a sample marker. In some embodiments, the binding portion comprises all four of a binder, a target marker, a presenting group, and a sample marker, or is composed of all four of a binder, a target marker, a presenting group, and a sample marker.

[0263] The terms “presenting group” and “receiving group” are used interchangeably herein to refer to a binding pair that can form a complex under suitable conditions. As used in the assay methods disclosed herein, the presenting group may be conjugated to a binder of the target analyte, and the receiving group may be coupled to a solid surface. Therefore, the binding between the presenting group and the receiving group allows the analyte to be trapped on the solid surface. In some embodiments, the bond formed between the presenting group and the receiving group is “releasable,” allowing the trapped binder to be released from the solid surface. In some embodiments, the bond formed between the presenting group and the receiving group is “regenerative,” allowing the binder to be recaptured on another solid surface coupled with the same receiving group. Similar to the binding pair of “binder” and “analyte” discussed above, the binding pair of “presenting group” and “receiving group” can take many forms. Examples of binding pairs of "presenting group" and "receiving group" include, but are not limited to, antigens and antibodies against the antigen (including fragments, derivatives, or mimics thereof), ligands and their receptors, complementary nucleic acid strands, biotin and avidin (or streptavidin or neutral avidin), lectins and carbohydrates, and reverse pairings of the above combinations. Other binding pairs of "presenting group" and "receiving group" include fluorescein and antifluorescein, digoxigenin / anti-digoxigenin, and DNP (dinitrophenol) / anti-DNP, and reverse pairings of the above combinations. In some embodiments, the binding pair of "presenting group" and "receiving group" is a complementary nucleic acid strand and is referred to as a "tag" and a "probe." In some embodiments, the binding pair of "presenting group" and "receiving group" is an antigen and antibody, or an antigen and antibody fragment.

[0264] The term "target marker" refers to a portion that facilitates the detection and identification of a target molecule. The term "sample marker" refers to a portion that facilitates the detection and identification of the sample source of the target. Markers applicable to target and sample markers include markers that provide an identifier that can be associated with a specific target or sample. Common markers that can be used for target and / or sample markers in the context of this disclosure are nucleotide sequences that can be associated with a target or sample by sequencing. In some embodiments, the target marker includes a target ID. In some embodiments, the target marker consists of a target ID. In other embodiments, the target marker is a target ID. In some embodiments, the sample marker includes a sample ID. In some embodiments, the sample marker consists of a sample ID. In other embodiments, the sample marker is a sample ID. Other markers applicable to target and sample markers in this disclosure include other molecules containing identifiable or relevant information, such as fluorescent molecules or combinations or sequences of fluorescent molecules, and / or colorimetric portions or combinations or sequences of colorimetric portions. Other markers considered in this disclosure include luminescent markers, light scattering markers, radionuclides, substrates, cofactors, inhibitors, chemiluminescent portions, magnetic particles, etc. Patents teaching the use of such markings include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many of these markings are commercially available and may be used in the context of this invention.

[0265] The term "identification barcode" or "ID," when used to refer to a target or sample, refers to a molecule or series of molecules that can be used to directly or indirectly identify a target or sample through identification information contained within the molecule or series of molecules. Such an ID can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a fluorescent label sequence, a colorimetric label sequence, or any other molecule or combination of molecules, provided that the molecule or combination of molecules used as an ID can identify or otherwise distinguish a particular target or sample from other targets or samples, and is associated with the intended target or sample. Nucleic acid molecules used as such IDs are also called barcode sequences. Such IDs can also be additional derived molecules containing information derived from but different from the original ID, provided that such derived molecules or derived information can identify or otherwise distinguish a particular target or sample from other targets or samples, and is associated with the intended target or sample. For example, a nucleic acid ID may include the original nucleic acid barcode sequence and / or its inverse complementary sequence, as both can distinguish the target or sample and are associated with the intended target or sample. A barcode sequence can be any natural or non-natural sequence that does not exist if it is not introduced as a barcode sequence into the intended sample, intended target, or any part of the intended sample or target, enabling the barcode sequence to identify and be associated with the sample or target. The barcode sequence may be unique to a single nucleic acid species within a population, or it may be shared by several different nucleic acid species within the population. Each nucleic acid probe in the population may contain a barcode sequence different from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in the population may contain a barcode sequence different from some or most of the other nucleic acid probes in the population. For a specific example, all reporter molecules generated from an immune complex of a sample may have the same sample barcode sequence (sample ID). For another example, all reporter molecules generated from an immune complex of the same sample may have different target barcode sequences (target IDs). Furthermore, all reporter molecules generated from the same sample, targeting the same target, and using the same binding agent with an immune complex may have the same target barcode sequence (target ID).

[0266] The terms “desktop” or “compact” when used to refer to a system, instrument, or housing or enclosure mean a system, instrument, or housing or enclosure with a base depth (from front to back) not exceeding 36 inches, 32 inches, 30 inches, 28 inches, 26 inches, 24 inches, 22 inches, or 20 inches. For example, in some embodiments, the benchtop system or instrument provided herein may have a base depth (from front to back) not exceeding 36 inches, 32 inches, 30 inches, 28 inches, 26 inches, 24 inches, 22 inches, or 20 inches, and a height (from top to base) not exceeding 50 inches, 48 ​​inches, 46 inches, 44 inches, 42 inches, 40 inches, 38 inches, 36 inches, 34 inches, 32 inches, or 30 inches.

[0267] In some implementations, the base depth (from front to back) of the desktop system or instrument provided herein may not exceed 36 inches, 32 inches, 30 inches, 28 inches, 26 inches, 24 inches, 22 inches, or 20 inches; the height (from top to base) may not exceed 50 inches, 48 ​​inches, 46 inches, 44 inches, 42 inches, 40 inches, 38 inches, 36 inches, 34 inches, 32 inches, or 30 inches; and the width (from left to right) may not exceed 50 inches, 48 ​​inches, 46 inches, 44 inches, 42 inches, 40 inches, 38 inches, 36 inches, 34 inches, 32 inches, or 30 inches.

[0268] The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0269] The assay method presented herein overcomes the limitations of existing immunoassays and enables single-molecule detection of immune complexes via nucleic acid-based signal amplification. The immune complex comprises a specific analyte bound between a first antibody or fragment of a first antibody in a first portion and a second antibody or fragment of a second antibody in a second portion, wherein the second portion may be pre-selected as paired or complementary to the first portion. Each of the first and second antibodies binds a non-overlapping target epitope on the target analyte. Thus, the analyte is “sandwiched” between the two antibodies. The method presented herein reduces background signal through a capture-and-release mechanism. Accordingly, an assay method for detecting analytes in a sample incorporating a capture-and-release mechanism is provided. In some embodiments, the capture-and-release mechanism is based on the hybridization and dissociation of nucleic acid pairs.

[0270] The assay method presented herein uses a capture-and-release mechanism to reduce nonspecific background signal. The method of capturing immune complexes to a solid surface and releasing them back into solution can be applied to the different assay formats disclosed herein.

[0271] In some embodiments, the assay methods provided herein utilize a capture-and-release mechanism involving two binders, each capable of being captured by two receiving groups on two solid surfaces. In some embodiments, the assay methods provided herein for detecting analytes in a sample include the following steps: (1) A first binder, a second binder and a sample are mixed in a solution, wherein the first binder and the second binder bind non-interfering epitopes on the analyte and form an immune complex, wherein the immune complex is captured on a first solid surface in contact with the solution by binding between a first presenting group conjugated to the first binder and a first receiving group coupled to the first surface. (2) Clean the surface of the first solid to remove unbound molecules; (3) By disrupting the binding between the first presenting group and the first receiving group, an immune complex is released from the first solid surface; (4) Introduce a second solid surface and recapture the immune complex through the binding between the second presenting group conjugated to the second binder and the second receiving group coupled to the second solid surface; (5) Clean the surface of the second solid to remove unbound molecules; and (6) Detect immune complexes.

[0272] The capture / release of the first binder (“Binder 1”) and / or from the first solid surface (“Surface 1”), and the capture / release of the second binder (“Binder 2”) and / or from the second solid surface (“Surface 2”), are achieved through two bioorthogonal bonds (i.e., each independent and specific) between the presenting group (“PG”) and the receiving group (“RG”). The bond between the first presenting group (“PG1”) and the first receiving group (“RG1”) (i.e., the first bond (“Bond 1”)) is releasable. In some embodiments, the bond between the second presenting group (“PG2”) and the second receiving group (“RG2”) (i.e., the second bond (“Bond 2”)) is also releasable, and the immune complex can be detected on or after release from Surface 2. In some embodiments, Bond 2 is not releasable, and the immune complex can be detected on Surface 2.

[0273] Those skilled in the art will understand that an additional round or more of capture / release will further reduce nonspecific background signals. In some embodiments, bond 1 is regenerable and at least one additional round of capture / release can be performed by binder 1. Specifically, the immune complex released from surface 2 can be recaptured by the new surface 1 by forming another bond between PG1 on binder 1 and RG1 on the new surface 1. In some embodiments, bond 2 is regenerable and at least one additional round of capture / release can be performed by binder 2. Specifically, the immune complex released from surface 1 or surface 2 can be recaptured by the new surface 2 by forming another bond between PG2 on binder 2 and RG2 on the new surface 2. In some embodiments, both bond 1 and bond 2 are regenerable and more than one recapture cycle can be performed by bond 1, bond 2, or both. In some embodiments, both bond 1 and bond 2 are non-regenerable and only one capture / release cycle is performed.

[0274] In some embodiments, the releasable and regenerable bond is formed via nucleic acid hybridization, wherein the presenting group and the receiving group comprise complementary nucleic acids. In some embodiments, the presenting group is a nucleic acid that can bind to a receiving group that serves as a DNA / RNA-specific protein or aptamer binding partner (e.g., US5,312,730). In some embodiments, the receiving group is a nucleic acid that can bind to a presenting group that serves as a DNA / RNA-specific protein or aptamer binding partner.

[0275] After the immune complex is formed, the first capture is performed.

[0276] In some embodiments, the benchtop system or instrument provided herein may perform a first capture of at least one immune complex of multiple immune complexes after multiple immune complexes have been formed in at least one or more samples by introducing a plurality of first paramagnetic beads into at least one of multiple samples, into at least two of multiple samples, into at least three of multiple samples, into at least four of multiple samples, into at least five of multiple samples, into at least ten of multiple samples, into at least twenty of multiple samples, into at least fifty of multiple samples, and incubating at least one of multiple samples, into at least two of multiple samples, into at least three of multiple samples, into at least four of multiple samples, into at least five of multiple samples, into at least ten of multiple samples, into at least twenty of multiple samples, into at least fifty of multiple samples with the plurality of first paramagnetic beads for less than 90 minutes, less than 80 minutes, less than 70 minutes, less than 65 minutes, less than 60 minutes, less than 55 minutes, less than 50 minutes, less than 45 minutes, or less than 40 minutes.

[0277] First capture & release: In some embodiments, the benchtop system or instrument provided herein can perform immune complex extraction from at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, or at least fifty of a plurality of samples after multiple immune complexes have been formed in at least one or more samples: by capturing at least one immune complex onto at least one of a plurality of first paramagnetic beads, and removing, washing, and reintroducing the captured one or more immune complexes from at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, or at least fifty of a plurality of samples into at least one, at least two, or at least three of a plurality of second solutions. In at least four, at least five, at least ten, at least twenty, or at least fifty of a plurality of second samples; wherein at least one or at least a portion of an immune complex is released from at least one of a plurality of first paramagnetic beads, and at least one of the first paramagnetic beads is removed from at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, or at least fifty of a plurality of second solutions; for a duration of less than 130 minutes, less than 120 minutes, less than 110 minutes, less than 100 minutes, less than 90 minutes, less than 85 minutes, less than 80 minutes, less than 75 minutes, less than 70 minutes, less than 65 minutes, less than 60 minutes, less than 55 minutes, less than 50 minutes, or less than 45 minutes.

[0278] Second capture: In some embodiments, the benchtop system or instrument provided herein may perform a second capture of at least one immune complex of multiple immune complexes after at least one of a plurality of second solutions has been formed (the second solution contains at least one immune complex of multiple immune complexes, and at least one of a plurality of first paramagnetic beads has been removed from the second solution): by means of at least one of the plurality of second solutions, at least two of the plurality of second solutions, at least three of the plurality of second solutions, at least four of the plurality of second solutions, at least five of the plurality of second solutions, at least ten of the plurality of second solutions, or to the plurality of second solutions. Introduce a plurality of second paramagnetic beads into at least twenty of a plurality of second solutions, or into at least fifty of a plurality of second solutions, and incubate at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, or at least fifty of a plurality of second solutions together with the plurality of second paramagnetic beads for a duration of less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes, less than 8 minutes, or less than 5 minutes.

[0279] The assay method disclosed herein includes step (1): mixing a first binding agent, a second binding agent, and a sample in a solution to form an immune complex, wherein the first and second binding agents bind non-interfering epitopes on the analyte, and wherein the immune complex is captured on a first solid surface in contact with the solution via binding between a first presenting group conjugated to the first binding agent and a first receiving group coupled to a first surface. In some embodiments, the first presenting group is a nucleic acid tag, and the first receiving group is a nucleic acid capture probe, wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof.

[0280] As disclosed herein, the binding agent used in the assay method can be any molecule or part of a molecule that binds to a specific target analyte. Therefore, the binding agent can comprise any protein, peptide, nucleic acid, carbohydrate, lipid, or small molecule. In some embodiments, the binding agent comprises an antibody. In some embodiments, the binding agent comprises an antibody fragment. In some embodiments, the binding agent comprises an antibody mimic. In some embodiments, the binding agent comprises a small molecule.

[0281] The binding agents used in the assays disclosed herein can be conjugated to a presenting group (e.g., a nucleic acid tag). The binding agent and the presenting group can be directly linked together by a bond or indirectly linked together by a linker group. When a linker group is used, it can be selected to provide covalent attachment of the presenting group to the binding agent and to maintain the required binding affinity of the binding agent to its target analyte. The linker group can vary depending on the binding agent. The linker group, if present, is generally bioinert. Various linker groups are known to those skilled in the art and can be used in the assays disclosed herein. In some embodiments, the linker group comprises a spacer group having reactive functionality at either end capable of covalently bonding with the presenting group or the binding agent.

[0282] The binding agent / presenting group conjugates used in the assay methods disclosed herein can be prepared using any method known in the art. In some embodiments, the presenting group (e.g., a nucleic acid tag) can be conjugated to the binding agent directly or via a linking group. Components can be covalently bonded to each other via functional groups, as is known in the art, wherein such functional groups can be present on the component or introduced onto the component using one or more steps (e.g., oxidation, reduction, cleavage, etc.). Functional groups that can be used to covalently bond components together include hydroxyl, thiol, amino, etc. Different components can be selectively modified to provide specific covalently linked portions such that the desired binding affinity of the component to the target analyte is not significantly adversely affected. Where necessary and / or desired, blocking groups can be used to protect certain portions of the component, as is known in the art, see, for example, Green & Wuts, Protective Groups in Organic Synthesis (John Wley & Sons) (1991); U.S. Patent No. 5,733,523.

[0283] The presenting group and the receiving group can be any binding pair disclosed herein or otherwise known in the art, including but not limited to antigens and antibodies against such antigens (including fragments, derivatives, or mimics thereof), ligands and their receptors, complementary nucleic acid strands, biotin and avidin (or streptavidin or neutral avidin), lectins and carbohydrates, and reverse pairings of the above combinations. Additional binding pairs of the "presenting group" and "receiving group" include fluorescein and antifluorescein, digoxigenin / anti-digoxigenin, and DNP (dinitrophenol) / anti-DNP, and reverse pairings of the above combinations. In some embodiments, the binding pair of the "presenting group" and "receiving group" is a complementary nucleic acid strand and is referred to as a "tag" and a "probe." In some embodiments, the binding pair of the "presenting group" and "receiving group" is an antigen and antibody, or an antigen and antibody fragment.

[0284] As described above, the samples measurable in the assay methods disclosed herein can be materials or mixtures of materials containing one or more components of interest. In some embodiments, the samples are derived from biological sources. For example, the samples can be obtained from an object, which can be biological tissues or body fluids obtained, collected, or harvested in vivo or in situ. Exemplary samples include biological body fluids, such as blood samples, urine samples, plasma samples, saliva samples, cerebrospinal fluid samples, semen samples, sputum samples, mucus samples, dialysis fluid samples, intestinal fluid samples, synovial fluid samples, and serous fluid samples. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a cerebrospinal fluid sample. Exemplary samples include tissue samples. Tissue samples can be liquid tissue samples. Tissue samples can be homogenized tissue samples. Tissue samples can be obtained from diseased tissue. In some embodiments, the sample is a cancer sample.

[0285] The solid surface may also include any support known in the art for molecular immobilization. In some embodiments, the solid surface may be any surface suitable for attaching nucleic acids and facilitating assay steps. Examples of solid surfaces include beads (e.g., magnetic heads, xMAPs). ® Magnetic heads, particles, colloids, single surfaces, test tubes, chips, multiwell plates, microtiter plates, glass slides, membranes, cuvettes, gels, and resins are all examples of solid surfaces. Exemplary solid surfaces may include the surface of magnetic particles and the pores of a microtiter plate. When the solid phase is a particulate material (e.g., beads), it may be distributed in the pores of the multiwell plate to enable parallel processing. In some embodiments, the solid surface is the surface of magnetic beads. Magnetic beads may be coupled with presenting groups. In some embodiments, the magnetic beads may be carboxylate-modified magnetic beads, amine-blocked magnetic beads, oligodeoxythymidine (Oligo(dT))-coated magnetic beads, streptavidin-coated magnetic beads, protein A / G-coated magnetic beads, or silica-coated magnetic beads. In some embodiments, the solid surface is the pores of a microtiter plate. In some embodiments, the first solid surface and the second solid surface are the same. In some embodiments, the first solid surface and the second solid surface are different. In some embodiments, the first solid surface and the second solid surface used in the assay methods disclosed herein are both surfaces of magnetic particles. In some embodiments, the first solid surface and the second surface used in the determination method disclosed herein are both surfaces of a microtiter plate.

[0286] As described above, the analyte measured in the assay methods disclosed herein can be any biomolecule. In some embodiments, the analyte is a protein analyte. In some embodiments, the analyte is a peptide analyte. In some embodiments, the analyte is a complex comprising at least two molecules. In some embodiments, the analyte is a protein complex comprising at least two proteins. In some embodiments, the analyte is a binding pair of two proteins. In some embodiments, the analyte is a macromolecular complex comprising at least one protein and at least one nucleic acid. In some embodiments, the analyte is a nucleic acid analyte.

[0287] In some embodiments, the first presenting group is a first nucleic acid tag (“first tag”), and the first receiving group is a first nucleic acid capture probe (“first probe”), wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof. In some embodiments, the second receiving group is a second nucleic acid capture probe (“second probe”), wherein the probe or a fragment thereof is complementary to the tag or a fragment thereof.

[0288] The analyte can be a nucleic acid molecule (e.g., DNA and RNA). In some embodiments, the analyte is a DNA molecule. In some embodiments, the analyte is an RNA molecule. The assay methods provided herein can directly detect nucleic acid molecules in samples (such as plasma and urine) without nucleic acid separation. The nucleic acid analyte can hybridize and be captured to a first surface, released into solution, and recaptured to a second surface, while the target-probe complex remains intact throughout the assay procedure. Therefore, in some embodiments, the assay methods provided herein can be used to detect nucleic acid analytes. For example, an assay method for detecting nucleic acid analytes in a sample is provided herein, comprising the following steps: (1) A first binding agent, a second binding agent, and a sample are mixed in a solution, wherein the first binding agent and the second binding agent bind to non-interfering epitopes on the nucleic acid analyte and form an immune complex, and wherein the immune complex is captured on a first solid surface in contact with the solution by binding between a first presenting group conjugated to the first binding agent and a first receiving group coupled to the first surface; wherein the first binding agent and the second binding agent of the nucleic acid analyte are nucleic acids complementary to different fragments of the nucleic acid analyte; (2) Clean the surface of the first solid to remove unbound molecules; (3) By disrupting the binding between the first presenting group and the first receiving group, an immune complex is released from the first solid surface; (4) Introduce a second solid surface and recapture the immune complex through the binding between the second presenting group conjugated to the second binder and the second receiving group coupled to the second solid surface; (5) Clean the surface of the second solid to remove unbound molecules; and (6) Detect immune complexes.

[0289] In some embodiments, the benchtop system or instrument provided herein can perform immune complex formation after multiple samples are dispensed and set into an assay plate: introducing multiple first binding portions and multiple second binding portions into at least one of multiple samples, into at least two of multiple samples, into at least three of multiple samples, into at least four of multiple samples, into at least five of multiple samples, into at least ten of multiple samples, into at least twenty of multiple samples, into at least fifty of multiple samples, and incubating at least one of multiple samples, into at least two of multiple samples, into at least three of multiple samples, into at least four of multiple samples, into at least five of multiple samples, into at least ten of multiple samples, into at least twenty of multiple samples, into at least fifty of multiple samples with the multiple first binding portions and multiple second binding portions for less than 100 minutes, less than 95 minutes, less than 90 minutes, less than 85 minutes, less than 80 minutes, less than 75 minutes, less than 70 minutes, less than 65 minutes, less than 60 minutes, or less than 45 minutes.

[0290] In some embodiments, the detectable biomarker is a nucleic acid, which can be amplified and detected by polymerase chain reaction (PCR). ImmunoPCR technology can be used, which leverages the advantages of nucleic acid technology to perform protein detection by converting the protein analyte detection into a nucleic acid reporter molecule (e.g., US 5,665,539). A nucleic acid segment pre-conjugated to a detection binder can be used as a reporter molecule for an immune complex, and the reporter molecule is amplified using PCR to generate a detectable signal. In some embodiments, a nucleic acid tag for capture / release is detectable, and no additional detectable biomarker is required. In some embodiments, a first tag is used for detection by PCR. In some embodiments, a second tag is used for detection by PCR.

[0291] Nucleic acid reporter molecules can take many forms. For example, a first tag and a second tag can be linked to form a nucleic acid reporter molecule. Those skilled in the art will understand that any reporter molecule generation method disclosed herein or otherwise known in the art can be used for this step, including methods such as ligation, polymerization extension, or co-hybridization. The reporter molecule comprises a first nucleic acid target marker in a first portion linked to a second nucleic acid target marker in a second portion, wherein the second portion may be pre-selected to be paired with or complementary to the first portion. These nucleic acid target markers can be linked directly or indirectly (by directly linking the first and second nucleic acid target markers together, partially linking them together, and / or using a linker sequence) to generate the reporter molecule. The linker sequence may also have a sample-specific target marker, which may be used, for example, for next-generation sequencing.

[0292] In some implementations, the second capture need not be releasable, and a nucleic acid reporter molecule can be generated while the immune complex is captured on the second surface. Alternatively, the immune complex can be released back into solution before the nucleic acid reporter molecule is generated.

[0293] Proximity connectivity assays (PLA) and proximity extension assays (PEA) are known in the art (e.g., US 6,511,809, US 6,878,515, US 7,306,904, US 9,777,315, US 10,174,366, WO9700446, Greenwood C, etc.). Biomol. Det. & Quan. 4 (2015) 10-16). Proximity-based detection differs from immunoPCR in that it relies on the simultaneous recognition of the target analyte by two nucleic acid conjugates to trigger the formation of an amplifiable product. Therefore, a single nucleic acid conjugate that is not part of the immune complex will not generate a reporter molecule, thus avoiding background from a single nonspecifically bound conjugate. In some embodiments, proximity ligation is used to generate nucleic acid reporter molecules, wherein a first tag and a second tag are placed sufficiently close to each other to ligate upon immune complex formation, and a fragment of the ligation product (containing fragments of the first tag and the second tag) is used as an amplicon to generate a detection signal. In some embodiments, proximity extension is used to generate nucleic acid reporter molecules, wherein a first tag and a second tag are placed sufficiently close to each other to interact and form a duplex upon immune complex formation, such that the 3' end of at least one nucleic acid tag in the duplex can be extended to generate an extension product, which can be used as an amplicon to generate a detection signal. In some embodiments, co-hybridization is used to generate nucleic acid reporter molecules, wherein a first tag and a second tag are placed sufficiently close to each other to interact and form a hybridization product upon immune complex formation, which can be used as an amplicon to generate a detection signal.

[0294] Although some assays use proximity linkage, as described above, proximity extension, co-hybridization, or other methods known in the art can also be used to generate nucleic acid reporter molecules for detection.

[0295] The reporter molecule generated in the final step can be detected using any existing nucleic acid detection technology, including but not limited to PCR, quantitative PCR (qPCR), digital PCR (dPCR), or next-generation sequencing (NGS). In some embodiments, the detection is qualitative. In some embodiments, the detection is quantitative. In some embodiments, the nucleic acid reporter molecule is detected by qPCR. In some embodiments, the nucleic acid reporter molecule is detected by dPCR. In some embodiments, the nucleic acid reporter molecule is detected by NGS.

[0296] Each analyte is assigned a unique ID. Therefore, the assay methods provided herein include the simultaneous detection of at least two analytes in a sample by simultaneously detecting the unique target ID associated with each analyte. In some embodiments, the assay methods provided herein involve the simultaneous detection of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, or at least one hundred analytes in a sample by simultaneously detecting the unique target ID associated with each analyte.

[0297] In some embodiments, the analyte is a protein. In other embodiments, the analyte includes at least one protein and at least one nucleic acid. The nucleic acid can be DNA or RNA. The fact is that the assay methods provided herein can be used to analyze proteins, DNA, and RNA, making it an ideal platform for multi-omics analysis.

[0298] In the assay method presented herein, incorporating the ID into the nucleic acid reporter molecule can also help improve the specificity of the assay. Each of the first and second binding agents is associated with a unique ID, and only the signal generated by the reporter molecule containing both binding agent IDs is considered a true signal. This scheme can be used to reduce or eliminate false positive signals caused by cross-reactivity or non-specific binding between different binding pairs. Therefore, this paper provides an assay method for detecting analytes in a sample by co-detecting the first target ID and the second target ID associated with the first and second binding agents, respectively.

[0299] In the assay methods provided herein, incorporating IDs into nucleic acid reporter molecules can also be used to detect interactions between molecules (e.g., protein-protein interactions). Co-detection and quantification of reporter molecules containing two binding agent IDs designed for different molecules can indicate the interaction and affinity of these two related molecules under assay conditions. In some embodiments, the assay methods provided herein detect protein-protein interactions. Therefore, this document provides an assay method for detecting analytes in a sample, wherein the analyte is a binding pair of two different proteins; wherein a first binding agent binds one protein, and a second binding agent binds the other protein in the binding pair; wherein the binding pair is detected by co-detection of the first target ID and the second target ID.

[0300] In addition to an analyte-specific “target ID,” the nucleic acid reporter molecules generated in the assay methods provided herein may also include a sample-specific “sample ID.” When the assay methods provided herein are performed on a specific sample, a sample ID can be introduced during the reporter molecule generation step or an assay prior to that step to identify the sample. Incorporating such a sample ID into the reporter molecule allows for the merging of reporter molecules from multiple samples and parallel reading via NGS. In some embodiments, the sample ID may be carried on a nucleic acid independent of the binding agent tag and incorporated into the reporter molecule during the ligation step. Thus, in some embodiments, the nucleic acid reporter molecule formed in each sample contains an ID as a sample ID, wherein the sample ID is inserted between a first tag or a substitute thereof and a second tag or a substitute thereof.

[0301] In some embodiments, the nucleic acid reporter molecule generated in the assay method provided herein may contain both a target ID and a sample ID. In some embodiments, the nucleic acid reporter molecule contains: (a) a target ID in a first tag or a first substitute, or in a second tag or a second substitute; and (b) a sample ID that (1) is inserted between the first tag or its substitute and the second tag or its substitute, (2) is contained in the first substitute or the second substitute, or (3) is linked to the first tag or its substitute, or the second tag or its substitute.

[0302] In some implementations, the assay method provided herein detects at least three analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter molecule of each sample.

[0303] In some implementations, the assay method provided herein detects at least five analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter molecule of each sample.

[0304] In some implementations, the assay method provided herein detects at least ten analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting a unique sample ID and a unique target ID in the nucleic acid reporter molecule of each sample.

[0305] In some implementations, the assay method provided herein detects at least twenty analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting the unique sample ID and unique target ID in the nucleic acid reporter molecule of each sample.

[0306] In some implementations, the assay method provided herein detects at least fifty analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting the unique sample ID and unique target ID in the nucleic acid reporter molecule of each sample.

[0307] In some implementations, the assay method provided herein detects at least eighty analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting the unique sample ID and unique target ID in the nucleic acid reporter molecule of each sample.

[0308] In some implementations, the assay method provided herein detects at least one hundred analytes simultaneously in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 samples by simultaneously detecting the unique sample ID and unique target ID in the nucleic acid reporter molecule of each sample.

[0309] Because NGS is a single-molecule detection and counting method, sequencing instruments have an upper limit on the total number of molecules that can be sequenced. For example, Illumina's MiSeq system can generate 25 million reads per run, limiting the total number of molecules that can be sequenced in a single run to 25 million. In most applications where targets are present at low concentrations or even close to the limit of detection (“LOD”), this limitation is not restrictive. However, in multiplex assays, some targets are known to have expression levels several orders of magnitude higher than others, consuming sequencing bandwidth without providing useful clinical / biological information. Therefore, there is a need to specifically reduce the signal generated by these high-abundance analytes while maintaining sensitivity to the remaining analytes in multiplex assays.

[0310] The assay method provided herein further meets this need and offers related advantages. In some embodiments, this paper provides an assay method in which the number of reporter molecules generated from high-concentration target analytes is reduced at a precise and known ratio, so as to efficiently allocate limited detection bandwidth to different target analytes. The assay method disclosed herein captures immune complexes to a solid surface using a receiving group (e.g., a nucleic acid capture probe) before reporter molecule generation, which provides a unique opportunity to reduce the signal of high-abundance analytes by selectively capturing only a portion of the immune complexes generated therein to the surface.

[0311] For example, in some embodiments, the binder may be conjugated with or without its presenting group (e.g., a nucleic acid tag) in a known proportion. For instance, if a binder conjugated with a presenting group is mixed with the same binder without a presenting group at a concentration of 1%, only 1% of the immune complex will be captured on the surface, and the ratio of the report molecule to the target analyte will be 1%. Non-functional presenting groups, i.e., presenting groups that do not bind to receiver groups, can also be used. For example, if only 0.1% of the first binder is conjugated to a functional first presenting group (e.g., a first tag), and the remaining 99.9% of the first binder is conjugated to a first presenting group (e.g., a first tag) that cannot be captured by a first receiver group (e.g., a first probe), the ratio of the report molecule to the immune complex will be 1:1000, effectively reducing the signal generated by the analyte to 1 / 1000.

[0312] An alternative to this partial capture method can be used, which introduces a known proportion of nonfunctional receiver groups (e.g., nucleic acid capture probes). For example, in indirect capture methods, a certain proportion of first capture probes may be included, which do not have segments complementary to universal capture probes or are not biotinylated. As a result, the same proportion of immune complexes cannot be captured onto the surface, and therefore no nucleic acid reporter molecule for detection can be generated. For example, if the receiver group contains only 0.1% functional molecules that can be coupled to a solid surface (the remaining 99.9% being nonfunctional dummy molecules), the ratio of reporter molecule to immune complex will also be 1:1000, reducing the signal generated by the analyte to 1 / 1000.

[0313] Accordingly, this document also provides a method for determining the amount of analyte detected by assay by proportionally reducing the amount detected by assay through the addition of a nonfunctional binding agent to the solution in step (1), wherein the nonfunctional binding agent competes with a first binding agent for binding of the analyte, but is unconjugated, or conjugated to a presenting group that does not bind to a first receiving group. In some embodiments, the nonfunctional binding agent is unconjugated. In some embodiments, the nonfunctional binding agent is conjugated to a presenting group that does not bind to a first receiving group. In some embodiments, the nonfunctional binding agent is conjugated to a nucleic acid tag that cannot hybridize with a first probe coupled to a first solid surface.

[0314] Accordingly, this article also provides a method for determining the amount of analyte detected by measurement by adding a nonfunctional receiving group to the solution in step (1), wherein the nonfunctional receiving group competes with the first receiving group for binding to the first presenting group, but cannot couple with the first solid surface.

[0315] In some embodiments, this document also provides a method for proportionally reducing the amount of analyte detected by the assay by adding a nonfunctional binder to the solution in step (1), wherein the nonfunctional binder competes with a first or second binder for binding the analyte but forms an undetectable immune complex. In some embodiments, the assay provided herein detects the immune complex by detecting a detectable biomarker conjugated to a first or second binder, and the nonfunctional binder does not conjugate to the detectable biomarker, or conjugates to a defective detectable biomarker that fails to generate a detection signal. In some embodiments, the immune complex is detected by a nucleic acid reporter molecule, and the nonfunctional binder may conjugate to a nucleic acid tag lacking the appropriate segment required to generate a nucleic acid reporter molecule. Those skilled in the art will understand that various variations of the methods disclosed herein can be employed to proportionally reduce the signal generated by high-abundance analytes in a sample, thereby enabling the simultaneous detection of multiple analytes present at concentration differences of even multiple orders of magnitude.

[0316] IDs include original ID molecules and derived ID molecules (containing information derived from but different from the original ID), provided that such derived molecules or derived information can identify or otherwise distinguish a particular target or sample from other targets or samples, and can be associated with the intended target or sample.

[0317] This disclosure further provides that step (4) of the method provided in section 4.2.5 further includes PCR amplification of a nucleic acid reporter molecule. As will be clear from this disclosure, such PCR may be any PCR provided in this section and other sections (such as section 4.2.3 (including 4.2.3.1 and 4.2.3.2), section 4.2.3 and section 4.2.5), and any suitable PCR known and practiced in the art.

[0318] The binder can indirectly bind the analyte via an intermediate (e.g., a primary antibody against the analyte). Therefore, this is the case in some embodiments of the methods provided herein.

[0319] The first and second binders can bind to epitopes on the analyte that are allowed to bind simultaneously, thereby improving the specificity of the detection. In some embodiments, the first and second binders bind to non-interfering epitopes on the analyte. In other embodiments, the first and second binders bind to non-overlapping epitopes on the analyte. In other embodiments, the first and second binders bind to different epitopes on the analyte. In other embodiments, the first and second binders bind to separate epitopes on the analyte. In still other embodiments, the first and second binders bind to two epitopes on the analyte, with each binder binding to both epitopes simultaneously and separately without any steric hindrance.

[0320] Furthermore, such a first tag, first probe, second tag, and second probe can be provided in a variety of combinations as a front-end. Thus, in one embodiment, the first probe is a protein that specifically binds to the first tag, and the second probe is a protein that specifically binds to the second tag. In another embodiment, the first probe is a protein that specifically binds to the first tag, and the second probe is a protein and nucleic acid complex that specifically bind to the second tag. In yet another embodiment, the first probe is a protein that specifically binds to the first tag, and the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof. In a further embodiment, the first probe is a protein that specifically binds to the first tag, and the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof hybridizes with the second tag or a fragment thereof.

[0321] Furthermore, in one embodiment, the first probe is a protein-nucleic acid complex that specifically binds to a first tag, and the second probe is a protein that specifically binds to a second tag. In another embodiment, the first probe is a protein-nucleic acid complex that specifically binds to a first tag, and the second probe is a protein-nucleic acid complex that specifically binds to a second tag. In yet another embodiment, the first probe is a protein-nucleic acid complex that specifically binds to a first tag, and the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof is complementary to the second tag or a fragment thereof. In a further embodiment, the first probe is a protein-nucleic acid complex that specifically binds to a first tag, and the second probe is a nucleic acid molecule, wherein the second probe or a fragment thereof hybridizes to the second tag or a fragment thereof.

[0322] In one specific aspect, this article provides a method for determining an analyte in a sample, comprising: (1) A first binding portion comprising a first binder and a first presenting group, a second binding portion comprising a second binder and a second presenting group, and a sample are mixed in a solution, wherein: (i) The first and second binders bind the analyte and form an immune complex; (ii) The immune complex is captured on the first solid surface in contact with the solution through the binding between the first presenting group and the first receiving group coupled to the first solid surface; (iii) The first binding portion further includes a first target marker containing a first identification barcode (“ID”) (“target ID”) specific to the analyte, and the second binding portion further includes a second target marker containing a second target ID; (2) Clean the surface of the first solid to remove unbound molecules; (3) By disrupting the binding between the first presenting group and the first receiving group, an immune complex is released from the first solid surface; (4) Introduce a second solid surface and recapture the immune complex onto the second solid surface by binding between the second presenting group and the second receiving group coupled to the second solid surface; (5) Clean the surface of the second solid to remove unbound molecules; (6) Combining a sample tag containing a sample-specific ID (“sample ID”) to (i) a first target tag, (ii) a second target tag, or (iii) both a first target tag and a second target tag; (7) Based on the proximity between the first target marker and the second target marker, a nucleic acid reporter molecule is generated from the immune complex, wherein the nucleic acid reporter molecule contains the first target ID, the second target ID and the sample ID; (8) By disrupting the binding between the second presenting group and the second receiving group, an immune complex is released from the second solid surface; and (9) Analytes are detected by detecting nucleic acid reporter molecules through qPCR.

[0323] In another specific aspect, this paper provides a method for detecting an analyte in at least two samples, comprising: (1) A first binding portion comprising a first binder and a first presenting group, a second binding portion comprising a second binder and a second presenting group, and a sample are mixed in a solution, wherein: (i) The first and second binders bind the analyte and form an immune complex; (ii) The immune complex is captured on the first solid surface in contact with the solution through the binding between the first presenting group and the first receiving group coupled to the first solid surface; (iii) The first binding portion further includes a first target marker containing a first identification barcode (“ID”) (“target ID”) specific to the analyte, and the second binding portion further includes a second target marker containing a second target ID; (2) Clean the surface of the first solid to remove unbound molecules; (3) By disrupting the binding between the first presenting group and the first receiving group, an immune complex is released from the first solid surface; (4) Introduce a second solid surface and recapture the immune complex onto the second solid surface by binding between the second presenting group and the second receiving group coupled to the second solid surface; (5) Clean the surface of the second solid to remove unbound molecules; (6) Combining a sample tag containing a sample-specific ID (“sample ID”) to (i) a first target tag, (ii) a second target tag, or (iii) both a first target tag and a second target tag; (7) Based on the proximity between the first target marker and the second target marker, a nucleic acid reporter molecule is generated from the immune complex, wherein the nucleic acid reporter molecule contains the first target ID, the second target ID and the sample ID; (8) Combine nucleic acid reporter molecules from at least two samples; (9) By disrupting the binding between the second presenting group and the second receiving group, an immune complex is released from the second solid surface; (9) Amplify nucleic acid reporter molecules; (10) Purification of nucleic acid reporter molecules; and (11) Analytes are detected by detecting nucleic acid reporter molecules through next-generation sequencing (NGS).

[0324] This document also provides a system for performing the assays disclosed herein. The systems disclosed herein can be used to detect analytes in a sample. The systems disclosed herein can also be used to detect multiple analytes in a sample, one analyte from multiple samples, or multiple analytes from multiple samples. In some embodiments, the systems provided herein are used to perform a qualitative assay for detecting analytes in a sample. In some embodiments, the systems provided herein are used to perform a quantitative assay for detecting analytes in a sample. In some embodiments, the systems provided herein are included in a kit.

[0325] The system disclosed herein may include any detection marker known in the art. In some embodiments, the detection marker is a colorimetric, fluorescent, or chemiluminescent detection reagent. In some embodiments, the colorimetric detection reagent includes PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid)), or OPD (o-phenylenediamine). In some embodiments, the fluorescent detection reagent includes QuantaBlu™ or QuantaRed™ (ThermoScientific, Waltham, MA). In some embodiments, the luminescent detection reagent includes luminol or fluorescein. In some embodiments, the detection reagent includes a trigger (e.g., H₂O₂) and a tracer (e.g., isoluminol conjugate).

[0326] For any system implementation involving nucleic acid binding or hybridization provided herein, for the purpose of binding a presenting group (e.g., a first tag and / or a second tag) to a receiving group (e.g., a first probe and / or a second probe), or binding a receiving group (e.g., a first probe and / or a second probe) to a solid surface (e.g., a first solid surface and / or a second solid surface), the complementary fragments of the two nucleic acids may be complementary sequence pairs rich in A and / or T, or complementary sequence pairs rich in G and / or C.

[0327] Furthermore, this disclosure provides sample tags comprising a sample ID, wherein, in various embodiments of the methods provided herein, the sample tag binds to an immune complex. The sample tag may bind to any component of the binding portion, an immune complex formed by two binding portions (e.g., a first binding portion and a second binding portion), a receiving group, or a component coupled to a solid surface. In one embodiment, the sample tag binds to a target tag. In another embodiment, the sample tag binds to a presenting group (e.g., a presenting group as a nucleic acid molecule). In a further embodiment, the sample tag binds to a binding agent. In yet another embodiment, the sample tag binds to a receiving group. In one embodiment, the sample tag binds to a first target tag. In another embodiment, the sample tag binds to a first presenting group (e.g., a presenting group as a nucleic acid molecule). In yet another embodiment, the sample tag binds to a first tag. In a further embodiment, the sample tag binds to a first binding agent. In yet another embodiment, the sample tag binds to a first receiving group. In one embodiment, the sample tag binds to a second target tag. In another embodiment, the sample tag binds to a second presenting group (e.g., a presenting group as a nucleic acid molecule). In yet another embodiment, the sample tag binds to a second tag. In a further embodiment, the sample label is bound to a second binder. In yet another embodiment, the sample label is bound to a second receiving group.

[0328] In various embodiments of the methods provided herein, when the sample label is a double-stranded nucleic acid molecule with one or two protrusions, the sample label can hybridize through its protrusions with any nucleic acid component of the binding portion, an immune complex formed by the two binding portions, a receiving group, or a nucleic acid component coupled to a solid surface. In various embodiments of the methods provided herein, when the sample label is a single-stranded nucleic acid molecule, the sample label can hybridize with any nucleic acid component of the binding portion, an immune complex formed by the two binding portions, a receiving group, or a nucleic acid component coupled to a solid surface.

[0329] Those skilled in the art will understand that when a double-stranded sample marker hybridizes with two items, in some embodiments, the double-stranded sample marker hybridizes with the first item through its 5' protrusion and with the second item through its 3' protrusion. In other embodiments, the double-stranded sample marker hybridizes with the first item through its 3' protrusion and with the second item through its 5' protrusion. In still a further embodiment, the double-stranded sample marker hybridizes with the first item through its 3' protrusion and with the second item through its 3' protrusion. When a double-stranded sample marker hybridizes with one item, in some embodiments, the double-stranded sample marker hybridizes with that item through its 5' protrusion. In some embodiments, the double-stranded sample marker hybridizes with that item through its 3' protrusion. When the sample marker is a single-stranded sample marker, such a sample marker can hybridize with any part or its nucleic acid sequence (including either end, both ends (5' and 3' ends), and any internal sequence) with any item or both items.

[0330] Because the system provided herein can associate each analyte with one or more target IDs provided herein, the system can simultaneously detect at least two analytes in a sample by simultaneously detecting the target IDs associated with each analyte and associating the target IDs with the analytes. Therefore, in some embodiments, the system provided herein can simultaneously detect at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, or at least one hundred analytes in a sample by simultaneously detecting the unique target IDs associated with each analyte. In a further implementation, the system provided herein detects approximately three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, or one hundred analytes in a sample simultaneously by detecting unique target IDs associated with each analyte. In some implementations, the system provided herein detects at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 analytes in a sample simultaneously by detecting the unique target ID associated with each analyte. In a further implementation, the system provided herein simultaneously detects approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 analytes in a sample by simultaneously detecting the unique target ID associated with each analyte.

[0331] In one aspect, this article provides a system for detecting an analyte in a sample, comprising: (1) a first binding portion comprising a first binding agent, a first presenting group, and a first target marker, the first target marker comprising an analyte-specific first identification barcode (“ID”) (“target ID”); (2) a second binding portion comprising a second binding agent, a second presenting group, and a second target marker, the second target marker comprising a second target ID; (3) a first receiving group, a first solid surface, a second receiving group, and a second solid surface; (4) a ligation reagent and a sample marker comprising a sample-specific ID (“sample ID”); and (5) quantitative qPCR. The reagent; wherein (i) a first binder and a second binder bind the analyte and form an immune complex; (ii) a first receiver group is coupled to a first solid surface and configured to capture a first presenting group; (iii) a second receiver group is coupled to a second solid surface and configured to capture a second presenting group; (iv) a first target label is directly or indirectly bound to the first binder, and a second target label is directly or indirectly bound to the second binder; (v) a first presenting group is directly or indirectly bound to the first binder, and a second presenting group is directly or indirectly bound to the second binder; and (vi) a sample label is bound to both the first target label and the second target label.

[0332] This document typically uses affirmative language to describe various embodiments of the invention. The invention also specifically includes embodiments that exclude certain subject matter, such as substances or materials, method steps and conditions, schemes, procedures, measurements, or analyses. Therefore, although not generally described herein as encompassing aspects not explicitly included in the invention, these aspects are disclosed herein.

[0333] This document describes systems, apparatus, and methods for performing fluid and sample manipulations. In some embodiments, these systems, apparatus, and methods are configured for determinations related to the detection and / or quantification of analyte molecules or particles in a sample fluid. In some cases, these systems, methods, and apparatus are automated. The subject matter of this invention relates in some cases to related products, alternative solutions to specific problems, and / or a variety of different uses of one or more systems and / or articles of manufacture.

[0334] These systems, apparatuses, and methods may include at least a portion thereof, configured for analyzing sample fluids containing a variety of analyte molecules and particles. In some embodiments, these systems, apparatuses, and methods are designed to determine the concentration of analyte molecules or particles in a sample fluid. Various aspects or portions of the apparatuses and systems may include one or more of the following: at least one mechanical gantry (“gantry”), reagent / consumable storage rack (“storage rack”), plate stage (“stage”), magnetic bead processor and mixer (“mixer”), plate washer (“washer”), high-capacity fluid station (“high-capacity station”), and / or computer control system. Furthermore, the apparatuses and systems may also include a readout unit (“reader”), incubator and sealer (“incubator sealer”), and / or other components, examples of which are provided herein. In some embodiments, automated apparatuses and systems enable rapid and / or accurate sample input compared to non-automated systems, and / or reduce errors or variability due to human error and / or sample handling.

[0335] In some embodiments, the assay performed by the apparatus or system described herein may include at least the following steps. First, a sample fluid containing multiple analyte molecules or particles (i.e., molecules and / or particles whose quantity and / or presence need to be determined) is provided. The sample fluid is contacted with multiple beads, wherein at least a portion of the analyte molecules (or particles) in the sample fluid associates with the beads. In some cases, the ratio of beads to analyte molecules is such that statistically zero or one analyte molecule associates with one bead, as described herein. In some cases, the ratio of beads to analyte molecules is such that statistically multiple analyte molecules associate with one bead, as described herein. The beads are then processed. In some cases, the beads are magnetic or can be induced to be magnetic (e.g., paramagnetic). The beads may be probed or analyzed (e.g., using a quantitative PCR (qPCR) module) to determine the concentration of at least one analyte molecule or particle. The qPCR module, and other components of the system included in some embodiments, may be associated with a computer control system capable of analyzing data obtained by the qPCR module. Measurements of analyte molecule concentrations may be determined based at least in part on the output of the qPCR module.

[0336] The publicly disclosed instruments are Figure 1The illustrated integrated component of the assay ecosystem includes a cloud-based application (“APP”), fully automated assay instruments, and reagents / consumables. Users first use the APP on any internet- or intranet-connected computing device to design experiments, set up assay protocols, and schedule assay runs on the instrument. The APP also helps users identify or optionally purchase the necessary reagents and consumables. Users manually load reagents, consumables, and samples into the instrument. The instrument scans the barcodes of the reagents and consumables to confirm correct loading, and then executes the assay protocol to generate test data. The test data is automatically sent to the APP via the internet or intranet, where users can view and analyze the results. Furthermore, instrument status, assay run logs, and error messages are uploaded to the APP to enable remote diagnostics, troubleshooting, and other customer support services.

[0337] In some implementations, for example, the instrument user uses the app to download a assay "recipe" from a remote server. In some implementations, for example, the downloaded recipe contains information about required reagents and consumables, analytical protocols, and / or experimental data analysis algorithms. In some implementations, for example, the downloaded recipe is both computer-readable and human-readable. In some implementations, for example, the instrument verifies the presence of all required reagents and consumables based on the information in the downloaded recipe before executing the assay protocol. In some implementations, for example, the instrument will not execute the assay protocol in the downloaded recipe unless all required reagents and consumables are present. In some implementations, for example, the app analyzes the experimental data according to the algorithm contained in the downloaded recipe. In some implementations, for example, the app uploads the experimental data to a remote server for analysis and downloads the analysis results to the user. In some implementations, for example, data transmission with the app is encrypted to ensure security.

[0338] Figure 2 The illustration depicts an exemplary conceptual hardware architecture of the disclosed instrument, comprising multiple modules, each performing key procedures or functions commonly used in biochemical assays. These modules may include a mechanical gantry (“gantry”) 2004, a reagent / consumable storage rack (“storage rack”) 2006, a plate stage (“stage”) 2008, a plate washer (“washer”) 2010, a magnetic bead processor and mixer (“mixer”) 2012, an incubator and sealer (“incubator sealer”) 2014, a large-volume fluid station (“large-volume station”) 2016, and a reading unit (“reader”) 2018. The gantry 2004 is the core of the instrument, carrying multiple tools on its end effector 2002, optionally including a pipette, plate clamp, barcode scanner, and position sensor. Depending on the assay protocol, it integrates the functional modules into an automated assay system by transferring fluid between plates and timely transporting assay plates to different modules.

[0339] For example, in some embodiments, the disclosed instrument has a benchtop housing 3000 equipped with a touchscreen display 3302, a user-accessible large-capacity reagent chamber 3306, and a set of user-accessible compartments 3308. The benchtop housing may include a controller 3018, a mechanical gantry 2004 equipped with an end effector 2002 movable in three degrees of freedom (X, Y, and Z axes), and includes a pipette 6002, a plate holder 6004, a laser position sensor 6006, and a barcode scanner 6008. The benchtop housing may include a storage rack 2006 containing a set of plate compartments for receiving and accommodating at least one universal reagent kit 29016 and a consumable carrier 30012. The benchtop housing may include a stage 2008 providing up to six plate positions, wherein the stage is movable along the Y-axis (from front to back within the instrument housing); a washer 2010 for cleaning plates; a mixer 2012, which serves as a magnetic bead processor and mixer, comprising multiple assay plate platforms 11006 stacked perpendicularly to each other; an incubation sealer 2014 for incubating and sealing at least one plate; a high-capacity fluid station 2016 for receiving and containing high-capacity materials, comprising a first buffer container 18018, a second buffer container 18020, a third buffer container 18022, and a waste container 18016; and a reader 2018 containing a qPCR unit for qPCR reading or preparation of mixed libraries for next-generation sequencing (NGS).

[0340] In some embodiments, the instrument and / or process includes a target kit comprising multiple pre-selected paired binding portions for binding a specific analyte. The paired binding portions include a first portion and a second portion. The first portion includes a first antibody or first antibody fragment pre-selected for binding the specific analyte, a first nucleic acid target marker containing a first identifier having analyte specificity, and a first nucleic acid tag. The second portion includes a second antibody or second antibody fragment pre-selected for binding the same specific analyte as the first antibody or antibody fragment in the first portion of the paired binding portions, a second nucleic acid target marker containing a second identifier having analyte specificity, and a second nucleic acid tag.

[0341] The instrument and / or process also includes a detection kit comprising multiple wells, wherein at least one of the multiple wells contains a first substrate solution containing multiple first substrates, wherein at least one of the multiple wells contains a second substrate solution containing multiple second substrates; and wherein at least one of the multiple wells contains a ligation reagent and an optional nucleic acid sample-specific label.

[0342] In some embodiments, in a portion of the plurality of first wells of the first plate 8012, the gantry 2004 introduces a portion of a plurality of multiple multiple paired binding portions from target kits 29012, 29014 in parallel with a portion of a plurality of biological samples to form multiple immune complex forming solutions in a portion of the plurality of first wells of the first plate 8012. The gantry 2004 and the substrate extractor / mixer 2012 incubate the multiple immune complex forming solutions in a portion of the plurality of first wells of the first plate 8012 in parallel to form multiple multiple immune complexes, wherein the immune complexes comprise a first antibody or first antibody fragment (binding a specific analyte) in a first portion of the paired binding portions and a second antibody or second antibody fragment (binding the specific analyte) in a second portion of the paired binding portions; this may also be referred to as an immune complex sandwich structure.

[0343] The gantry 2004 combines a portion of a first substrate solution containing multiple first substrates from the test kit with a multiple immune complex forming solution in a portion of a plurality of first wells of the first plate 8012. The gantry 2004 and the substrate extractor / mixer 2012 bind a first nucleic acid tag of a first portion of a multiple paired binding portion to a portion of the first substrate in a portion of a plurality of first wells of the first plate 8012. The substrate extractor / mixer 2012 extracts multiple multiple immune complexes in parallel from the multiple immune complex forming solution through a portion of the multiple first substrates to form a multiple first immune complex purification solution in at least a portion of a plurality of second wells of the second plate 8014, wherein a portion of the multiple first substrates elutes from the multiple multiple immune complexes in the multiple first immune complex purification solution.

[0344] The substrate extractor / mixer 2012 extracts portions of multiple first substrates in parallel from multiple first immune complex purification solutions. The gantry 2004 combines a portion of a second substrate solution containing multiple second substrates from the test kit with multiple first immune complex purification solutions from portions of multiple second wells of the second plate 8014. The gantry 2004 and the substrate extractor / mixer 2012 bind a portion of a second nucleic acid tag of a second portion of a paired binding portion to a portion of a second substrate from a portion of multiple first immune complex purification solutions from portions of multiple second wells of the second plate 8014.

[0345] The substrate extractor / mixer 2012 extracts portions of the second substrate in parallel from multiple first immune complex purification solutions in portions of multiple second wells of the second plate 8014 via multiple portions of the second substrate, to form multiple multiplex analyte-specific reporter molecules in portions of multiple third wells of the third plate 8016 by parallel linking multiple first nucleic acid target tags (directly or indirectly) of the first portion of the pair-binding portion with multiple second nucleic acid target tags of the second portion of the pair-binding portion. The plate washer 2010 washes the recently used second plate in preparation for its reuse in subsequent steps.

[0346] The substrate extractor / mixer 2012 extracts multiple multiple analyte-specific reporter molecules in a portion of a plurality of third wells of a third plate 8016 in parallel back to a portion of a plurality of second wells of a second plate 8014 through a portion of a plurality of second substrates, so as to elute multiple second substrates in parallel from multiple multiple analyte-specific reporter molecules in a portion of a plurality of second wells of a second plate 8014.

[0347] The gantry 2004 and incubator sealer 2014 prepare a fourth plate 8018 with multiple multiplex analyte-specific reporter molecules and seal the plate. A thermal cycler / qPCR (reader) 2018 replicates the multiple multiplex analyte-specific reporter molecules. The qPCR (reader) 2018 detects the replicated multiple multiplex analyte-specific reporter molecules to identify (and quantify) specific analytes in multiple biological samples.

[0348] In another embodiment, the gantry 2004 introduces a portion of multiple multiple paired binding portions from target kits 29012, 29014 in parallel with a portion of multiple biological samples into a portion of multiple first wells of the first plate 8012 to form multiple immune complex formation solutions in the portion of the multiple first wells of the first plate 8012. The gantry 2004 and the substrate extractor / mixer 2012 incubate the multiple immune complex formation solutions in the portion of the multiple first wells of the first plate 8012 in parallel within the incubation sealer 2014 to form multiple multiple immune complexes.

[0349] The gantry 2004 combines a portion of a first substrate solution containing multiple first substrates from the test kit with a multiple immune complex formation solution in a portion of a plurality of first wells of the first plate 8012. After incubation in the incubation sealer 2014, the gantry 2004 and the substrate extractor / mixer 2012 bind a first nucleic acid tag of a first portion of a multiple paired binding portion to a portion of a first substrate in a portion of a plurality of first wells of the first plate 8012. The substrate extractor / mixer 2012 extracts multiple multiple immune complexes in parallel from the multiple immune complex formation solution through a portion of the multiple first substrates to form a multiple first immune complex purification solution in at least a portion of a plurality of second wells of the second plate 8014, wherein a portion of the multiple first substrates elutes from the multiple multiple immune complexes in the multiple first immune complex purification solution.

[0350] The substrate extractor / mixer 2012 extracts portions of multiple first substrates in parallel from multiple first immune complex purification solutions. The gantry 2004 combines a portion of a second substrate solution containing multiple second substrates from the test kit with multiple first immune complex purification solutions in portions of multiple second wells of the second plate 8014. The gantry 2004 and the substrate extractor / mixer 2012 bind a portion of a second nucleic acid tag of a second portion of a paired binding portion to a portion of a second substrate in multiple first immune complex purification solutions in portions of multiple second wells of the second plate 8014.

[0351] The substrate extractor / mixer 2012 extracts portions of multiple second substrates in parallel from portions of multiple first immune complex purification solutions in portions of multiple second wells of the second plate 8014, to form multiple multiplex analyte-specific reporter molecules in portions of multiple third wells of the third plate 8016 by parallel linking multiple first nucleic acid target tags of the first portion of the paired binding portion, multiple second nucleic acid target tags of the second portion of the paired binding portion, and multiple sample-specific target tags (directly or indirectly). The plate washer 2010 washes the recently used second plate in preparation for its reuse in subsequent steps.

[0352] The substrate extractor / mixer 2012 extracts multiple multiple analyte-specific reporter molecules from a portion of a plurality of third wells of a third plate 8014 in parallel back into a portion of a plurality of second wells of the second plate 8014 through a portion of a plurality of second substrates, so as to elute multiple second substrates in parallel from multiple multiple analyte-specific reporter molecules in a portion of a plurality of second wells of the second plate 8014.

[0353] The gantry 2004 and incubator sealer 2014 were used to prepare the fourth plate 8018 with multiple multiplex analyte-specific reporter molecules, and the plate was sealed. The thermal cycler / qPCR (reader) 2018 was used to replicate the multiple multiplex analyte-specific reporter molecules. The gantry 2004 was used to combine solutions and prepare for next-generation sequencing (NGS).

[0354] It should be noted that in some embodiments, one or more modules or their functions may be integrated into a single module. In some embodiments, a module having multiple functions may be separated into multiple modules. For example, in some cases, two or more functions of an incubator seal may be combined into a single module of the system or separated into two modules. Therefore, references to any module herein, unless specifically stated otherwise, do not preclude that the module performs other functions of the system. Similarly, references to an instrument system comprising a series of separately listed components do not require that these components be physically distinct structural elements unless specifically illustrated or described as such (e.g., multiple components may share the same structural element or have a common structural element but are configured as multiple components of the overall instrument system). Furthermore, multiple copies of some components of the instrument system may exist.

[0355] Before the assay, the user loads the sample, along with all necessary reagents and drying consumables, into the storage rack. The user also needs to load large-volume fluids (such as washing buffers, rinsing buffers, etc.) into the large-volume station. If necessary, the user can also remove, empty, and reload the waste container. The instrument performs a self-test to ensure all functional modules are functioning correctly. Additionally, the gantry uses its barcode scanner to scan the barcodes printed on reagent and consumable labels to ensure the correct items are used for the assay. After the self-test, the instrument performs the assay step-by-step according to the assay protocol. These steps may include adding reagents to the sample, mixing, capturing the analyte onto magnetic beads, incubating at a set temperature, washing to remove unbound molecules, and releasing the analyte from the beads. The assay plate may need to be sealed before reading or storage. All these steps can be performed through the functional modules in the system. The gantry uses clamps to transport the assay plate from one functional module to another as needed for specific assay steps. At the end of the assay, the assay plate is moved to the reading module. The reading results, along with the operation log, can be stored, displayed on the interface, or sent to the user via the internet or intranet. In addition, any physical products measured, such as nucleic acid reporter molecules or NGS (next-generation sequencing) libraries or waste reagents / consumables, can be removed from the instrument.

[0356] It should be understood that for the instrument system described herein, each module (e.g., gantry, washer, and incubator sealer) may operate simultaneously or substantially simultaneously, thus performing functions at different spatial locations at approximately the same time. For example, in some embodiments, the gantry may be applying reagent fluid to a microtiter plate on the stage, while the washer is rinsing a different plate. For example, in some embodiments, the readout module may be analyzing the contents of a microtiter plate via qPCR, while the incubator sealer is incubating the microtiter plate for the next assay run.

[0357] The disclosed instrument optionally includes devices to prevent the accumulation of biocontamination, including a UV lamp and a HEPA air filtration subsystem. Additionally, the instrument optionally includes an inactivating buffer as one of the high-volume fluids. It utilizes a plate washer and / or pipette to clean all used consumables after each assay run.

[0358] A key advantage of this modular architecture is the ability to modify or replace one or more modules, enabling the instrument to adapt to measurements requiring different functional modules not mentioned above. Furthermore, if any module fails in the field, it can be quickly replaced, maximizing uptime for the end user.

[0359] Another important feature of the instrument of this invention is the separation of the sample container (“sample plate”) from the assay plate. In many prior art instruments, the user must set up the initial assay plate before loading it into the instrument, which may include setting up controls (including dilution profiles and sample pre-dilution). This step can be very cumbersome and error-prone. In the instrument of this invention, the sample is contained in its original state in the sample plate. The instrument can automatically set up the assay plate before executing the assay protocol.

[0360] Many existing instruments dump used consumables into waste containers, a major source of contamination or batch-to-batch cross-contamination. In publicly available instruments, used consumables are washed using a plate washer and returned to the storage rack, where they are disposed of by the user before the next measurement run.

[0361] Figure 3A -C illustrates an exemplary design implementation of the disclosed system. Figure 3A A photo showing a recently released prototype of the instrument. Figure 3B The front view of the instrument is shown, with the instrument housing 3000 removed, exposing the large-capacity reagent 3002, storage rack 3004, plate washer 3006, mixer 3008, and incubator seal 2010. Figure 3C The rear perspective view of the instrument is shown, with the instrument housing removed, revealing the stage 3012, gantry 3014, reader 3016, and controller 3018. Detailed descriptions of the key functional modules in the system are as follows.

[0362] storage rack The storage rack in the instrument is a user / instrument interface module that facilitates the loading of samples, reagents, and other drying consumables into the instrument. It incorporates mechanisms to ensure user safety during such loading. Simultaneously, the storage rack provides access to the instrument's mechanical components for: a) checking labels on loaded items to confirm correct loading; and b) moving reagents and / or consumables to the appropriate functional modules to perform assay procedures according to the protocol. The storage rack optionally includes a refrigeration zone to cool the internal temperature to a lower temperature below ambient temperature for the preservation of temperature-sensitive samples and / or reagents. The set lower temperature can be 4°C, 0°C, or even -20°C.

[0363] Figure 4A and 4B A specific design implementation of the storage rack is shown. Figure 4A The storage rack behind the instrument's front panel is shown; the front door of the top compartment opens to reveal the loaded reagents and consumables. Figure 4B The rear perspective view of the storage rack is shown, with the rear door of the refrigerated section 4012 in the top compartment open. Figure 4B The rear door of the ambient temperature zone 4010 in the lower compartment is also shown. The storage rack contains three structurally identical "compartments," each providing shelf space for a set of reagents and consumables to perform assay runs. Guide structures are provided on the shelves to help users correctly position reagents and consumables. Furthermore, each compartment contains two areas: a refrigerated area for storing temperature-sensitive samples and reagents that require cooling before use; and an ambient temperature area for storing items suitable for room temperature (including desiccant plates and other consumables). Further, each compartment has a front door facing the user on the instrument's front panel and a rear door facing the gantry inside the instrument housing. The front door opens to allow the user to load reagents and consumables, at which point the rear door closes for user safety. The rear door opens as needed according to the assay protocol, allowing the gantry to fully access the reagents and consumables. When the mechanical gantry reaches a specific storage rack compartment, the front door of that compartment locks to ensure user safety. Figure 4A In the exemplary compartment shown, sample plate 4004, test kit, and target kit 4002 have been loaded into the refrigerated area of ​​the compartment. Consumable carrier 4006 (disclosed in detail later) and two pipette tip boxes 4008 have been loaded into the RT area of ​​the compartment.

[0364] For example, in some embodiments, the storage rack (supply storage shelf) in the instrument system comprises a single shelf space compartment. For example, in some embodiments, the storage rack in the instrument system comprises multiple shelf space compartments with identical structures. For example, in some embodiments, the storage rack in the instrument system comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 such compartments.

[0365] exist Figure 4A and 4B In this system, the compartments are stacked vertically to form independent shelves. Alternatively, the compartments can be arranged side by side. Each compartment is located in an independent drawer, which can be pulled out to allow the user to load from the top. When the drawer is closed, the gantry shelf is also accessible from the top for reagents and consumables.

[0366] Gantry A gantry crane is a type of robotic arm whose end effector carries one or more tools required to perform measurements. Figure 5 A specific implementation of the gantry design is shown, which enables the end effector 5008 to move in three degrees of freedom: X, Y, and Z. The Z-axis 5002 is driven by a ball screw mechanism, while the X-axis 5004 and Y-axis 5006 are driven by piezoelectric motors. Figure 6 A specific embodiment of the end effector is shown, on which are mounted two air displacement pipettes (ADP) 6002, a plate clamp 6004, a laser distance sensor 6006, and a barcode scanner 6008. The gantry is the core of the instrument, responsible not only for transferring fluids but also for moving the measurement plate from one module to another to perform the measurement steps. In alternative embodiments, the gantry may comprise two or more smaller gantry units, with different tool combinations carried on separate smaller gantry units. A gantry with only two degrees of freedom may also be used, with the third degree of freedom provided by an additional mechanism in other functional modules.

[0367] Another innovative feature of the instrument is the integration of a position sensor into the end effector of the gantry. For robust and reliable operation, pipettes and grippers must achieve sub-millimeter positioning accuracy relative to functional modules in multiple spatial dimensions. Many existing instruments, such as fluid transfer robots sold by Hamilton or Tecan, achieve this performance through position calibration (or “teaching”) after instrument installation. This teaching process is typically very time-consuming and must be repeated after each instrument movement, and may even require periodic adjustments even when the instrument is not moved. The disclosed instrument addresses this problem by placing a position target at the module requiring calibration and integrating a position sensor into the end effector of the gantry. Position calibration between the end effector and the module can be achieved automatically by sensing the relative position of the target and sensor as needed or periodically. Any sensor capable of measuring the relative position between the sensor and the target in three-dimensional space can be used for this application. A specific implementation of such a position sensor is the Keyence IA-030 (https: / / www.keyence.com / products / sensor / positioning / ia / models / ia-030 / ). This low-cost sensor was originally designed for high-precision measurement of the distance between the sensor and the surface, so the relative position of the target and the sensor can only be determined in one-dimensional space. Figure 7A -B describes a specific implementation where the relative position between such a sensor and a specifically designed target can be determined in all three dimensions. The gantry first moves the sensor close to the front surface of the target block ( Figure 7A The relative position in the Y-axis direction is determined by measuring the distance between the sensor and the front surface. Then, the gantry scanning sensor passes through the upper edge of the target block, and the precise position of the upper edge is detected by monitoring sudden changes in the measured distance. Figure 7B The relative position in the Z-axis direction is determined. The relative position in the X-axis direction can be determined in a similar manner by scanning the sensor through the vertical edge of the target block.

[0368] For any automated assay instrument, controlling all reagents and consumables used in the instrument is crucial to ensure robust and reliable operation. Many prior art instruments require the user to manually scan the barcodes marked on the reagents and consumables during loading. In disclosed instruments, reagents and consumables are placed in a storage rack after loading. A barcode scanner is mounted on the end effector of the gantry. Before the assay run begins, the instrument moves the barcode scanner to the rear opening of the storage rack and checks each barcode in the set of reagents and consumables to ensure the correct items are loaded and, where appropriate, are in the correct position. Optionally, the instrument may combine a position sensor and a barcode scanner to detect potential positioning deviations of reagents and consumables.

[0369] For example, in some embodiments, a suitable computer-readable identifier tag may be used instead of a barcode. Non-limiting examples of suitable identifier tags include barcodes or radio frequency identification (RFID) chips. Identifier tags can be used for a variety of purposes, such as determining the authentication and / or identification, type, batch number, expiration date, etc., of consumables and / or their contents. Authentication can be achieved, for example, by an optical scanner or an RFID proximity reader (depending on the type of identifier tag used). In some embodiments, information from the identifier may be stored for future reference and record-keeping purposes. Any suitable identifier may be used, such as an RFID tag, serial number, color tag, fluorescent or optical tag (e.g., using quantum dots), chemical compound, radio tag, or magnetic tag. Detection of the identifier can be achieved by a variety of methods known to those skilled in the art. Detection methods depend in part on the specific identifier and may include, for example, imaging, fluorescence detection, spectroscopy, microscopy, etc. In one embodiment, an RFID tag is used as the identifier. The RFID tag may include an integrated circuit (e.g., for storing and processing information, modulating and demodulating radio frequency (RF) signals) and an antenna for receiving and transmitting signals. RFID tags can be passive, semi-passive (e.g., battery-assisted), or active. It should be understood that RFID tags are known in the art, and any suitable RFID tag can be incorporated into the components of the measurement consumables described herein.

[0370] Platform The plate stage contains multiple platforms on which plates and reagent kits are placed as fluid is transferred from one plate to another. It also provides temporary storage space for plates awaiting the next assay step. Pipette tip holders are also placed on the platforms for pipettes to pick up tips from the gantry rack.

[0371] For example, in some embodiments, the instrument system can internally accommodate more than 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 boards. For example, in some embodiments, the instrument system can internally accommodate 3 to 10, 4 to 9, or 5 to 8 boards. For example, in some embodiments, the instrument system can internally accommodate 8 to 10, 7 to 9, 6 to 8, 5 to 7, 4 to 6, or 3 to 5 boards. For example, in some embodiments, the instrument system has an internal storage capacity of 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 boards.

[0372] Figure 8A-Figure B illustrates a specific embodiment of stage 8010, which provides six plate positions. Figure 8A shows a perspective view of stage 8010, including a motor 8002 that drives stage 8010 along the Y-axis, a target block 8004, a tip holder 8006, and a weighing sensor unit 8008. The entire stage is movable along one axis (Y-axis). This feature helps reduce the instrument's footprint in the Y-axis direction. Obviously, the pipette must be able to reach all the holes at each plate position on the stage. Additional free space is required around the stage to avoid physical collisions between other tools (such as clamps) and other objects. Moving the stage solves this interference without increasing the instrument's footprint. Optionally, a weighing sensor is mounted below one or more plate positions to monitor the force applied to the ADP during tip pickup. This ensures operational robustness of tip pickup and ejection.

[0373] Another important function of the stage is to improve the positioning accuracy of consumables, thereby enhancing the operational reliability of automated instruments. As mentioned earlier, reagents and consumables are manually loaded into the storage rack by the user. Due to the manual operation, the positioning accuracy and consistency of these items are not high. The instrument first transports the reagent kit or consumable from the storage rack to the stage. Each plate position on the stage is provided with a groove to accommodate the plate, and this groove is large enough to accommodate positioning deviations in the storage rack. Once the plate is in its groove, the "corner pusher" mechanism is activated, pushing the plate towards a corner of the groove, thereby forcing the plate to align with the groove in a highly repeatable manner. Figure 9A- Figure B illustrates a specific implementation of the corner pusher mechanism, where each pusher 9002 is spring-loaded to a "closed" position. When the stage moves to a specific position where the plate is placed into or removed from the groove, the cam mechanism 9004 opens the pusher 9002 (Figure 9A). After the stage moves away from the plate loading position, the spring closes the pusher 9002, pushing the loaded plate towards a corner of the groove, thereby precisely aligning the plate with the stage. Figure 9B ).

[0374] Another way to ensure the board is placed accurately on the platform is to ensure the relative position between the gripper and the board to be gripped is accurate when the gantry approaches the board to remove it from the storage rack. This can be achieved by using a position sensor on the end effector to measure the distance (Y-axis) between the gripper and the board. Furthermore, the gripping action naturally centers the board relative to the gripper, ensuring positioning accuracy in the X-axis direction.

[0375] The stage may optionally include a mechanism to prevent the plate from moving upwards when placed in the plate position. This is important because some plate or reagent containers are sealed with a sealing membrane. When a pipette tip punctures the membrane, the membrane can easily adhere to the tip, causing the plate to move upwards when the pipette is withdrawn. Figure 10A-C indicates a specific implementation of the mechanism, in which components are mounted on the edge of the plate placement groove, forming a "dangling" structure above the flange of the microtiter plate to prevent the plate from moving upwards. When removing the plate from this groove, the gantry uses clamps to grip the plate, first sliding it laterally to disengage it from the hanging structure, and then moving it upwards out of the groove.

[0376] mixer Magnetic beads are widely used as solid surfaces in biochemical assays to selectively bind specific types of molecules from mixtures of different molecules in a sample, achieving separation, purification, or signal enhancement. There are two main methods for handling magnetic beads during assays: 1) fluid transfer method, where after completing one assay step, the beads are left in the reaction vessel, the used fluid is removed, and new reagents are added if necessary; 2) bead transfer method, where beads are captured from the solution in the vessel and released into another vessel containing new reagents for the next assay. Disclosed instruments optionally include a dedicated module for handling magnetic beads, employing either of the above methods.

[0377] In most magnetic bead processing units employing the bead transfer method, the bead capture step is achieved by immersing a sleeved magnetic pole into a first reaction vessel, causing beads in the solution to adhere to the sleeve. The sleeve / magnetic pole is then moved to the next vessel. By withdrawing the magnetic pole from the sleeve, the beads are released into the new reagent. To process multiple samples contained in multiple vessels within a plate, a magnetic head with multiple magnetic poles is used. Accordingly, multiple sleeves are integrated into a single component called a comb. The KingFisher™ series instruments manufactured by ThermoFisher Scientific are typical prior art devices employing the bead transfer method. Their operating mechanisms are covered in granted U.S. Patents 6040192, 6207463, 6447729, 6448092, and 6596162.

[0378] Compared to fluid transfer methods, one advantage of bead transfer methods for handling magnetic beads is that the bead processing module also functions as a stirrer and mixer, which is crucial in almost all biochemical assays. A key requirement for modules employing bead transfer is minimizing the time between the beads leaving the first reagent and re-immersing them in the next to reduce the risk of bead drying. In many instruments, this is achieved by placing a set of pre-filled reagent plates on a rotating or translating platform. As soon as the comb rises from the first reagent, the platform immediately moves the next plate below the comb, causing the comb to immediately descend and re-immerse the beads in the solution. One problem with this configuration is the significant floor space required to house the pre-filled reagent plates, especially for complex assays involving multiple plates.

[0379] This paper discloses a more compact magnetic bead processor configuration based on bead transfer.

[0380] like Figure 11A-11C As shown, the disclosed mixer places pre-filled test plates on multiple platforms stacked perpendicularly to each other. The cannulated comb first contacts the test plate on the upper platform. Figure 11B After completing one measurement step, the platform slides laterally, allowing the magnetic head to reach the measurement plate on the next platform. Figure 11C If necessary, the used test plate can be removed manually or by mechanical clamp and replaced with a new plate containing fresh reagents. After completing the relevant test steps on the lower platform, the magnetic head / comb moves to its original position above the upper platform, the upper platform slides back to its original position above the lower platform, and then the magnetic head / comb descends again to touch the new plate. By repeating the above steps, the device can perform an unlimited number of test steps for bead processing using at least two vertically placed platforms. Figure 11A The design described in -C will occupy the footprint of at least two microtiter plates. More specifically, Figure 11A A front view of the structure is depicted, showing a magnetic head 11002 with magnetic poles, a plastic comb 11004 with a sleeve, and a measuring plate placed on a vertical positioning platform 11006. Figure 11B The illustration shows a bead handling device when the bead touches the plate on the upper platform, wherein the magnetic pole is inserted into the sleeve 11008. Figure 11C A bead handling device is shown when the bead touches the plate on the lower platform.

[0381] Figure 12A -C illustrates a design that further reduces the footprint, where the upper measurement plate rests on a "detachable" platform. After a measurement step is completed, the used measurement plate is first removed from the upper platform using a mechanical clamp or plate stacker. The upper platform is then detached, allowing the magnetic head and / or sleeve comb to access the next plate placed on the lower platform. Figure 11A The design concept described in -C is similar; as long as all upper platforms except the lowest platform can be disassembled, there can be multiple such vertical positioning platforms. And as described in the concept of 11A-C, two platforms can be used alternately to perform an unlimited number of measurement steps.

[0382] Figure 13A and 13BAnother concept is illustrated, featuring two or more fixed and vertically positioned measuring plate platforms. The upper platform has a large opening at its center, covering the perforated area of ​​the measuring plate. When the measuring plate is placed on the upper platform, the magnetic poles / sleeves can directly contact the measuring plate as described above. When the measuring plate is placed on the lower platform, the magnetic poles and sleeves pass through the opening to contact the plate on the lower platform. This device is greatly simplified, eliminating the need to move or disassemble the upper platform. Although the total number of platforms in this design is limited by the length of the magnetic poles / sleeves, as previously mentioned, it can also utilize two platforms alternately to perform an unlimited number of bead processing steps in a single measurement.

[0383] Figure 14A- Figure 14 shows an exemplary detailed design of a concept with two fixed plate platforms. Figures 14A and 14B show the front and side views of the design, respectively, including a motor 14002, a magnetic head carrier 14004, a sleeve comb carrier 14006, a magnetic head 14008 with an inserted sleeve comb, a micro-tipping plate 14010, an upper platform 14012, and a lower platform 14014. Figure 14C This is a perspective view showing guide rail 14016. Figure 14D The image shows the magnetic head 14018 held in an elevated position, with the comb-like element immersed in a plate on the lower support for mixing or bead release. Figure 14E The illustration shows the magnetic head being inserted into the comb-like component, with the magnetic head carrier mounted on the comb-like component carrier, and the magnetic head / comb-like component being immersed in a plate on a lower support for bead collection. Figure 14F The image shows the magnetic head 14018 held in an elevated position, with the comb-like element immersed in the deep-hole plate 14002 on the upper support for bead release or mixing.

[0384] As mentioned earlier, the core operations of any bead processing are: 1) collecting beads from the solution onto a sleeve; and 2) releasing the beads from the sleeve into another solution. Bead collection is achieved by inserting a magnetic pole into the sleeve and moving the magnetic pole / sleeve combination up and down within a container containing the bead solution. This up-and-down movement is typically slow, allowing time for the beads to be collected onto the magnetic pole. Bead release is achieved by withdrawing the magnetic pole from the sleeve containing the beads and then moving the empty sleeve up and down within a container containing another solution. The frequency of this up-and-down movement must be high to effectively detach the beads from the sleeve surface.

[0385] To achieve these operations, the magnetic head and the comb must be mounted on two separate, movable support units. In existing devices, the magnetic head support unit is mounted as a sub-assembly on the comb support unit, allowing the magnetic head to insert into or withdraw from the comb. When the comb support unit moves, the magnetic head support unit moves along with the magnetic head, enabling the magnetic head and comb assembly to move up and down together in the container to collect the beads. The problem with this design is that during bead release, the comb support unit must move at high frequency with the heavy magnetic head, generating significant vibration. Existing bead processors use large, heavy bases to mitigate vibration. Furthermore, the magnetic head support unit and the comb support unit are driven by separate motors, resulting in higher cost and complexity.

[0386] In the mixer design disclosed herein (as shown in the example in Figure 14), the head carrier and the comb carrier are mounted on two separate carriages (sliding on the same guide rail 14016), with the head carrier 14004 positioned above the comb carrier 14006. The comb carrier 14006 is driven by a motor 14002 and moves up and down along the guide rail 14016, while the head carrier 14004 is unpowered. During bead collection, the head carrier sits on the comb carrier under its own weight, with the magnetic poles inserted into their respective sleeves. The comb carrier drives the head / comb assembly to move slowly up and down within the container, adsorbing the beads onto the sleeves. During bead release, the comb carrier first moves the head carrier mounted on it to a higher position. An activation mechanism is then used to hold the head carrier in this higher position. Then, the comb-shaped support device moves downwards with only the comb, inserting the sleeve into the container and circulating it up and down at high frequency, causing the beads to fall from the sleeve surface into the fresh reagent in the container. Because the comb is very light, this high-frequency oscillation does not cause severe vibration. Furthermore, in the design of this invention, the device is greatly simplified because the magnetic head support device is passive and does not require a power transmission chain.

[0387] Another crucial task in the bead processor is aligning the magnetic poles with the sleeve. The magnetic poles are made of rare-earth magnet materials and are very fragile. Unlike the magnetic head, which is permanently mounted on the head carrier, the sleeve comb is a plastic consumable and is temporarily attached and secured by the comb carrier. The most common combs have "ear-like" structures at the four corners of their top plate. Figure 15AIn existing bead processors (such as KingFisher™), the comb carrier has two rotating components on either side, each with two "finger-like" appendages that engage with four "ear-like" structures on the comb to align and secure it to its carrier. A problem with this prior art design is that if any of the comb's "ear-like" structures is damaged during transport, the comb carrier cannot properly secure the comb, causing the read / write head to collide with it. The detailed design of the rotating components in the processor of this invention is as follows... Figure 15B As shown in & 15C (some parts are omitted or set to transparent for clarity).

[0388] The two rotating parts 15006 on both sides of the comb-shaped support device 15004 each have two finger-shaped parts 15010 to engage with the ear-shaped structure 15002 on the comb-shaped part, aligning the comb-shaped part with its support device 15004. Figure 15B As the rotating part 15006 rotates further ( Figure 15C The baffle 15012 behind the "finger" 15010 engages with the underside of the comb substrate to secure the comb to its carrier 15004. The required reliable attachment of the comb to its carrier 15004 can be achieved as long as any two of the four ear-like structures 15002 are undamaged. Operational robustness is greatly improved.

[0389] cleaner Cleaning microtiter plates is one of the most common assay steps, in which reagents (such as washing buffers) are dispensed into the wells of the plate and, after optional soaking or shaking, are aspirated from the wells.

[0390] There are two types of plate washers on the market: strip washers, which dispense fresh reagent into one well (a column or row) of the plate each time and aspirate used reagent; and matrix washers, which dispense and aspirate reagent into all wells of the plate in parallel. Generally, strip washers are simpler and cheaper, but take longer to complete a plate washing cycle. More importantly, strip washers can cause cross-contamination between wells because the aspiration needle must be inserted into the fluid within the well, limiting their applicability. Matrix washers, on the other hand, are faster and have the lowest risk of cross-contamination between wells on the plate. However, they are more complex and more expensive. Another problem with matrix washers is their large dead volume and the significant waste of reagent during filling or switching to different reagent stages.

[0391] This article discloses a plate washer that avoids the drawbacks of strip and matrix washers.

[0392] Figure 16The illustration shows the basic conceptual structure of the plate washer of the present invention, which includes one or more striped dispensing heads 16004 and a matrix-type aspiration head 16002. Dispensing head 16004 comprises one or more hollow dispensing needles aligned with a column or row of wells. During a dispensing cycle, reagent is pumped into a manifold connected to the dispensing needles to fill a column or row of wells, and then a microtiter plate 16006 moves below dispensing head 16004, allowing reagent to be dispensed into the next column or row of wells until all wells of plate 16006 are filled. Aspiration head 16002 comprises a 2D matrix of aspiration needles aligned with each well of microtiter plate 16006. During an aspiration cycle, a relative vacuum is applied to the manifold connected to the aspiration needles, and aspiration head 16002 descends, inserting the aspiration needles into the fluid within the well and aspirating the fluid. Because the dispensing needles do not need to descend to contact the liquid within the well, the risk of inter-well contamination is minimal. Figure 16 An exemplary configuration designed for a 96-well plate is described, wherein the dispensing head 16004 has 8 needles (one), and the aspiration head 16002 has 96 (8×12) needles. The same concept can be readily applied to other plate sizes, including 24-well (4×6), 384-well (16×24), and even 1536-well (32×48). Using the disclosed plate washer, the microtiter plates used in the disclosed instrument system can be cleaned and reused in multiple steps of the same assay without concern for cross-contamination between wells, making the instrument system more versatile for a wider range of assays.

[0393] Many applications require washers to dispense multiple different reagents. In existing plate washers, this is achieved by connecting a dispensing manifold to different reagents via a multi-way selector valve. Multiple reagents are dispensed one at a time through the same set of needles. The problem with this design is that there is always residual volume in the fluid path, and some reagents are incompatible and cannot be mixed. Another significant advantage of the disclosed “strip dispensing + matrix aspiration” washer architecture is that the dispensing head can optionally contain multiple dispensing needles. Each needle is fluidly separate and has its own manifold connected to a dedicated reagent source. Optionally, multiple reagents can be dispensed in parallel.

[0394] Parallel suction of fluid from all the holes on the plate requires a very powerful pump, which inevitably results in a large size and high noise level. For example... Figure 17 As shown, the matrix aspiration head provided herein also optionally includes partitioned manifolds above the needle matrix, with each manifold connecting to a portion of the needles throughout the matrix. Using a three-way selector valve 17002, a portion of the orifices in a microtiter plate can be aspirated at once using a smaller, quieter pump 17004.

[0395] Figure 18An exemplary system architecture of the disclosed cleaner is depicted, comprising two distribution strips, wherein distribution strip #1 (18012) is shared by buffers 1 & 2 (18018 & 18020) and selected via a three-way valve 18008, and distribution strip #2 (18014) is dedicated to buffer 3 (18022). Each distribution strip is connected to a distribution pump 18010. The suction manifold is partitioned into two separate sections 18004 and 18006, each connected to a waste container 18016 via a suction pump 18002.

[0396] In existing plate washer designs, such as Figure 19A As shown, the suction pump is connected to one side of the suction manifold. Sometimes, a small amount of fluid becomes trapped at the distal end of the manifold during suction, and this fluid may drip from the nearest needle after the suction pump stops. This dripping can cause cross-contamination between orifices. Similarly, the pump power must be significantly increased to completely remove the trapped volume. More specifically, Figure 19A This shows that when liquid is drawn from one end or the center of the manifold, residue 19002 is produced in the "dead zone" section of the manifold. Figure 19B This shows “residue-free” aspiration of liquid from both ends of the manifold.

[0397] The publicly disclosed plate washer connects both ends of the suction manifold to the same pump via a "T" connector. Figure 19B This solves the problem by eliminating trapped fluid in manifold dead zones without increasing pump power.

[0398] Figure 20A -B illustrates a specific implementation of the publicly disclosed system-level piping and instrumentation diagram (PID) for a plate washer. Figure 21A- Figure 21B shows details of this design embodiment, which includes an optional soaking tray 21010 so that the needles of the dispensing head 21006 can be soaked in the dispensing gap to prevent clogging. Furthermore, Figures 21A and 21B depict a reservoir 21002 for dispensing fluid, a matrix-style suction head 21004, two striped dispensing heads 21006, a vertically movable plate platform 21008, and a dispensing needle soaking tray 21010.

[0399] For example, in some embodiments, the instrument system consumes only a small amount of cleaning buffer when cleaning the plate during a NULISA assay cycle. For example, in some embodiments, the instrument system dispenses no more than 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL, or 200 μL of cleaning buffer to each well of the plate when cleaning it. For example, in some embodiments, the instrument system dispenses 100 to 200 μL, 110 to 190 μL, 120 to 180 μL, 130 to 170 μL, or 140 to 160 μL of cleaning buffer to each well of the plate when cleaning it. For example, in some embodiments, when cleaning the instrument system plate, 100 to 120 μL, 110 to 130 μL, 120 to 140 μL, 130 to 150 μL, 140 to 160 μL, 150 to 170 μL, 160 to 180 μL, 170 to 190 μL, or 180 to 200 μL of cleaning buffer are dispensed to each well of the plate.

[0400] For example, in some embodiments, the instrument system flushes its internal fluid lines with a decontamination solution at the end of each NULISA assay cycle. For example, in some embodiments, the instrument system cleans each used plate with a decontamination solution at the end of each NULISA assay cycle; this decontamination solution neutralizes infectious agents or hazardous biological materials that pose a risk or potential risk to human, animal health, or the environment, allowing the used plates to be disposed of in a conventional non-hazardous waste recycling stream. For example, in some embodiments, the decontamination solution is a bleach solution. For example, in some embodiments, the decontamination solution is an aqueous sodium hypochlorite solution. For example, in some embodiments, the decontamination solution is an aqueous sodium hypochlorite solution with a concentration of 0.1%, 0.2%, 0.5%, 1%, 2%, or 3%.

[0401] Incubation seal Incubation is one of the most common assay steps, in which the reaction solution is kept at a specific temperature for a predetermined period of time. Because incubation temperatures are usually much higher than ambient temperatures and the sample volume is small, the incubator must be equipped with devices to prevent liquid evaporation and condensation of the evaporating liquid.

[0402] At the end of the measurement, sealing the test plate with a multilayer film is a common procedure before sending it for reading or storage. Based on the sealing mechanism, these sealing films are divided into two categories: 1) pressure-sensitive adhesive (PSA) films, which are adhered to the plate by pressure; and 2) heat-sealing films, which are welded to the microtiter plate by a heating block.

[0403] Plate incubation and sealing are typically performed by two separate devices. This paper discloses a compact incubator / sealer that can perform both incubation and sealing functions on microtiter plates in a fully automated process.

[0404] Figure 22 illustrates the basic conceptual structure of the disclosed module, comprising three sub-components: 1) a top block 22002, the temperature of which is controllable at a set value suitable for incubation. The set temperature can also be a temperature suitable for welding the sealing film to the sample plate, typically in the range of 150°C to 200°C; 2) a bottom block 22006, also controllable at the set temperature, and optionally containing a surface profile consistent with the bottom hole profile of the measurement plate to maximize heat transfer efficiency; 3) a thermally insulating housing 22004 surrounding the top and bottom blocks. Optionally, the housing 22004 can also be controllably heated to a temperature suitable for incubation. The top block 22002 and bottom block 22006 are movable vertically to clamp the measurement plate between them. A pressure sensor is placed on either the top block 22002 or the bottom block 22006 to measure the clamping force. By synchronously controlling the movement of the top block 22002 and the bottom block 22006, the entire sandwich structure (top block 22002, measuring plate and bottom block 22006) can move relative to the outer shell while maintaining the clamping force.

[0405] Figures 23A and 23B depict the use of the disclosed module for incubation. The bottom block 23004 is controllably heated and maintained at a set temperature suitable for incubation, and the top block 23002 is controllably heated and maintained at a temperature slightly above the set incubation temperature. The higher temperature of the top block 23002 is to prevent fluid condensation on its surface. A measuring plate is placed on the bottom block 23004 (Figure 23A), wherein the bottom block 23004 is optionally removable from the housing for placement. The top block 23002 is lowered to clamp the measuring plate with sufficient pressure to seal the plate and prevent evaporation during incubation. The sandwich structure formed by the top block 23002, the measuring plate, and the bottom block 23004 is moved into the housing together and held there during incubation. Figure 23B Optionally, the outer casing is also heated to improve temperature uniformity on the measuring plate during incubation. After incubation, the sandwich structure is removed from the outer casing, and the top block 23002 and the bottom block 23004 separate to release the measuring plate.

[0406] For example, in some embodiments, the incubator sealing module in the instrument system can maintain the reaction solution within a set temperature range of 30 to 40°C, 35 to 40°C, 35 to 39°C, 36 to 38°C, or 36.5 to 37.5°C. For example, in some embodiments, the incubator sealing module in the instrument system can maintain the reaction solution at a set temperature point of 25°C, 30°C, 33°C, 35°C, 37°C, 39°C, or 40°C. For example, in some embodiments, the incubator sealing module in the instrument system is programmable to maintain the reaction solution at any temperature between room temperature and 100°C. For example, in some embodiments, the incubator sealing module in the instrument system is programmable to maintain the reaction solution at low and high temperature points, wherein the low temperature point is 25°C, 30°C, 33°C, 35°C, 37°C, 39°C, or 40°C, and the high temperature point is 80°C, 83°C, 85°C, 87°C, 89°C, 90°C, 93°C, 95°C, 97°C, or 100°C. For example, in some embodiments, the incubator sealing module in the instrument system is programmable to maintain the reaction solution at a low temperature point and a high temperature point, wherein the low temperature point is 37°C and the high temperature point is 95°C.

[0407] Figure 24A -E illustrates the use of the disclosed module to seal the test plate with a sealing membrane pre-assembled onto a rigid frame. Details of this framed sealing membrane (“FSF”) are disclosed in the “Consumables” section. The test plate is placed on the bottom block after ( Figure 24A The FSF is placed on a separate platform, with the membrane suspended directly above the measurement plate. Figure 24B The top block descends to set pressure, pressing the sealing membrane against the top of the measuring plate. Figure 24C If the FSF uses a heat-sealing film, the top block also needs to be preheated to the set sealing temperature (usually 150-200°C). If the FSF uses a PSA sealing film, the set pressure must be sufficient to ensure a seal. After the film is attached to the test plate by pressure or heat, the top and bottom blocks move downwards through the opening of the FSF frame at a synchronized speed, maintaining the clamping force on the test plate. As the frame remains in place, the sealing film is peeled off the frame. Figure 24D After sealing, the used frame (without membrane) and the sealed measuring plate can be removed from the module. Figure 24E ).

[0408] Figure 25 illustrates an exemplary design of the disclosed module. More specifically, Figure 25A shows a perspective view of the module. Figure 25B A side view of the module is shown when a framed sealing membrane (FSF) is used. Figure 25C This shows a cross-sectional side view of the module when the FSF is out of frame. Figure 25D A cross-sectional side view of the module is shown when the incubation plate is in use. Figure 25A-B shows the top block 25002 and bottom block 25008, the heat insulation shell 25004, and the framed sealing membrane 25006.

[0409] High-capacity stations Large volume fluids typically refer to certain commonly used reagents consumed in large quantities during assays, and may include washing buffers, rinsing buffers, and disinfecting fluids. Waste liquids generated during assay procedures can also be considered large volume fluids. Disclosed instruments optionally include large volume fluid stations for containing and managing these large volume fluids.

[0410] Figures 26A and 26B illustrate a specific embodiment of the large-capacity station, including a large-capacity station door, large-capacity nests 26006, and a tubular manifold 26004. The large-capacity station also includes suction tubing 26002 and a linkage 26010 connecting the manifold to the large-capacity station door. The large-capacity station door rises, exposing multiple large-capacity nests 26006, allowing the user to load large-capacity bottles 26008 into their designated nests. Nests 26006 are equipped with a locking mechanism to hold the large-capacity bottles 26008 in place, and a weight sensor below to monitor the fluid volume in the large-capacity bottles 26008. After manual loading, the large-capacity station door closes, and the tubular manifold 26004 descends to insert a set of suction tubing 26002 into the large-capacity bottles 26008. The tubing 26002 connects to a plate washer module or other fluid management device in the instrument.

[0411] Figure 26C -E illustrates the safety switch mechanism for a large-capacity station door. (For example...) Figure 26C The mechanism, as depicted, includes an upper support 26102 fixed to a movable lifting frame, a housing 26104, two push-button switches 26106, two switch actuators 26108, a sliding rod 26010 that moves with the door, and a lower support 26112 connected to the door. A tubular manifold support contains the top of the foldable linkage of the safety switch mechanism. The high-capacity station door is suspended by gravity at the bottom of the safety switch mechanism. The safety switch mechanism integrates two push-button switches that are triggered when an obstacle obstructs the high-capacity station door from closing downwards. Switch triggering causes the high-capacity station door to stop closing, but the trigger signal is ignored within the final closing distance (where there is no potential risk of pinching fingers or causing other injury to the operator). Figure 26D As shown, gravity pulls the door and slider rod downwards (26114). Figure 26E As shown, the obstacle pushes the door and slider rod upward (26116).

[0412] Figure 26F and 26GThis diagram shows two possible locations for the cap on the large-volume bottle. The large-volume bottle has two threaded configurations to accommodate the screw cap—one at the top opening and one at the front closure. The cap can be removed from the top opening and stored at the front closure, or vice versa. The large-volume station is equipped with a cap detector to prevent the large-volume bottle from being installed with the cap at the top opening, and to prevent damage to the suction tubing and / or mechanism during operation.

[0413] For example, in some embodiments, the instrument system contains sufficient reagents and other consumables to run multiple assay cycles without requiring the user to reload them. For example, in some embodiments, the instrument system contains sufficient reagents and other consumables to run more than 5, 6, 7, 8, 9, 10, 11, or 12 assay cycles. For example, in some embodiments, the instrument system contains sufficient reagents and other consumables for continuous operation over extended periods without requiring reloading. For example, in some embodiments, the instrument system contains sufficient reagents and other consumables for continuous operation for 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, or 30 days.

[0414] Reader The reading module in the publicly available instrument can be any measurement reading device based on various detection principles, including qPCR units, ddPCR units, flow cytometers, fluorescence or luminescent plate readers, mass spectrometers, etc.

[0415] For example, in some embodiments, the NULISA assay results are quantified using quantitative polymerase chain reaction (qPCR) at the end of each NULISA assay cycle. For example, in some embodiments, the user removes a microtiter plate containing the NULISA assay product from the benchtop instrument and places it in a separate thermal cycler for qPCR reaction. For example, in some embodiments, the microtiter plate containing the NULISA assay product is automatically transferred from the benchtop instrument to a separate thermal cycler for qPCR reaction via a robotic arm, for example. For example, in some embodiments, the benchtop instrument runs the qPCR reaction in an internally integrated qPCR module. For example, in some embodiments, the internally integrated qPCR module has a shape factor of approximately 13 inches wide × 22 inches deep × 14 inches high. For example, in some embodiments, the internally integrated qPCR module is a commercially available off-the-shelf qPCR system, such as the Bio-Rad Opus 96.

[0416] Consumables The disclosed instrument uses multi-container plates to perform measurements on multiple samples in parallel, including standard 24-well, 96-well, and 384-well microtiter plates. In its mixer module, it optionally uses plastic-sleeved comb-shaped transfer beads.

[0417] For example, in some implementations, the microtiter plates used in the instrument system may conform to the standards published in January 2004 by the Society for Laboratory Automation and Screening (SLAS) and the American National Standards Institute (ANSI) (ANSI SLAS1-2004 to 4-2004). For example, in some implementations, the microtiter plates have a footprint of approximately 127.76 mm (5.0299 inches) long and 85.48 mm (3.3654 inches) wide.

[0418] For example, in some embodiments, the multi-container support plate used in the instrument system is a conventional multi-container support plate containing 96 wells. For example, in some embodiments, the multi-container support plate used in the instrument system contains multiple wells exceeding 96 wells to increase the throughput of the instrument system's NULISA assays. For example, in some embodiments, the multi-container support plate used in the instrument system may have any well array configuration, but is not limited to, that described in Table 1.

[0419] Table 1

[0420] In addition to commonly used consumables, the disclosed instruments also employ the following disclosed proprietary consumables to facilitate fully automated measurement operations and / or improve the user experience.

[0421] Framed sealing film Sealing assay plates or sample plates with biochemically compatible sealing films is a widely used procedure that can be performed during an assay (before incubation or amplification via a PCR target) or at the end of an assay (to prepare for long-term storage). Sealing films typically comprise multiple layers of material, with the layer facing the assay plate facilitating adhesion and other layers providing structure or protection. Some films have a metallic layer to prevent evaporation and / or block light. For the purposes of this disclosure, all such films or foils are referred to as “sealing films.”

[0422] Integrating plate sealing into a fully automated testing process is challenging because the sealing membrane is flexible and stretchable, making it difficult to handle in automated instruments. Current methods typically involve producing the membrane in large rolls and using specialized mechanical and pneumatic mechanisms to pull the membrane from the rolls, lay it flat on the plate, seal it, and then cut it to a specific size. Such devices are bulky, complex, and costly, and are only suitable for applications requiring the sealing of a large number of plates.

[0423] U.S. Patent Application Publication No. US20120058516 discloses a different method in which a sealing membrane is pre-mounted on a rigid frame, allowing the framed seal membrane (FSF) assembly to be manipulated using mechanical clamps commonly found in automated measurement instrument systems. After sealing, the frame is typically separated from the membrane (“deframed”) so that the sealed plate can be used for subsequent measurement procedures. US20120058516 describes several methods for constructing and deframed FSFs, all of which appear theoretically feasible but struggle to achieve the robustness and reliability required for automated operation. The applicant of US20120058516 announced the launch of a commercial product, “FrameSeal,” based on this patent application in 2017, but later withdrew the product from the market, and the aforementioned application was subsequently abandoned.

[0424] We disclose a different FSF that has been proven to operate robustly in fully automated environments. Section 4.6 (Incubator Sealr) of this application describes a functional module designed for sealing and unpacking using the disclosed FSF.

[0425] Similar to the prior art described above, the frame can be made of any material with sufficient rigidity, and the membrane can be mounted on the frame by a variety of methods, including mechanical clamping, thermal welding, laser welding, epoxy resin bonding, pressure-sensitive bonding, etc. Figure 27 The illustration depicts a specific embodiment of the frame design, which includes a mechanical structure that can be stacked on a standard microtiter plate 27006, such that a pre-installed membrane 27002 lies flat over the top surface of the test plate to be sealed. It may optionally include a self-stacking structure. In one embodiment, the frame 27004 is molded from glass fiber reinforced polypropylene. This material provides sufficient mechanical rigidity to the frame 27004 while allowing most of the sealing membrane to be thermally welded to the frame 27004 along the weld line 27008.

[0426] Unlike existing technologies, the sealing membrane is pre-cut with a specific perforation pattern using die-cutting or other cutting techniques. The perforations consist of a series of openings and connections. The location and length of each perforation, as well as the width of the openings and connections within the perforation pattern, are carefully designed to ensure the membrane adheres to the frame with sufficient strength, guaranteeing safe handling of the FSF during manufacturing, transportation, and instrument operation. Furthermore, once the central portion of the membrane is sealed onto the measurement plate, as described in Section 4.5, the membrane can be easily peeled off the frame by pressing down on the central portion of the sealing membrane with the sealing block.

[0427] Figure 28A specific embodiment of the perforation design is shown, in which the membrane is welded to the frame on both sides, and the perforation line 28004 is positioned adjacent to the weld line 28002 (closer to the center of the membrane). Furthermore, the opening portion of the perforation extends to the edges of all four corners of the membrane. This design ensures that during frame removal, the tear line will reliably originate from these corners and form an opening 28006 along the perforation line 28004 until the membrane is completely separated from the frame.

[0428] Existing FSFs disclosed in the art only use a sealing film through heat-welded sealing plates, and most release methods are achieved by heat-softening the film. However, the FSF disclosed herein can use both heat-sealed films and PSA films. In many applications involving heat-sensitive reagents, PSA sealing films have advantages over heat sealing.

[0429] Universal reagent kit Biochemical assays typically consume a set of reagents, which are usually assembled into one or more "kits." In automated assay instruments, it is highly beneficial to handle these kits in a uniform manner. However, because the quantity, type, and volume of reagents in a kit are assay-specific, designing an instrument capable of performing a variety of different assays is challenging.

[0430] This paper discloses a "universal" reagent kit that is designed to be flexible enough to accommodate a variety of different reagents to meet assay requirements, while also having the footprint and size to be suitable for processing in fully automated assay instruments.

[0431] Figure 29A -C illustrates a specific design embodiment of box 29016, which includes a base 29008, a lid 29010, and various different types of reagent containers 29002 (e.g., MONO 29006 and TRIB 29004). The base 29008 contains multiple positions arranged in a two-dimensional spatial array. Each position is provided with a "slot" for securing a container 29002. Different types of containers 29002 have the same external profile to match the shape of the slots, so that they can be secured in a specific position by the base 29008. On the other hand, different container types have different volumetric capacities, and they may contain one or more containers or openings. Figure 29AThe illustrated implementation comprises two types of containers, one being a single container (29006) with a capacity of approximately 2.7 ml. The other (29004) comprises three containers. The two side containers each have a volumetric capacity of approximately 300 μl, and the central container has a volumetric capacity of approximately 200 μl. Each reagent in the kit is placed in one or more suitable containers 29002, sealed with a membrane or foil, and assembled onto the base 29008 of the kit. A cap 29010 is then placed on the base 29008, securely clamping the container 29002 within the kit. The cap 29010 has a set of holes on its top surface, aligned with the container posit...

Claims

1. A compact, fully automated instrument for performing dual capture and release multiplex immunoassays, comprising: A) a benchtop housing, comprising: 1) a controller; 2) a mechanical gantry capable of movement in three degrees of freedom; 3) a storage rack containing multi-container carrier plates accessible to both the user and the mechanical gantry; 4) a controllable stage providing multiple multi-container carrier plate positions, wherein the stage is capable of movement along the Y axis (front to back within the instrument housing); 5) a mixer serving as a magnetic bead processor and mixer, comprising multiple multi-container carrier plate platforms stacked vertically one on top of the other; 6) a washer for washing multi-container carrier plates; and 7) a reader.

2. A compact instrument designed to perform multi-step, high sensitivity immunoassays involving sequential capture / release processes, comprising: A) a benchtop housing with a touch screen display, comprising: 1) a controller; 2) a mechanical gantry capable of movement in three degrees of freedom; 3) a storage rack containing multi-container carrier plates accessible to both the user and the mechanical gantry; 4) a controllable stage providing multiple multi-container carrier plate positions, wherein the stage is capable of movement along the Y axis (front to back within the instrument housing); 5) a mixer serving as a magnetic bead processor and mixer, comprising multiple multi-container carrier plate platforms stacked vertically one on top of the other; 6) a washer for washing multi-container carrier plates; 7) an incubation sealer for incubating and sealing at least one multi-container carrier plate; and 8) a reader.

3. A fully automated, high throughput precision proteomics instrument for performing ultra-high sensitivity analysis at a range of multiplexed assay levels to support broad biomarker profiling and translation of validated biomarkers, comprising: A) a benchtop housing with a touch screen display, a user accessible large capacity reagent chamber, and a user accessible set of compartment chambers, comprising: 1) a controller; 2) a controllable mechanical gantry equipped with an end effector comprising a pipettor, a multi-container carrier plate gripper, a laser position sensor, and a barcode scanner, the mechanical gantry capable of moving the end effector in three degrees of freedom in the X, Y, and Z axes; 3) a storage rack comprising a set of multi-container carrier plate compartments for receiving and housing at least one universal kit and consumable carrying device; 4) a controllable stage providing multiple multi-container carrier plate positions, wherein the stage is capable of movement along the Y axis (front to back within the instrument housing); 5) a controllable mixer serving as a magnetic bead processor and mixer, comprising multiple multi-container carrier plate platforms stacked vertically one on top of the other; 6) a controllable washer for washing multi-container carrier plates; 7) an incubation sealer for incubating and sealing at least one multi-container carrier plate; 8) a large capacity fluid station comprising a plurality of containers; and 9) a controllable reader.

4. A benchtop instrument for performing a plurality of multiplexed oligonucleotide conjugated antibody proximity ligation assays in parallel on a plurality of biological samples automatically, comprising: A) a benchtop housing with a touch screen display, a user accessible large capacity reagent chamber, and a user accessible set of compartment chambers, comprising: 1) a controller; 5) a controllable mixer serving as a magnetic bead processor and mixer, comprising multiple multi-container carrier plate platforms stacked vertically one on top of the other; 6) a controllable washer for washing multi-container carrier plates; 7) an incubation sealer for incubating and sealing at least one multi-container carrier plate; and 8) a controllable reader. 2) a mechanical gantry equipped with an end effector comprising a pipette, a multi- container carrier plate gripper, a laser position sensor, and a bar code scanner, the mechanical gantry capable of moving the end effector in three degrees of freedom in the X, Y, and Z axes; 3) a storage rack comprising a set of multi-container carrier plate compartments for receiving and housing at least one universal cartridge and consumable carrier device; 4) a stage providing a plurality of multi-container carrier plate positions, wherein the stage is capable of moving along the Y axis (within the instrument housing from front to back); 5) a mixer for use as a magnetic bead processor and mixer comprising a plurality of multi- container carrier plate platforms stacked vertically on top of one another; 6) a washer for washing multi-container carrier plates; 7) a reader.

5. The instrument and / or method of any one of the preceding claims, wherein at least one of the multi-container carrier plates comprises a set of pair-wise binding moieties preselected for binding to a particular analyte, wherein the pair-wise binding moieties comprise: (i) a first moiety comprising a first antibody or a first antibody fragment preselected for binding to a particular analyte, a first nucleic acid target tag label comprising a first identity specific to the particular analyte, and a first nucleic acid tag; (ii) a second moiety comprising a second antibody or a second antibody fragment preselected for binding to the same particular analyte as the first antibody or antibody fragment of the first moiety of the pair-wise binding moiety, a second nucleic acid target tag label comprising a second identity specific to the particular analyte, and a second nucleic acid tag.

6. A benchtop automated instrument for performing biochemical assays on a biological sample comprising a plurality of analytes, comprising: a) a controller; b) a controllable gantry; c) a controllable stage; d) a set of 5 multi-container carrier plates; e) a controllable quantitative PCR unit; f) a controllable plate washer; g) a controllable plate sealer; h) a controllable plate incubator; i) a controllable mixer comprising a paramagnetic bead extractor comprising magnetic poles and a comb subassembly; j) a storage rack accessible to the gantry, comprising: 1) a plurality of first moieties comprising a first antibody or a capture antibody fragment conjugated to a first nucleic acid tag that hybridizes to a first nucleic acid target tag label and is not covalently attached to the first antibody or first antibody fragment; 2) a plurality of second moieties comprising a second antibody or a second antibody fragment conjugated to a second nucleic acid target tag label that hybridizes to a second nucleic acid tag and is not covalently attached to the second antibody or second antibody fragment; 3) a first set of first substrates; 4) a second set of second substrates; 5) a set of buffers; 6) a plurality of double-stranded nucleic acid sample labels comprising a 5' overhang and a 3' overhang; 7) a frame comprising a sealing film; and k) a controller comprising a processor and a non-transitory machine-readable storage medium comprising instructions executable by the processor to provide controllable operation of unit operations within the instrument, operations controlled by the controller comprising: 1) forming a first solution in a first multi-container carrier plate by mixing: i) a portion of at least one sample, ii) a portion of the plurality of first moieties, and iii) a portion of the plurality of second moieties; 2) incubating the first solution to form an immunocomplex comprising one of the first moieties in a portion of the plurality of first moieties, one of the second moieties in a portion of the plurality of second moieties, and the analyte in at least one sample; 3) performing a sequential capture and release mechanism on the immunocomplex by introducing: i) a portion of the first set of first substrates to capture at least a portion of the immunocomplex from the first solution in the first multi-well carrier plate, wherein the first solution in the first multi-well carrier plate is incubated and the first set of paramagnetic beads is transferred to the second multi-well carrier plate by the mixer to form a second solution, thereby releasing at least a portion of the immunocomplex, and the first set of first substrates is removed by the mixer, and ii) a portion of the second set of second substrates to capture at least a portion of the immunocomplex from the second solution in the second multi-well carrier plate, wherein the second solution is incubated and the second set of second substrates is removed by the mixer to form a third solution in a third multi-well plate; 4) introducing at least a portion of the plurality of double-stranded nucleic acid sample labels into the third solution to form at least one multiplexed analyte-specific reporter molecule comprising the first nucleic acid target label associated with one of the first antibodies in at least one of the immunocomplexes and at least one of the second nucleic acid target labels conjugated to one of the second antibodies in at least one of the immunocomplexes, and a sample-specific identification barcode hybridized to one of the first nucleic acid target labels associated with one of the first antibodies in at least a portion of the immunocomplexes by one overhang and to the second nucleic acid target label conjugated to one of the second antibodies in at least a portion of the immunocomplexes by another overhang to complete proximity ligation between the first nucleic acid target label and the nucleic acid sample label and between the second nucleic acid target label and the nucleic acid sample label to generate at least one multiplexed analyte-specific reporter molecule conjugated to the second antibody or second antibody fragment and not covalently attached to the first antibody or first antibody fragment; 5) collecting the at least one multiplexed analyte-specific reporter molecule in a fourth multi-well carrier plate, followed by pooling the multiplexed analyte-specific reporter molecules to form a pooled sample for detection of the analyte with amole sensitivity by NGS.

7. A benchtop automated instrument for performing biochemical assays on a biological sample comprising a plurality of analytes, comprising: a) a controller; b) a controllable gantry; c) a controllable stage; d) a set of 5 multi-well carrier plates; e) a controllable quantitative PCR unit; f) a controllable plate washer; g) a controllable plate sealer; h) a controllable plate incubator; i) a controllable mixer comprising a paramagnetic bead extractor comprising magnetic poles and a comb subassembly; j) a gantry-accessible storage rack comprising: 1) a plurality of first moieties comprising a first antibody or a capture antibody fragment conjugated to a first nucleic acid tag that hybridizes to a first nucleic acid target label and is not covalently attached to the first antibody or first antibody fragment; 2) a plurality of second moieties comprising a second antibody or a second antibody fragment conjugated to a second nucleic acid target label that hybridizes to a second nucleic acid tag and is not covalently attached to the second antibody or second antibody fragment; 3) a first set of first substrates; 4) a second set of second substrates; 5) a set of buffers; 6) a plurality of double-stranded nucleic acid sample labels comprising a 5' overhang and a 3' overhang; 7) a frame containing a sealing membrane; and k) a controller for controlling unit operations of the instrument, the controller comprising a non-transitory computer readable storage medium having executable files therein, the executable files executable by the instrument controller to cause unit operations of the instrument to perform operations, the instrument operations comprising: 1) forming a first solution in a first multi-vessel carrier plate by mixing: i) a portion of at least one sample, ii) a portion of the plurality of first moieties, and iii) a portion of the plurality of second moieties; 2) incubating the first solution to form an immunocomplex comprising one of the first moieties in the portion of the plurality of first moieties, one of the second moieties in the portion of the plurality of second moieties, and the analyte in the at least one sample; 3) performing a sequential capture and release mechanism on the immunocomplex to remove contaminants and improve sensitivity by introducing: i) a portion of the first set of first substrates to capture at least a portion of the immunocomplex from the first solution in the first multi-vessel carrier plate, wherein the first solution in the first multi-vessel carrier plate is incubated, and a first set of paramagnetic beads is transferred by the mixer to a second multi-vessel carrier plate to form a second solution, thereby releasing at least a portion of the immunocomplex, and the first set of first substrates is removed by the mixer, and ii) a portion of the second set of second substrates to capture at least a portion of the immunocomplex from the second solution in the second multi-vessel carrier plate, wherein the second solution is incubated, and the second set of second substrates is removed by the mixer to form a third solution in a third multi-vessel carrier plate; 4) introducing at least a portion of the plurality of double-stranded nucleic acid sample labels into the third solution to form at least one multiplexed analyte-specific reporter molecule comprising the first nucleic acid target label associated with at least one of the first antibodies in the immunocomplex and at least one of the second nucleic acid target label conjugated to at least one of the second antibodies in the immunocomplex, and a sample-specific identification barcode hybridized to at least a portion of the first nucleic acid target label associated with at least one of the first antibodies in the immunocomplex by one overhang and to at least a portion of the second nucleic acid target label conjugated to at least one of the second antibodies in the immunocomplex by another overhang to complete the proximity ligation between the first nucleic acid target label and the nucleic acid sample label and between the second nucleic acid target label and the nucleic acid sample label to generate at least one multiplexed analyte-specific reporter molecule conjugated to the second antibody or second antibody fragment and not covalently attached to the first antibody or first antibody fragment; 5) collecting the at least one multiplexed analyte-specific reporter molecule in a fourth multi-well carrier plate, followed by pooling the multiplexed analyte-specific reporter molecules to form a pooled sample for detection of the analyte at an amole level of sensitivity by NGS.

8. A benchtop automated instrument for performing biochemical assays to detect an analyte in at least a first biological sample and a second biological sample, comprising: a) a gantry; b) a stage; c) a plurality of multi-well carrier plates, including a first plate, a second plate, a third plate, and a fourth plate; d) a quantitative PCR unit; e) a plate washer; f) a plate sealer; g) a plate incubator; h) a mixer, including a paramagnetic bead extractor comprising magnetic poles and a comb subassembly; i) a storage rack, comprising: 1) a plurality of first portions comprising a first antibody or a capture antibody fragment conjugated with a first nucleic acid tag that hybridizes to a first nucleic acid target label and is not covalently attached to the first antibody or first antibody fragment; 2) a plurality of second portions comprising a second antibody or a second antibody fragment conjugated with a second nucleic acid target label that hybridizes to a second nucleic acid tag and is not covalently attached to the second antibody or second antibody fragment; 3) a first set of first substrates; 4) a second set of second substrates; 5) a set of buffers; 6) a plurality of double-stranded nucleic acid sample labels comprising a 5' overhang and a 3' overhang; 7) a frame comprising a sealing film; and j) a controller comprising a processor and a non-transitory machine-readable storage medium containing instructions executable by the processor to provide controllable operation of unit operations within the instrument, operations controlled by the controller including: 1) for each of a first assay reaction comprising the first biological sample and a second assay reaction comprising the second biological sample, forming a first solution in the first multi-well carrier plate by mixing: i) at least a portion of the sample, ii) a portion of the plurality of first portions, and iii) a portion of the plurality of second portions. iii) One of the plurality of second parts; 2) Incubate the first solution of the first assay reaction and the second assay reaction to form an immune complex with the analyte; 3) To remove contaminants and improve sensitivity, the immune complexes in each of the first and second assay reactions are subjected to a dual capture and release mechanism via the following methods: i) Introducing a portion of the first set of first substrates into each of the first assay reaction and the second assay reaction to capture at least a portion of the immune complex from a first solution in the first multi-container support plate, wherein the first solution in the first multi-container support plate is incubated, and the first set of paramagnetic beads is transferred to a second multi-container support plate by the mixer to form a second solution, thereby releasing at least a portion of the immune complex, and removing the first set of first substrates by the mixer; ii) Introduce a portion of the second set of second substrates into each of the first assay reaction and the second assay reaction to capture at least a portion of the immune complex from the second solution in the second multi-container support plate, wherein the second solution is incubated and the second set of second substrates is removed by the mixer to form a third solution in the third multi-container support plate; 4) Introducing at least a portion of the plurality of double-stranded nucleic acid sample markers into the third solution of each of the first and second assay reactions to form at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule comprising a first nucleic acid target marker associated with at least one first antibody in at least one of the immune complexes and at least one second nucleic acid target marker conjugated to at least one second antibody in at least one of the immune complexes, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of the first antibody in the immune complexes through one protrusion and with the second nucleic acid target marker conjugated to at least a portion of the second antibody in the immune complexes through another protrusion to complete proximity linkage between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule being conjugated to the second antibody or second antibody fragment and not covalently attached to the first antibody or first antibody fragment; 5) Collect the at least one multiplex analyte-specific reporter molecule in the fourth multi-container support plate, and then combine the multiplex analyte-specific reporter molecule of the first assay reaction with the multiplex analyte-specific reporter molecule of the second assay reaction to form a pooled sample for detection of the analyte by NGS at an amolar sensitivity.

9. A method for automatically performing parallel assays of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: A) In a first plate, a portion of a plurality of multiple pairwise binding moieties is introduced in parallel into a plurality of biological samples to form a plurality of immune complex forming solutions, wherein the multiple pairwise binding moieties comprise different pre-selected pairwise binding moieties for binding different specific analytes, the pairwise binding moieties comprising: i) The first portion of the paired binding portion includes a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker including a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion includes a second antibody or second antibody fragment pre-selected for binding to a specific analyte that is the same as the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker containing a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first portion of the pair-binding portion is preselected for binding to a first substrate that is the same for all different pair-binding portions in multiplex assays, and the second nucleic acid tag of the second portion of the pair-binding portion is preselected for binding to a second substrate that is the same for all different pair-binding portions in multiplex assays; B) Incubate the multiple immune complex forming solutions in parallel to form multiple multiple immune complexes; C) For the first time, a portion of the first substrate solution containing multiple first substrates is combined with the multiple immune complex forming solution; D) For the first time, the first nucleic acid tag of the first portion of the paired binding portion binds to a portion of the first substrate; E) Firstly, through a portion of the plurality of first substrates, the plurality of multiple immune complexes are extracted in parallel from the plurality of immune complex forming solutions to form a plurality of first immune complex purification solutions in a second plate; F) Elute the multiple first substrates in parallel from the multiple multiple immune complexes in the purification solution of the multiple first immune complexes; G) Remove the plurality of first substrates in parallel from the purified solutions of the plurality of first immune complexes; H) The second step involves combining a portion of the second substrate solution containing multiple second substrates with the multiple first immune complex purification solutions in parallel. I) The second nucleic acid tag of the second portion of the paired binding portion is bound to a portion of the second substrate for the second time; J) The plurality of multiple immune complexes are extracted in parallel from the plurality of first immune complex purification solutions through a portion of the plurality of second substrates to form a plurality of second immune complex purification solutions in the third plate; K) Connecting multiple first nucleic acid target markers (directly or indirectly) of the first portion of the pair-binding portion to the second nucleic acid target marker of the second portion of the pair-binding portion in parallel to form multiple multiplex analyte-specific reporter molecules; L) A third extraction of the multiple multiple immune complexes (or the multiple multiple analyte-specific reporter molecules) from the multiple second immune complex purification solutions in parallel through a portion of the multiple second substrates to form multiple third immune complex purification solutions in the second plate; M) Further elute the multiple second substrates in parallel from the multiple multiple analyte-specific reporter molecules in the purification solution of the multiple third immune complexes; N) Replicate the multiple multiplex analyte-specific reporter molecules; and O) Detect the replicated multiple multiplex analyte-specific reporter molecules to identify (and quantify) the specific analyte in the multiple biological samples.

10. A method for automatically performing parallel assays of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: A) In a first multi-container carrier plate, a portion of a plurality of multiple multiple paired binding portions is introduced in parallel into a plurality of biological samples to form a plurality of immune complex forming solutions; The multiple pairwise binding portions of the plurality of multiple pairwise binding portions include different pre-selected pairwise binding portions for binding different specific analytes, the pairwise binding portions including: i) The first portion of the paired binding portion includes a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker including a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion includes a second antibody or second antibody fragment pre-selected for binding to a specific analyte that is the same as the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker containing a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first portion of the pair-binding portion is preselected for binding to a first substrate that is the same for all different pair-binding portions in multiplex assays, and the second nucleic acid tag of the second portion of the pair-binding portion is preselected for binding to a second substrate that is the same for all different pair-binding portions in multiplex assays; B) The multiple immune complex forming solutions are incubated in parallel to form multiple multiple immune complexes in a first multi-container support plate; C) For the first time, a portion of a first substrate solution containing multiple first substrates is combined with the multiple immune complexes formed in a first multi-container carrier plate; D) For the first time, the first nucleic acid tag of the first portion of the paired binding portion is bound to a portion of the first substrate in the first multi-container carrier plate; E) For the first time, the multiple multiple immune complexes are extracted in parallel from a first multi-container plate through a portion of the multiple first substrates and transferred to a second multi-container plate; F) Clean the multiple multiple immune complexes in the second multi-container carrier plate; G) Extract the plurality of multiple immune complexes from the second multi-container plate for the second time and transfer them to the third multi-container plate; H) Elution of the multiple first substrates in parallel from multiple multiple immune complexes in the third multi-container carrier plate; I) Remove the plurality of first substrates in parallel from the third multi-container support plate; J) A portion of the second substrate solution containing multiple second substrates is combined in parallel with the multiple multiple immune complexes in the third multi-container carrier plate; K) The second nucleic acid tag of the second portion of the paired binding portion is secondly bound to a portion of the second substrate of the third multi-container carrier plate; L) A third time, through a portion of the plurality of second substrates, the plurality of multiple immune complexes are extracted in parallel from the third multi-container plate and transferred to the second multi-container plate; M) In the second multi-container carrier plate, multiple first nucleic acid target markers (directly or indirectly) of the first portion of the paired binding portion on the multiple multiple immune complexes are connected in parallel with the second nucleic acid target markers of the second portion of the paired binding portion to form multiple multiple analyte-specific reporter molecules; N) The multiple multiple immune complexes carrying analyte-specific reporter molecules are extracted in parallel from the second multi-container plate through a portion of the multiple second substrates for the fourth time and transferred to the third multi-container plate; O) Elute the multiple second substrates in parallel from the multiple multiple immune complexes carrying analyte-specific reporter molecules in the third multi-container carrier plate; P) Replicate the multiple multiplex analyte-specific reporter molecules; and Q) Detect the replicated multiple multiplex analyte-specific reporter molecules to identify (and quantify) the presence of the specific analyte in multiple biological samples.

11. A method for automatically performing parallel assays of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: A) In a first multi-container carrier plate, a portion of a plurality of multiple multiple paired binding portions is introduced in parallel into a plurality of biological samples to form a plurality of immune complex forming solutions; The multiple pairwise binding portions of the plurality of multiple pairwise binding portions include different pre-selected pairwise binding portions for binding different specific analytes, the pairwise binding portions including: i) The first portion of the paired binding portion includes a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker including a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion includes a second antibody or second antibody fragment pre-selected for binding to a specific analyte that is the same as the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker containing a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first portion of the pair-binding portion is preselected for binding to a first substrate that is the same for all different pair-binding portions in multiplex assays, and the second nucleic acid tag of the second portion of the pair-binding portion is preselected for binding to a second substrate that is the same for all different pair-binding portions in multiplex assays; B) The multiple immune complex forming solutions are incubated in parallel to form multiple multiple immune complexes in a first multi-container support plate; C) For the first time, a portion of a first substrate solution containing multiple first substrates is combined with the multiple immune complexes formed in a first multi-container carrier plate; D) For the first time, the first nucleic acid tag of the first portion of the paired binding portion is bound to a portion of the first substrate in the first multi-container carrier plate; E) For the first time, the multiple multiple immune complexes are extracted in parallel from a first multi-container plate through a portion of the multiple first substrates and transferred to a second multi-container plate; F) Clean the multiple multiple immune complexes in the second multi-container carrier plate; G) Extract the plurality of multiple immune complexes from the second multi-container plate for the second time and transfer them to the third multi-container plate; H) Elution of the multiple first substrates in parallel from multiple multiple immune complexes in the third multi-container carrier plate; I) Remove the plurality of first substrates in parallel from the third multi-container support plate; J) A portion of the second substrate solution containing multiple second substrates is combined in parallel with the multiple multiple immune complexes in the third multi-container carrier plate; K) The second nucleic acid tag of the second portion of the paired binding portion is secondly bound to a portion of the second substrate of the third multi-container carrier plate; L) The plurality of multiple immune complexes are extracted in parallel from the third multi-container plate through a portion of the plurality of second substrates for the third time and transferred to the fourth multi-container plate; M) In the fourth multi-container carrier plate, multiple first nucleic acid target markers (directly or indirectly) of the first portion of the paired binding portion on the multiple multiple immune complexes are connected in parallel with the second nucleic acid target markers of the second portion of the paired binding portion to form multiple multiple analyte-specific reporter molecules; N) The multiple multiple immune complexes carrying analyte-specific reporter molecules are extracted in parallel from the fourth multi-container plate through a portion of the multiple second substrates for the fourth time and transferred to the third multi-container plate; O) Elute the multiple second substrates in parallel from the multiple multiple immune complexes carrying analyte-specific reporter molecules in the third multi-container carrier plate; P) Replicate the multiple multiplex analyte-specific reporter molecules; and Q) Detect the replicated multiple multiplex analyte-specific reporter molecules to identify (and quantify) the presence of the specific analyte in multiple biological samples.

12. A method for performing a biochemical assay on a biological sample to detect at least one analyte on a benchtop automated system, comprising: 1) Transfer at least one sample from the sample multi-container support plate to the first multi-container support plate; 2) Dilute the sample in the first multi-container support plate; 3) A first solution is formed in the second multi-container support plate by mixing the following substances: i) at least a portion of a sample, ii) A portion of the plurality of first portions includes a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker including a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag, and iii) A portion of the plurality of second portions includes a second antibody or second antibody fragment pre-selected for binding to the same specific analyte as the first antibody or antibody fragment of the first portion of the paired binding portion, a second nucleic acid target marker including a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; 4) Incubate the first solution to form an immune complex comprising a first portion of a plurality of first portions, a second portion of a plurality of second portions, and at least one analyte from the sample; 5) To remove contaminants and improve sensitivity, the immune complexes are subjected to a dual capture and release mechanism via the following methods: i) Add a portion of the first substrate to capture at least a portion of the immune complex from the first solution in the second multi-container support plate; ii) Incubate the first solution in the second multi-container carrier plate to immobilize the immune complex on the surface of the first substrate; iii) The first substrate is transferred to a third multi-container support plate via a mixer to form a second solution with the elution buffer; iv) Clean the first substrate to remove unbonded impurities; v) Incubate the second solution in the third multi-container support plate to release at least a portion of the immune complex; vi) Remove the first substrate using the mixer; vii) Adding a portion of the second substrate to the second solution in the third multi-container support plate; viii) Incubate the second solution in the second multi-container carrier plate to immobilize the immune complex on the surface of the second substrate; ix) The second substrate is transferred through the mixer to a third multi-container support plate to form a third solution; x) Clean the second substrate to remove unbonded impurities; 6) Add a connecting reagent to the third solution in the fourth multi-container support plate; 7) Introducing at least a portion of the plurality of double-stranded nucleic acid sample markers into a third solution in the fourth multi-container support plate to form at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule comprising a first nucleic acid target marker associated with a first portion of at least one of the immune complexes and at least one second nucleic acid target marker conjugated to a second portion of at least one of the immune complexes, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of the first portion of the immune complexes through one protrusion and with the second nucleic acid target marker conjugated to a second portion of the immune complexes through another protrusion to complete proximity linkages between the first nucleic acid target marker and the nucleic acid sample markers and between the second nucleic acid target marker and the nucleic acid sample markers, to generate at least one multiplex analyte-specific reporter molecule, the multiplex analyte-specific reporter molecule being conjugated to the second antibody or second antibody fragment and not covalently attached to the first antibody or first antibody fragment; 8) Collect the at least one multiplex analyte-specific reporter molecule in the first multi-container support plate and combine the solutions of the multiplex analyte-specific reporter molecule in the first multi-container support plate; as well as 9) Transfer the combined multiplex analyte-specific reporter molecule solution from the first plate to a qPCR plate and detect the analytes by NGS with atmolar sensitivity.

13. A rapid, compact instrument for automatically performing proximity-to-conjugate assays of multiple multiple oligonucleotide conjugated antibodies on multiple biological samples, comprising: a) Target kit, comprising: Multiple pre-selected paired binding portions for binding a specific analyte, the paired binding portions including: i) The first part includes a pre-selected first antibody or first antibody fragment for binding to a specific analyte, a first nucleic acid target marker including a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second part comprises a second antibody or second antibody fragment pre-selected for binding to a specific analyte identical to the first antibody or antibody fragment of the first part of the paired binding portion, a second nucleic acid target marker comprising a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; and b) A gantry equipped with an end effector with pipettes and plate clamps, an incubator, and a substrate extractor / mixer for performing dual capture and release of immune complex purification using a portion of multiple paired binding parts.

14. A benchtop automated biochemical assay instrument for preparing multiplex analyte-specific reporter molecules in a sample, comprising: a) A set of multi-container support plates, each containing multiple holes; b) Target kit containing multiple wells; 1) In the target kit, at least one of the multiple wells, the first well includes a first portion, the first portion including a first antibody or a first antibody fragment that specifically binds to the analyte, a first nucleic acid tag conjugated to the first antibody or the first antibody fragment, and a first nucleic acid target label that hybridizes with the first nucleic acid tag; 2) The second well of at least one of the plurality of wells comprises a second portion, the second portion comprising a second antibody or second antibody fragment that specifically binds to the analyte, a second nucleic acid label conjugated to the second antibody or second antibody fragment, and a second nucleic acid tag that hybridizes with the second nucleic acid label.

15. A rapid, compact instrument for automatically performing multiple multiple oligonucleotide conjugated antibody proximity assays on multiple biological samples, comprising: a) Target kit, comprising: Multiple pre-selected paired binding portions for binding a specific analyte, the paired binding portions including: i) The first part includes a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker including a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; ii) The second part includes a second antibody or second antibody fragment pre-selected for binding to the same specific analyte as the first antibody or antibody fragment of the first part of the paired binding portion, a second nucleic acid target marker containing a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; b) Test kit, containing: i) a first substrate solution comprising a plurality of first substrates; and ii) A second substrate solution comprising multiple second substrates; and iii) Ligation reagents and optional nucleic acid sample-specific markers c) Microtiter plate containing at least 96 wells; d) A gantry frame equipped with an end effector with pipettes and plate clamps, an incubator, and a substrate extractor / mixer for performing dual capture and release of immune complex purification using a portion of a plurality of paired binding moieties and a first substrate solution and a second substrate solution, and additionally using a ligation reagent to ligate (directly or indirectly) a portion of a first nucleic acid target label of the plurality of paired binding moieties to a portion of a second nucleic acid target label of the plurality of paired binding moieties to form a plurality of analyte-specific reporter molecules.

16. A benchtop instrument for automatically performing parallel assays of multiple multiple oligonucleotide conjugated antibody proximity on multiple biological samples, comprising: 1) A multi-container support plate containing multiple biological samples; 2) Storage rack, including: a) Target kit, comprising: Multiple multiple pairwise binding portions, the multiple pairwise binding portions comprising different pre-selected pairwise binding portions for binding different specific analytes, the pairwise binding portions including: i) The first portion of the paired binding portion, the first portion comprising a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker comprising a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion, the second portion comprising a second antibody or second antibody fragment pre-selected for binding a specific analyte identical to the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker comprising a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first portion of the pair-binding portion is preselected for binding to a first substrate that is the same for all different pair-binding portions in multiplex assays, and the second nucleic acid tag of the second portion of the pair-binding portion is preselected for binding to a second substrate that is the same for all different pair-binding portions in multiplex assays; b) Test kit, containing: i) a first substrate solution comprising a plurality of first substrates; and ii) A second substrate solution comprising multiple second substrates; 3) Gantry crane, equipped with end effector with pipette and plate clamp; 4) Incubator; 5) Substrate extractor / mixer; 6) qPCR; 7) A first plate containing multiple first holes; 8) A second plate containing multiple second holes; 9) A third plate containing multiple third holes; and 10) Controller, of which a) The controller controls the gantry to introduce, in parallel, a portion of the plurality of multiple pairwise binding portions of the target kit and a portion of the plurality of biological samples into a portion of a plurality of first wells of the first plate to form a plurality of immune complex forming solutions in a portion of a plurality of first wells of the first plate; b) The controller controls the gantry and the incubator to incubate the plurality of immune complex forming solutions in a portion of a plurality of first wells of the first plate in parallel to form a plurality of multiple immune complexes; c) The controller controls the gantry to combine a portion of a first substrate solution containing the plurality of first substrates in the test kit with the plurality of immune complex forming solutions in a portion of the plurality of first wells in the first plate; d) The controller controls the gantry and the incubator to bind the first nucleic acid tag of the first portion of the multiple paired binding portion to a portion of the first substrate in a portion of a plurality of first wells of the first plate; e) The controller controls the substrate extractor to extract the plurality of multiple immune complexes in parallel from the plurality of immune complex forming solutions through a portion of the plurality of first substrates to form a plurality of first immune complex purification solutions in at least a portion of a plurality of second wells of the second plate, wherein a portion of the plurality of first substrates elutes from the plurality of multiple immune complexes in the plurality of first immune complex purification solutions; f) The controller controls the substrate extractor to extract a portion of the plurality of first substrates in parallel from the plurality of first immune complex purification solutions; g) The controller controls the gantry to combine a portion of the second substrate solution containing the plurality of second substrates in the test kit with the plurality of first immune complex purification solutions in a portion of the plurality of second wells in the second plate; h) The controller controls the gantry and the incubator to bind a portion of the second nucleic acid tag of the second portion of the paired binding portion to a portion of the second substrate of a portion of the purified solution of the plurality of first immune complexes in a portion of a plurality of second wells of the second plate; i) The controller controls the substrate extractor to extract a portion of the second substrate in parallel in a portion of the purification solution of a plurality of first immune complexes in a portion of a plurality of second wells of the second plate through a portion of the plurality of second substrates, to form a plurality of multiplex analyte-specific reporter molecules in a portion of a plurality of third wells of the third plate by linking a plurality of first nucleic acid target tags (directly or indirectly) of the first portion of the pair-binding portion in parallel with a plurality of second nucleic acid target tags of the second portion of the pair-binding portion; j) The controller controls the substrate extractor to extract, through a portion of the plurality of second substrates, the plurality of multiple analyte-specific reporter molecules in a portion of a plurality of third wells of the third plate back in parallel to a portion of a plurality of second wells of the second plate, so as to elute the plurality of second substrates in parallel from the plurality of multiple analyte-specific reporter molecules in a portion of a plurality of second wells of the second plate; k) The controller controls the gantry and thermal cycler to replicate the plurality of multiplex analyte-specific reporter molecules; and l) The controller controls qPCR to detect replicated multiple multiplex analyte-specific reporter molecules to identify (and quantify) the specific analyte in multiple biological samples.

17. A method for automating dual capture and release multiplex immunoassays on multiple biological samples, comprising: Operating instruments, the instruments including: 1) Controller; 2) A mechanical gantry crane capable of moving in three degrees of freedom; 3) A storage rack that accommodates multiple container support plates, accessible to both the user and the mechanical gantry; 4) A mixer that serves as a magnetic bead processor and includes multiple multi-container carrier plate platforms; 5) Reader; and 6) The following components located within the storage rack: a) Multiple multi-container support plates, including a first plate with multiple first wells, a second plate with multiple second wells, a third plate with multiple third wells, a fourth plate with multiple fourth wells, and a multi-container support measurement plate containing the multiple biological samples; and b) Multi-container target delivery kit, comprising: Multiple multiple pairwise binding portions, the multiple pairwise binding portions comprising different pre-selected pairwise binding portions for binding different specific analytes, the pairwise binding portions including: i) The first portion of the paired binding portion, the first portion comprising a pre-selected first antibody or first antibody fragment for binding a specific analyte, a first nucleic acid target marker comprising a first identifier having analyte specificity for the specific analyte, and a first nucleic acid tag; and ii) The second portion of the pair-binding portion, the second portion comprising a second antibody or second antibody fragment pre-selected for binding a specific analyte identical to the first antibody or antibody fragment of the first portion of the pair-binding portion, a second nucleic acid target marker comprising a second identifier having analyte specificity for the specific analyte, and a second nucleic acid tag; The first nucleic acid tag of the first portion of the pair-binding portion is preselected for binding to a first substrate that is the same for all different pair-binding portions in multiplex assays, and the second nucleic acid tag of the second portion of the pair-binding portion is preselected for binding to a second substrate that is the same for all different pair-binding portions in multiplex assays; c) Multi-container test kit, comprising: i) a first substrate solution comprising a plurality of first substrates; and ii) A second substrate solution comprising multiple second substrates; Operate the instrument to control: a) The mechanical gantry is used to introduce, in parallel, a portion of a plurality of multiple multiple pairwise binding portions of the target kit and a portion of a plurality of biological samples in the assay plate into a portion of a plurality of first wells of the first plate, to form a plurality of immune complex forming solutions in a portion of a plurality of first wells of the first plate; b) The gantry and the substrate mixer are used to incubate the plurality of immune complex forming solutions in a portion of a plurality of first wells of the first plate in parallel to form a plurality of multiple immune complexes; c) The gantry for combining a portion of a first substrate solution containing a plurality of first substrates in the test kit with the plurality of multiple immune complexes in a portion of a plurality of first wells in the first plate; d) The gantry and the mixer, such that a portion of the first nucleic acid tag of the first portion of the multiple paired binding portion binds to a portion of the first substrate in a portion of a plurality of first wells of the first plate; e) The mixer is configured to extract, in parallel, a plurality of multiple immune complexes bound to the plurality of first substrates from a portion of a plurality of first wells in the first plate through a portion of the plurality of first substrates, and transfer them to a portion of a plurality of second wells in the second plate, and elute the plurality of first substrates from the plurality of multiple immune complexes in a portion of a plurality of second wells in the second plate; f) The mixer is used to extract a portion of the plurality of first substrates in parallel from at least a portion of the plurality of multiple immune complexes in a plurality of second wells of the second plate; g) The gantry for combining a portion of a second substrate solution containing the plurality of second substrates with the plurality of multiple immune complexes in a portion of a plurality of second wells of the second plate; h) The gantry and the mixer, such that a portion of the second portion of the paired binding portion of the second nucleic acid tag binds to a portion of the second substrate in a portion of a plurality of second wells of the second plate; i) The mixer is used to extract, in parallel, portions of the plurality of multiplex immune complexes bound to a portion of the second substrate in a portion of a plurality of second wells of the second plate through a portion of the plurality of second substrates, and to transfer them to a portion of a plurality of third wells of the third plate, and to form a plurality of multiplex analyte-specific reporter molecules by linking in parallel a plurality of first nucleic acid target markers (directly or indirectly) of the first portion of the pair-binding portion with a plurality of second nucleic acid target markers of the second portion of the pair-binding portion; j) The mixer is configured to extract, in parallel, the plurality of multiple analyte-specific reporter molecules bound to a portion of a portion of a plurality of third wells of the third plate through a portion of the plurality of second substrates, and transfer them back to a portion of a plurality of second wells of the second plate, and to elute the plurality of second substrates in parallel from the plurality of multiple analyte-specific reporter molecules in a portion of a plurality of second wells of the second plate; k) the gantry for transferring at least a portion of the plurality of multiplex analyte-specific reporter molecules from a portion of a plurality of second wells of the second plate to a portion of a plurality of fourth wells of the fourth plate; and l) The gantry frame is used to move the fourth plate to the reader.

18. A compact, fully automated instrument for performing dual capture and release multiplex immunoassays, comprising: A) Desktop chassis, including: 1) A mechanical gantry crane capable of moving in three degrees of freedom; 2) A storage rack that accommodates multiple container support plates, accessible to both the user and the mechanical gantry; 3) A controllable stage providing multiple multi-container support plate positions, wherein the stage is movable along the Y-axis (from front to back within the instrument housing); and 4) Mixer, which serves as a magnetic bead processor and mixer, comprises multiple multi-container carrier plate platforms stacked vertically to each other.

19. The instrument and / or method according to any one of the preceding claims, wherein one of the first nucleic acid tag or the second nucleic acid tag comprises a nucleotide-rich sequence.

20. The instrument and / or method according to any one of the preceding claims, wherein one of the first nucleic acid tag or the second nucleic acid tag comprises one or more of the following nucleotide-rich sequences: poly-A, poly-T, poly-C, or poly-G sequences.

21. The instrument and / or method according to any one of the preceding claims, wherein the first nucleic acid tag comprises a poly-A or poly-T sequence.

22. The instrument and / or method according to any one of the preceding claims, wherein one of the first nucleic acid tag or the second nucleic acid tag comprises an immobilization reagent.

23. The instrument and / or method according to any one of the preceding claims, wherein one of the first nucleic acid tag or the second nucleic acid tag comprises one or more of the following immobilization reagents: biotin, streptavidin, EDC, DCC, NHS ester, imine ester, maleimide, haloacetyl, pyridyl disulfide, acylhydrazine, alkoxyamine, aryl azide, diazide, or a chemically selective linker.

24. The instrument and / or method according to any one of the preceding claims, wherein the second nucleic acid tag is conjugated with biotin.

25. The instrument and / or method according to any one of the preceding claims, wherein the first nucleic acid tag is not covalently attached to the first antibody or antibody fragment.

26. The instrument and / or method according to any one of the preceding claims, wherein the first nucleic acid target label is not covalently attached to the first antibody or the first antibody fragment.

27. The instrument and / or method according to any one of the preceding claims, wherein the second nucleic acid tag is not covalently attached to the second antibody or the second antibody fragment.

28. The instrument and / or method according to any one of the preceding claims, wherein the first antibody or first antibody fragment pairs with the second antibody or second antibody fragment to bind to a non-overlapping epitope of the analyte and form an immune complex.

29. The instrument and / or method according to any of the preceding claims, wherein the first antibody or first antibody fragment has a binding affinity to the analyte of at least 10 -4 M and the second antibody or second antibody fragment has a binding affinity to the analyte of at least 10 -4 M.

30. The instrument and / or method according to any one of the preceding claims, wherein the first nucleic acid target marker comprises a first identification barcode.

31. The instrument and / or method according to any one of the preceding claims, wherein the second nucleic acid target marker comprises a second identification barcode.

32. The instrument and / or method according to any one of the preceding claims, wherein the instrument further comprises a target kit.

33. The instrument and / or method according to any one of the preceding claims, wherein the target kit comprises a plurality of wells in a multi-container plate. (i) wherein the first well of at least one of the plurality of wells in the target kit comprises the first portion; and (ii) The second well in at least one of the plurality of wells in the target kit contains the second portion.

34. The instrument and / or method according to any one of the preceding claims, wherein the first hole and the second hole are the same holes of the multi-container plate.

35. The instrument and / or method according to any one of the preceding claims, wherein the first hole and the second hole are different holes in the multi-container plate.

36. The instrument and / or method according to any one of the preceding claims, wherein the target kit further comprises a plurality of wells of the multi-container plate. (i) wherein a third well of at least one of the plurality of wells in the target kit contains a first nonfunctional binder comprising a third antibody or a third antibody fragment that specifically binds to the analyte; and (ii) wherein the fourth well of at least one of the plurality of wells of the target kit contains a second nonfunctional binder, the second nonfunctional binder containing a fourth antibody or a fourth antibody fragment that specifically binds to the analyte.

37. The instrument and / or method according to any one of the preceding claims, wherein the first nonfunctional binder binds to the same epitope of the analyte in the same manner as the first portion.

38. The instrument and / or method according to any one of the preceding claims, wherein the third antibody or the third antibody fragment is identical to the first antibody or the first antibody fragment of the first portion.

39. The instrument and / or method according to any one of the preceding claims, wherein the fourth antibody or the fourth antibody fragment is identical to the second antibody or the second antibody fragment of the second portion.

40. The instrument and / or method according to any one of the preceding claims, wherein the third hole and the fourth hole are the same holes of the multi-container plate.

41. The instrument and / or method according to any one of the preceding claims, wherein the third hole and the fourth hole are different holes in the multi-container plate.

42. The instrument and / or method according to any one of the preceding claims, wherein the first hole, the second hole, the third hole and the fourth hole are the same holes of the multi-container plate.

43. The instrument and / or method according to any one of the preceding claims, wherein the first hole, the second hole, the third hole and the fourth hole are different holes of the multi-container plate.

44. The instrument and / or method according to any one of the preceding claims, wherein the first nonfunctional binder is mixed with the first portion in a predetermined ratio.

45. The instrument and / or method according to any one of the preceding claims, wherein the second nonfunctional binder is mixed with the second portion in a predetermined ratio.

46. ​​The instrument and / or method according to any one of the preceding claims, wherein the instrument further comprises a detection kit.

47. The instrument and / or method according to any one of the preceding claims, wherein the detection kit comprises a plurality of wells of a multi-container plate. (i) wherein the first pore of at least one of the plurality of pores contains a first substrate solution containing a plurality of first substrates; (ii) wherein the second pore of at least one of the plurality of pores comprises a second substrate solution containing a plurality of second substrates; and (iii) The third well of at least one of the plurality of wells contains a ligation reagent and an optional nucleic acid sample-specific label.

48. The instrument and / or method according to any one of the preceding claims, wherein the first substrate is a first paramagnetic bead.

49. The instrument and / or method according to any one of the preceding claims, wherein the first paramagnetic bead is coupled with a first binding sequence capable of binding to the first nucleic acid tag on the first antibody or a first antibody fragment.

50. The instrument and / or method according to any one of the preceding claims, wherein the first paramagnetic bead is coated with a first binding sequence capable of binding to the first nucleic acid tag on the first antibody or a first antibody fragment.

51. The instrument and / or method according to any one of the preceding claims, wherein the second substrate is a second paramagnetic bead.

52. The instrument and / or method according to any one of the preceding claims, wherein the second paramagnetic bead is coupled with a second binding group capable of binding to the second nucleic acid tag associated with the second antibody or the second antibody fragment.

53. The instrument and / or method according to any one of the preceding claims, wherein the second paramagnetic bead is coated with a second binding group capable of binding to the second nucleic acid tag associated with the second antibody or the second antibody fragment.

54. The instrument and / or method according to any one of the preceding claims, wherein the first binding sequence is preselected for hybridization with the first nucleic acid tag on the first antibody or the first antibody fragment.

55. The instrument and / or method according to any one of the preceding claims, wherein the first binding sequence coupled to the first paramagnetic bead is a poly-A or poly-T sequence.

56. The instrument and / or method according to any one of the preceding claims, wherein the poly-A sequence from the first nucleic acid tag hybridizes with the poly-T sequence from the first paramagnetic bead.

57. The instrument and / or method according to any one of the preceding claims, wherein the poly-T sequence from the first nucleic acid tag hybridizes with the poly-A sequence from the first paramagnetic bead.

58. The instrument and / or method according to any one of the preceding claims, wherein the second binding group coupled to the second paramagnetic bead is streptavidin or avidin.

59. The instrument and / or method according to any one of the preceding claims, wherein the biotin from the second nucleic acid tag binds to streptavidin from the second paramagnetic bead.

60. The instrument and / or method according to any one of the preceding claims, wherein the biotin from the second nucleic acid tag binds to avidin from the second paramagnetic bead.

61. The instrument and / or method according to any one of the preceding claims, wherein the bond between the first nucleic acid tag and the binding sequence is an orthogonal bond.

62. The instrument and / or method according to any one of the preceding claims, wherein the bond between the second nucleic acid tag and the binding group is an orthogonal bond.

63. The instrument and / or method according to any one of the preceding claims, wherein the first, second, and third wells of the multi-container plate of the detection kit are located in the multi-container plate of the target kit.

64. The instrument and / or method according to any one of the preceding claims, wherein the first and second wells of the target kit are located in the detection kit.

65. The instrument and / or method according to any one of the preceding claims, wherein the instrument further comprises an incubation sealer for incubating and / or sealing at least one multi-container support plate.

66. The instrument and / or method according to any one of the preceding claims, wherein the instrument further comprises a large-capacity fluid station containing multiple containers.

67. The instrument and / or method according to any one of the preceding claims, wherein the mechanical gantry further comprises an end effector, the end effector comprising a pipette, a multi-container support plate clamp, a laser position sensor and a barcode scanner, the mechanical gantry being capable of moving the end effector in three degrees of freedom of the X, Y and Z axes.

68. The instrument and / or method according to any one of the preceding claims, wherein the reader comprises a qPCR unit for qPCR reading.

69. The instrument and / or method according to any one of the preceding claims, wherein the reader comprises qPCR for preparing a mixed library that can be used for next-generation sequencing (NGS).

70. The instrument and / or method according to any one of the preceding claims, wherein the reader is capable of identifying and / or quantifying nucleic acid reporter molecules.

71. The instrument and / or method according to any one of the preceding claims, wherein the housing further includes a touch screen display.

72. The instrument and / or method according to any one of the preceding claims, wherein the storage rack comprises a set of multi-container compartments for receiving and accommodating at least one universal reagent kit and consumable carrier.

73. The instrument and / or method according to any one of the preceding claims, wherein the storage rack further comprises: a) The target reagent kit; b) The test kit described; as well as c) The at least one consumable carrying device includes: i) PCR multi-container plate, ii) Framed sealing film, iii) Multiple pipette tips, and iv) Multiple multi-container support plates.

74. The instrument and / or method according to any one of the preceding claims, wherein the framed sealing membrane has one or more perforated lines configured to allow easy and complete separation of the membrane from the frame by tearing.

75. The instrument and / or method according to any one of the preceding claims, wherein the framed sealing membrane seals the holes when it is thermally welded to the edge of the multi-container support plate.

76. The instrument and / or method according to any one of the preceding claims, wherein the framed sealing membrane comprises a metal layer.

77. The instrument and / or method according to any one of the preceding claims, wherein the framed sealing membrane can be punctured by a pipette tip.

78. The instrument and / or method according to any one of the preceding claims, wherein the controller includes a processor and a non-transitory machine-readable storage medium, the non-transitory machine-readable storage medium containing instructions executable by the processor to provide controlled operation of components within the instrument.

79. The instrument and / or method according to any one of the preceding claims, wherein the controller includes a processor and a non-transitory machine-readable storage medium, the non-transitory machine-readable storage medium containing pre-programmed instructions executable by the processor to perform dual capture and release multiplex immunoassays.

80. The instrument and / or method according to any one of the preceding claims, wherein the controller comprises a processor and a non-transitory machine-readable storage medium, the non-transitory machine-readable storage medium containing instructions executable by the processor to provide controllable operation of unit operations within the system, the operations controlled by the controller including: a) A first solution is formed in the first multi-container support plate by mixing the following substances: i) at least one part of a biological sample, ii) a portion of multiple first parts, and iii) One of multiple parts of Part II; b) Incubate the first solution in the incubator to form an immune complex comprising a first portion of a plurality of first portions, a second portion of a plurality of second portions, and an analyte from at least one biological sample; c) To implement a dual capture and release mechanism on the immune complex by introducing the following substances to remove unbound contaminants and improve sensitivity: i) A portion of the first paramagnetic bead is used to capture at least a portion of the immune complex from a first solution in the first multi-container support plate, wherein the first solution in the first multi-container support plate is incubated, and the first paramagnetic bead is transferred to a second multi-container support plate by the extractor / mixer to form a second solution, thereby releasing at least a portion of the immune complex, and the first paramagnetic bead is removed by the extractor / mixer; ii) A portion of the second paramagnetic beads to capture at least a portion of the immune complex from the second solution, wherein the second solution is incubated and the second set of paramagnetic beads is removed by the extractor / mixer to form a third solution in a third multi-container carrier plate.

81. The instrument and / or method according to any one of the preceding claims, wherein the contaminant may include an unbound first portion or an unbound second portion.

82. The instrument and / or method according to any one of the preceding claims, wherein the contaminant may include a non-target analyte.

83. The instrument and / or method according to any one of the preceding claims, wherein the contaminant may include unbound target analytes.

84. The instrument and / or method according to any one of the preceding claims, wherein the operation controlled by the controller further comprises: a) Clean the first solid surface of the first substrate to remove the unbound contaminants; as well as b) Clean the second solid surface of the second substrate to remove the unbonded contaminants.

85. The instrument and / or method according to any one of the preceding claims, wherein the operation controlled by the system controller further includes: a) Introducing at least a portion of a plurality of nucleic acid sample markers into the third solution to form at least one nucleic acid reporter molecule, the nucleic acid reporter molecule comprising a first nucleic acid target marker associated with at least one first portion of the immune complex and at least one second nucleic acid target marker conjugated to at least one second portion of the immune complex, and a sample-specific identification barcode, hybridizing with the first nucleic acid target marker associated with at least a portion of the first portion of the immune complex through one protrusion and with the second nucleic acid target marker conjugated to at least a portion of the second portion of the immune complex through another protrusion to complete proximity linkages between the first nucleic acid target marker and the nucleic acid sample marker and between the second nucleic acid target marker and the nucleic acid sample marker, to generate at least one nucleic acid reporter molecule conjugated to the first portion and not covalently attached to the second portion; as well as b) The at least one nucleic acid reporter molecule is collected in a fourth multi-container plate and then the nucleic acid reporter molecules are combined to form a pooled sample for detection of the analyte by NGS with atmolar sensitivity and a wide dynamic range.

86. The instrument and / or method according to any one of the preceding claims, wherein the operation controlled by the controller further comprises cleaning the multi-container plate prior to performing the capture and release mechanism with a multi-container plate cleaner and reintroducing the immune complex into the cleaned multi-container plate.

87. The instrument and / or method according to any one of the preceding claims, wherein the instrument is pre-configured to perform dual capture and release multiplex immunoassays.

88. The instrument and / or method according to any one of the preceding claims, wherein the instrument is an integrated instrument with an integrated qPCR instrument capable of processing sample determination from sample preparation to data or to a merged NGS library.

89. The instrument and / or method according to any one of the preceding claims, wherein the instrument is capable of atmospheric sensitivity detection.

90. The instrument and / or method according to any one of the preceding claims, wherein the instrument is capable of achieving a sensitivity detection of at least 5 atmore.

91. The instrument and / or method according to any one of the preceding claims, wherein the instrument, capable of achieving ammolar-level sensitivity detection, has a wide dynamic range of up to 12 orders of magnitude, the range including an upper limit for detection of a component in the target group and a lower limit for detection of said component in the target group.

92. The instrument and / or method according to any one of the preceding claims, wherein the instrument is capable of performing dual capture and release multiplex immunoassays on more than fifty samples in parallel.

93. The instrument and / or method according to any one of the preceding claims, wherein the instrument can perform a dual capture and release multiple immunoassay of multiple samples starting from the assay sample without human intervention.

94. The instrument and / or method according to any one of the preceding claims, wherein the instrument can perform dual capture and release multiple immunoassays of multiple samples starting from the assay sample in less than four hours without human intervention.

95. The instrument and / or method according to any one of the preceding claims, wherein the instrument is capable of performing a dual capture and release multiplex immunoassay of multiple samples from assay sample to qPCR reading in less than 6 hours without human intervention.

96. The instrument and / or method according to any one of the preceding claims, wherein the instrument automatically completes the assay of multiple oligonucleotide conjugated antibody proximity linkages on multiple biological samples, from sample preparation, purification via dual capture and release of immune complexes to the formation of multiple analyte-specific reporter molecules for multiple analytes in multiple biological samples, with a sensitivity as low as 50 atmore.

97. The instrument and / or method according to any one of the preceding claims, wherein the instrument completes the dual capture and release mechanism in less than 2 hours.

98. The instrument and / or method according to any one of the preceding claims, wherein the instrument contains a sufficient amount of reagents and other consumables for running at least three assay cycles without requiring the user to reload the reagents and other consumables.

99. The instrument and / or method according to any one of the preceding claims, wherein the instrument is a benchtop instrument.

100. The instrument and / or method according to any one of the preceding claims, wherein the instrument has a height of less than 50 inches (excluding the stage).

101. The instrument and / or method according to any one of the preceding claims, wherein the base of the instrument has a depth of less than 30 inches and a width of less than 50 inches.

102. The instrument and / or method according to any one of the preceding claims, wherein the instrument is capable of high-throughput measurement.

103. The instrument and / or method according to any one of the preceding claims, wherein the sample is a serum sample, a plasma sample, or a cerebrospinal fluid sample.

104. The instrument and / or method according to any one of the preceding claims, wherein the sample is derived from a cell sample or a tissue sample.

105. The instrument and / or method according to any one of the preceding claims, wherein the sample has more than 50 analytes.

106. The instrument and / or method according to any one of the preceding claims, wherein the sample volume is less than 20 microliters.

107. The instrument and / or method according to any one of the preceding claims, wherein the analyte is a protein or polypeptide.

108. The instrument and / or method according to any one of the preceding claims, wherein the multiplex assay detects at least 50 analytes.

109. An automated instrument for performing a dual capture and release immunoassay procedure, wherein the instrument consumes no more than five multi-container support plates for a single assay run.

110. An automated instrument for performing a dual capture and release immunoassay procedure, wherein the instrument consumes no more than two boxes of pipette tips per assay run.

111. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for carrying a solution containing the immune complex for capturing the immune complex on a substrate, and reuses the at least one plate for carrying the solution containing the immune complex for eluting the immune complex from the substrate.

112. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate to hold a solution containing the immune complex for capturing the immune complex on a substrate, washing the at least one plate, and reusing the at least one plate to hold the solution containing the immune complex for eluting the immune complex from the substrate.

113. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for carrying a solution containing an immune complex bound to the first substrate, and the at least one plate is reused for carrying a solution containing an immune complex bound to the second substrate.

114. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for carrying a solution containing an immune complex bound to the first substrate for eluting the first substrate from the immune complex, and uses the at least one plate for carrying a solution containing an immune complex bound to the second substrate for eluting the second substrate from the immune complex.

115. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for holding a solution containing the immune complex for eluting the first substrate from the immune complex, and reuses the at least one plate for holding a solution containing the immune complex for eluting the second substrate from the immune complex.

116. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for holding a solution containing the immune complex for eluting the first substrate from the immune complex, washing the at least one plate, and reusing the at least one plate for holding a solution containing the immune complex for eluting the second substrate from the immune complex.

117. The instrument and / or method according to any one of the preceding claims, wherein before capturing the immune complex on the second substrate, the instrument uses at least one plate for holding a solution containing the immune complex, and reuses the at least one plate for holding the solution containing the immune complex for eluting the second substrate from the immune complex.

118. The instrument and / or method according to any one of the preceding claims, wherein before capturing the immune complex on the second substrate, the instrument uses at least one plate for holding a solution containing the immune complex, and after capturing the immune complex on the second substrate, the at least one plate is reused for holding a solution containing the immune complex.

119. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for carrying a solution containing an immune complex bound to the first substrate, and uses the at least one plate for carrying a solution containing the immune complex for capturing the immune complex on the second substrate.

120. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for carrying a solution containing an immune complex bound to the first substrate, and reuses the at least one plate for carrying a solution containing the immune complex for eluting the immune complex from the second substrate.

121. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate to hold a solution containing an immune complex bound to the first substrate, and uses the at least one plate to hold a solution containing the immune complex for capturing the immune complex on the second substrate, and reuses the at least one plate to hold a solution containing the immune complex for eluting the immune complex from the second substrate.

122. The instrument and / or method according to any one of the preceding claims, wherein during elution of the first substrate, the instrument uses at least one plate to hold a solution containing an immune complex bound to the first substrate, and uses the at least one plate to hold a solution containing the immune complex for capturing the immune complex on the second substrate, and reuses the at least one plate to hold a solution containing the immune complex for eluting the immune complex from the second substrate.

123. The instrument and / or method according to any one of the preceding claims, wherein before capturing the immune complex on the second substrate, the instrument uses at least one plate to hold the solution containing the immune complex, washes the at least one plate, and after capturing the immune complex on the second substrate, reuses the at least one plate to hold the solution containing the immune complex.

124. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate to hold a solution containing an immune complex bound to the first substrate, washes the at least one plate, and reuses the at least one plate to hold the solution containing the immune complex for capturing the immune complex on the second substrate.

125. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate to hold a solution containing an immune complex bound to the first substrate, washes the at least one plate, and reuses the at least one plate to hold the solution containing the immune complex for eluting the immune complex from the second substrate.

126. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate to hold a solution containing an immune complex bound to the first substrate, washes the at least one plate, and reuses the at least one plate to hold a solution containing the immune complex for capturing the immune complex on the second substrate, and reuses the at least one plate to hold a solution containing the immune complex for eluting the immune complex from the second substrate.

127. The instrument and / or method according to any one of the preceding claims, wherein during the elution of the first substrate, the instrument uses at least one plate to carry a solution containing an immune complex bound to the first substrate, washes the at least one plate, and reuses the at least one plate to carry a solution containing the immune complex for capturing the immune complex on the second substrate, and reuses the at least one plate to carry a solution containing the immune complex for eluting the immune complex from the second substrate.

128. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for carrying an immune complex solution for capturing the immune complex on a substrate, then washes the at least one plate, and reuses the at least one plate for eluting the immune complex solution from the substrate.

129. The instrument and / or method according to any one of the preceding claims, wherein during cleaning, the instrument uses at least one plate to contain the immune complex solution, and during the formation of the reporter molecule, the at least one plate is reused to contain the immune complex solution.

130. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing a solution comprising an immune complex bound to the first substrate, and the at least one plate is reused for containing a solution comprising an immune complex bound to the second substrate.

131. The instrument and / or method according to any one of the preceding claims, wherein during cleaning, the instrument uses at least one plate to contain a solution comprising an immune complex bound to the first substrate, and during connection, the at least one plate is reused to contain a solution comprising an immune complex bound to the second substrate.

132. The instrument and / or method according to any one of the preceding claims, wherein prior to elution of the first substrate, the instrument uses at least one plate to contain a solution comprising immune complexes bound to the first substrate, and prior to elution of the second substrate, the at least one plate is reused to contain a solution comprising immune complexes bound to the second substrate.

133. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing an immune complex formation solution for forming the immune complex, and uses the at least one plate for capturing the immune complex on a substrate.

134. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing an immune complex forming solution for forming the immune complex, and uses the at least one plate for capturing the immune complex on a first substrate.

135. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing an immune complex forming solution for forming the immune complex, and uses the at least one plate for capturing the immune complex on a first substrate, and reuses the at least one plate for containing a solution comprising an immune complex solution bound to the first substrate.

136. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing an immune complex forming solution for forming the immune complex, and uses the at least one plate for capturing the immune complex on a first substrate, washing the at least one plate, and reusing the at least one plate for containing a solution comprising the immune complex solution bound to the first substrate.

137. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing an immune complex forming solution for forming the immune complex, and uses the at least one plate for capturing the immune complex on a first substrate, and reuses the at least one plate for containing a solution containing the immune complex solution bound to the first substrate for washing.

138. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing an immune complex forming solution for forming the immune complex, and uses the at least one plate for capturing the immune complex on a first substrate, washing the at least one plate, and reusing the at least one plate for containing a solution containing the immune complex solution bound to the first substrate for washing.

139. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least the same plate for containing the immune complex solution, for capturing the immune complex on a substrate, and for eluting the immune complex from the substrate.

140. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least the same plate for containing an immune complex forming solution for forming the immune complex, and uses at least the same plate for capturing the immune complex on the first substrate.

141. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least the same plate for containing the immune complex, for eluting the first substrate, and for capturing the immune complex on the first substrate.

142. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least the same plate for containing the immune complex solution for eluting the first substrate, and uses at least the same plate for capturing the immune complex on the second substrate.

143. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least the same plate for containing the first substrate solution and the second substrate solution.

144. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least the same plate to contain the first substrate solution, cleans at least the same plate, and reuses the same plate to contain the second substrate solution.

145. The instrument and / or method according to any one of the preceding claims, wherein the instrument reuses at least one plate for carrying a solution containing the target analyte from one step of the process to a subsequent step of the process.

146. The instrument and / or method according to any one of the preceding claims, wherein the instrument reuses at least one plate for carrying a solution containing the immune complex from one step of the process to a subsequent step of the process.

147. The instrument and / or method according to any one of the preceding claims, wherein the instrument uses at least one plate for containing a solution comprising at least one immune complex, the immune complex being a precursor of a target analyte, and the at least one plate is reused for carrying the solution comprising the immune complex from one step of the process to a subsequent step of the process.

148. The instrument and / or method according to any one of the preceding claims, wherein, from the formation of the immune complex through multiple process steps to the formation of the corresponding nucleic acid analyte reporter molecule, the instrument transfers the sample, i.e., a solution containing the target analyte or analyte captured by the immune complex, without any pipette tip contacting the sample.

149. The instrument and / or method according to any one of the preceding claims, wherein from the formation of the immune complex through multiple process steps to the formation of the corresponding nucleic acid analyte reporter molecule, the instrument transfers the sample, i.e., a solution containing the target analyte or analyte captured by the immune complex, without requiring any pipette tip to contact the sample.

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