Systems and methods for automated single cell processing

Abstract

A system and method for automated single cell capture and processing is described, wherein the system includes a platform that supports and positions a collection of sample processing elements; a stage for actuating tools for engaging with the collection of sample processing elements supported by the platform; and a base that supports a variety of processing subsystems and a control subsystem that communicates with the processing subsystems. The system can automatically execute workflows associated with single cell processing, including mRNA capture, cDNA synthesis, protein-related assays, and library preparation for next-generation sequencing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 844,470, filed May 7, 2019, and U.S. Provisional Application No. 62 / 866,726, filed June 26, 2019, which are both incorporated herein by this reference in their entirety. Technical Field

[0003] The present invention relates generally to the field of cell capture and cell processing, and more particularly to new and useful automated systems and methods for single cell capture and processing in the field of cell capture and cell processing.

[0004] background

[0005] With the increasing interest in cell-specific drug testing, diagnostics, and other assays, systems and methods that allow for individual cell isolation, identification, and recovery (retrieval) have become highly desirable. Single cell capture systems and methods have been shown to be particularly advantageous for these applications. However, the associated processes and protocols for single cell capture and subsequent analysis must typically be performed in a specific order and with high precision in order to properly maintain the cells. As a result, these processes can be time-consuming for the user, and if they are not properly performed (e.g., due to errors in pipetting, due to mixing of reagents, etc.), damage to the cells or other adverse results can result. In particular, these novel high-throughput single cell cytometry assays have tremendous utility in translational medicine, personalized therapy selection, clinical diagnostics, and / or other use applications, but the lack of automation hinders proper operation by novice users, thereby limiting throughput.

[0006] Therefore, there is a need in the field of cell capture and cell processing to create new and useful systems and methods for single cell capture and processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A-Figure 1D A schematic diagram depicting an embodiment of a system for automated single cell sample processing;

[0009] Figure 2A-2F Depicted Figure 1A-Figure 1D A view of a variation of the system shown in;

[0010] Figure 3A-3C Depicting variations of reagent cartridges associated with systems for automated single cell sample processing;

[0011] Figure 4A-4C depicts views of variations of a sample processing cartridge associated with a system for automated single cell sample processing;

[0012] Figure 5A-5C Depicted with Figure 4A-4C The operating mode of the lid-opening tool associated with the sample processing box shown in FIG.

[0013] Figure 6A-6B Depicted with Figure 4A-4C The operating modes of the valve and heating subsystem associated with the sample processing cartridge shown in FIG.

[0014] Fig. 7A and Figure 7B Depicting variations of tool containers and contents associated with systems for automated single cell sample processing;

[0015] Figure 8A-8I Depicting variations of features for holding components on a platform of a system for automated single cell sample processing;

[0016] Fig. 9 An example of a liquid level detection subsystem for a system for automated single cell sample processing is depicted;

[0017] Figure 10A-10C Depicting variations of a first set of molecules for material separation in a system for automated single cell sample processing;

[0018] Figure 11A-11B depicts variations of a second set of molecules for separation of materials in a system for automated single cell sample processing;

[0019] Figure 12A-12J depicting a mode of operation of a separation subsystem associated with a system for automated single cell sample processing;

[0020] Figures 13A-13D depicts views of components of a variation of a separation subsystem associated with a system for automated single cell sample processing;

[0021] Figure 14A-Figure 14C Depicting embodiments of functional couplings between components of a system for automated single cell sample processing;

[0022] Fig.15 A flow chart depicting an embodiment of a method for automated single cell sample processing;

[0023] Fig.16 A first variation of a method for automated single-cell sample processing is depicted;

[0024] Fig.17 A second variation of the method for automated single cell sample processing is depicted;

[0025] Fig.18 A third variation of a method for automated single cell sample processing is depicted; and

[0026] Fig.19 A third variation of a method for automated single-cell sample processing is described.

[0027] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The following description of the preferred embodiments of the invention is not intended to limit the invention to those preferred embodiments, but rather to enable any person skilled in the art to make and use the invention.

[0029] 1. Benefits

[0030] The present invention may confer several benefits over conventional systems and methods.

[0031] In particular, the present invention confers the benefit of enabling at least partial automation of protocols involved in single cell capture and subsequent processing, thereby optimizing run success and consistency. In more detail, the user can be removed from some or all of the methods (e.g., loading samples, capping, on-instrument lysis, reverse transcription processes, cDNA amplification, bead or cDNA product recovery, on-instrument library preparation and cleanup, etc.). In addition, relative to conventional systems and methods, the system and / or method can achieve better accuracy of the protocol (e.g., better accuracy in adding the correct reagents, better temperature control of reagents, rapid processing of key liquid handling steps, precise incubation times, optimal bead cleaning and separation, automatic barcode reading, etc.). In addition, the system and / or method can confer the benefit of preventing accidents (e.g., knocking the system, spillage of reagents, contamination of samples or instruments, etc.), which can typically occur during manual execution of the protocol.

[0032] In addition, by using limited use and / or preloaded and combined test kits, the system and / or method can give the benefit of providing a streamlined user experience with optimized quality control and design architecture to adapt to the continued development of determination and future applications. Therefore, the system gives the benefit of independent or near independent control of reagents or reagent groups. In the specific example of this variation, the system includes a test kit with any one or all of the following dedicated areas: room temperature area, cooling area, heating area, magnetic area (for example, overlapping with heating area), waste capture area, intermediate reagent parking area or any other suitable area. In related benefits, the system and / or method can give the benefit of enabling users to purchase reagents of smaller volumes, such as by distributing reagents to be used in a specific type and quantity according to a specific automated scheme. This can play a role in saving costs, reducing reagent waste or having any other suitable results.

[0033] In addition, by using fluid handling and separation elements (e.g., magnetic separation components), the system and / or method can confer the benefit of providing automated sample and library cleaning steps. Relatedly, the system and / or method can confer the benefit of establishing better fluid flow in the entire system. In the first instance, this is achieved by an automated liquid transfer system (e.g., a pipette, a stand, and a variety of pipette tips), which can monitor and / or guide fluid flow (e.g., to maintain optimal flow, to establish optimal reagent volume, etc.) without user intervention. Fluid handling system components for single cell preparation and / or other assays can include the use of both: (a) a liquid pipette, which is coupled to a stand for fluid distribution and pumping to a fluid channel or fluid reservoir (e.g., of a sample processing box); and / or (b) a built-in on-chip pressurizable waste chamber, which is connected and controlled by a valve integrated with a fluid network, as described in more detail below. Such a combined dual liquid handling system provides unprecedented control over the flow rate (e.g., microliters per second to tens of milliliters per second), delivery (e.g., 1 to 100,000 microliters), and residence time (e.g., milliseconds to hours) of reagents through the fluid system. Additionally or alternatively, the system can monitor and / or direct fluid flow with or without user intervention (e.g., with minimal user intervention, to encourage optimal user intervention, etc.).

[0034] Furthermore, through software and workflow improvements, the system and / or method may minimize the number of manual operations performed by the user and provide relevant system status reports to ensure smooth operation and sample processing.

[0035] In addition, with respect to sample processing disposables, the system and / or method can confer the benefit of merging multiple components in a manner that is scalable for disposables with a higher number of sample processing chambers. In addition, the system can confer the benefit of merging two or more conventionally separate processing platform components into a single unit, which can reduce the overall size of the system (e.g., enabling a benchtop model), reduce the overall footprint of the system's mechanisms (e.g., a pipette stand), enable more efficient transfer of materials between systems, or perform any other suitable function. In a specific example of this variation, the inlet, the collection of micropores, the outlet valve, the cover mechanism (e.g., a cover of the cover mechanism, a full cover mechanism, etc.), and the waste chamber are all confined to a single piece.

[0036] In addition, the present invention addresses the needs in low parameter flow applications, high parameter flow applications, large scale cytometry applications, proteogenomics applications, single cell RNA applications, protein detection applications, single cell multi-atom applications, and other applications by allowing standard users (e.g., novice, expert) with a variety of skill levels to operate platform components. Specific workflows enabled by embodiments of the system are described in more detail below.

[0037] Regarding performance, the systems and / or methods can process cells to produce purified libraries, perform next generation sequencing (NGS) preparation, and perform other processes in a streamlined process (e.g., through a collection of dedicated consumables that include efficient loading of reagent kits, sample processing cartridges, and containers for fluid handling disposables) within a single day.

[0038] In addition, the system also provides the benefit of three-dimensional mobility of components such as pipettes. In a specific example of this variation, the system includes a stage that provides XYZ mobility for the pipette, so that the pipette can perform multiple tasks (e.g., piercing the foil covering of a reagent tube, transferring materials between a collection of holes, etc.) in an automated manner.

[0039] Additionally or alternatively, the systems and / or methods may impart any other suitable benefits.

[0040] 2. System

[0041] like Figure 1A-Figure 1D As shown, an embodiment of a system 100 for automated single cell capture and processing includes: a platform 110 that supports and positions a collection of sample processing elements; a gantry 170 for actuating tools for interfacing with the collection of sample processing elements supported by the platform 110; and a base 180 that supports a variety of processing subsystems and a control subsystem that communicates with the processing subsystems, wherein the control subsystem controls the state of the platform 110, the collection of sample processing elements, and the gantry 170 to convert the system 100 between a variety of operating modes. Embodiments, variations, and examples of operating modes that provide a variety of workflows will be described in further detail in Section 3 below.

[0042] The embodiment of system 100 is used to realize any or all associated processing of automated single cell capture and captured cells. In more detail, the user can be removed from some or all methods (e.g., loading samples, covering the lid, lysis on the instrument, reverse transcription process, cDNA amplification, bead or cDNA product recovery, library preparation and cleaning on the instrument, etc.). The system can be used to improve the accuracy of cell capture and sample processing schemes (e.g., by minimizing manual processes) in addition or alternatively. In addition, by using limited-use and / or pre-loaded test kits, system 100 can provide a streamlined user experience with optimized quality control and design architecture to adapt to the continued development of assays and future applications. Therefore, the system confers the benefits of independent or nearly independent control of reagents or reagent groups. In a specific example of this variation, the system includes a test kit having any one or all of the following dedicated areas: a room temperature area, a cooling area, a heating / thermal cycle area, a magnetic area (e.g., overlapping with a heating area), an area providing a cell sample input, an area for preparing a library output, or any other suitable area. Among related benefits, the system and / or method can confer the benefit of enabling a user to purchase smaller volumes of reagents, such as by dispensing the reagents to be used in a protocol-specific type and quantity according to a specific automated protocol. This can serve to save costs, reduce reagent waste, or have any other suitable result.

[0043] In addition, by using fluid handling and separation elements (e.g., magnetic separation components), embodiments of system 100 can be used to provide automated sample and library cleaning steps. Relatedly, system 100 can impart the benefit of establishing better fluid flow in the entire system. In a first example, this is achieved by an automated liquid transfer system (e.g., a pipette, a stand, and various pipette tips) that can monitor and / or guide fluid flow (e.g., to maintain optimal flow, to establish optimal reagent volume, etc.) without user intervention or with minimal user intervention.

[0044] In addition, by allowing standard users with multiple skill levels (e.g., novice, expert) to operate the platform components, the system 100 can enable low parameter flow applications, high parameter flow applications, mass cytometry applications, proteogenomics applications, single cell RNA applications, protein detection applications, and other applications. In addition, with respect to performance, the system 100 can process cells or other biological materials to rapidly generate purified libraries, perform next generation sequencing (NGS) preparations, and perform other processes in a streamlined process (e.g., through a collection of dedicated consumables, which include highly efficient loading kits, sample processing boxes, and containers for fluid handling disposables).

[0045] In certain embodiments, the system 100 can meet usage requirements including one or more of the following: providing an automated process for nucleic acid library preparation, providing the ability to perform quality control at desired points in a run, providing complete and single-use kits for a variety of assays, providing validation and lockout protocols, providing alignment and retention of various system components, providing means for monitoring and controlling system operation (e.g., with a touch display), providing remote monitoring capabilities, providing sample processing within 24 hours, providing visual and / or audible system notifications, providing the ability to be cleaned with standard laboratory cleaners without disassembly, mounting on a standard laboratory workbench, providing ease of installation, providing assay materials with a stable shelf life, returning reports on maintenance history, providing data storage (e.g., on external storage media, on cloud storage, etc.), providing training, and providing other suitable functionality based on a variety of requirements.

[0046] As described above, about sample processing, the embodiment of system 100 can include or can be configured to process cells, cell-derived materials and / or other biomaterials (for example, cell-free nucleic acids).Cell can include any or all of the cells of mammalian cells (for example, human cells, mouse cells, etc.), embryos, stem cells, plant cells or any other suitable kind.Cell can include target material (for example, target lysate, mRNA, RNA, DNA, etc.), which derives from intracellular and is optionally captured by cell capture system for processing.In addition, the container comprising cells can be prepared by multiple samples (for example, 12 samples, 24 samples, 48 ​​samples, 96 samples, 384 samples, 1536 samples, samples of other quantities) comprising cells, wherein before multiple samples are mixed into a single container (or the container of reduced quantity), the multiple samples are hashed or barcoded.This feature can realize the automated processing of multiple samples in the same automated operation, for their corresponding single cell preparation and library preparation operations. Additionally or alternatively, the system 100 can be configured to interact with particles (e.g., beads, probes, nucleotides, oligonucleotides, polynucleotides, etc.), droplets, encapsulated cells, encapsulated biomarkers, reagents, or any other suitable material.

[0047] The system may further additionally or alternatively include any or all of the system components as described in the following applications: U.S. Application No. 16 / 048,104 filed on July 27, 2018; U.S. Application No. 16 / 049,057 filed on July 30, 2018; U.S. Application No. 15 / 720,194 filed on September 29, 2017; U.S. Application No. 15 / 430,833 filed on February 13, 2017; U.S. Application No. 15 / 821,329 filed on November 22, 2017; U.S. Application No. 15 / 720,194 filed on September 29, 2017; U.S. Application No. 15 / 430,833 filed on February 13, 2017; U.S. Application No. 15 / 821,329 filed on November 22, 2017; No. 15 / 782,270, filed on October 12, 2018; No. 16 / 049,240, filed on July 30, 2018; No. 15 / 815,532, filed on November 16, 2017; No. 16 / 115,370, filed on August 28, 2018, No. 16 / 564,375, filed on September 9, 2019, and No. 16 / 816,817, filed on March 12, 2020, each of which is incorporated by reference in its entirety.

[0048] 2.1 System: Platform

[0049] like Figure 1A-Figure 1D As shown, the platform (deck) 110 is used as a platform (platform) for supporting and positioning one or more components of the system 100 (e.g., on the top wide surface, on the top and bottom wide surfaces, on the side surfaces, etc.) for automated sample processing. In addition, the platform 110 can be used to position one or more components of the system 100 to align with or otherwise interface with a fluid processing subsystem, a heating subsystem, a separation subsystem (e.g., a magnetic separation subsystem) and / or other subsystems coupled to the stage 170 and / or the base 180, as described below. In this regard, the platform 110 can be stationary as a reference platform, while other components are actuated into position for engagement with elements of the platform 110. Alternatively, the platform 110 can be coupled to one or more actuators for positioning elements of the platform 110 for interfacing with other subsystems.

[0050] exist Figure 1A-Figure 1DIn the illustrated embodiment, the platform 110 provides a platform for supporting the collection of sample processing elements, wherein the sample processing elements may include disposable and / or reusable components, wherein the components include containers for containing sample processing materials and / or tools for processing samples (e.g., with respect to fluid handling, with respect to material separation, with respect to heating and cooling, etc.). In an embodiment, the platform 110 may support a collection of sample processing elements, which includes one or more of the following units: a reagent cartridge 120, a sample processing cartridge 130, a tool container 140, a heating and cooling subsystem 150, a pumping subsystem 157, a liquid level detection subsystem 159, and a separation subsystem 160. Additionally or alternatively, the platform 110 may include other suitable components (e.g., a fluorescence detection subsystem, a confocal microscopy subsystem, a spectral detection subsystem, a total internal reflection fluorescence (TIRF) subsystem, a nuclear magnetic resonance (NMR) subsystem, a Raman spectroscopy (RS) subsystem, etc.).

[0051] The sample processing elements may be supported in a coplanar manner by the platform 110, or alternatively may be supported in different planes. Preferably, the discrete elements supported by the platform are non-overlapping, but alternative embodiments of the platform 110 may support the sample processing elements in an overlapping manner (e.g., to save space, for operational efficiency, etc.).

[0052] like Figure 1A and Figure 1D As shown, the platform 110 may be accessible through the door 90, wherein the door 90 of the system 100 may be transitioned between an open mode and / or a closed mode to provide access to the platform 110 and elements supported by the platform 110. However, in other variations, the platform 110 may not be accessible through the door 90.

[0053] Details of embodiments, variations, and examples of elements supported by platform 110 are further described in Sections 2.1.1 through 2.1.5 below.

[0054] 2.1.1 Platform Support Components: Test Kits

[0055] The platform 110 includes at least one area 111 (in Figure 2A and Figure 2B 1 ), a unit for supporting a reagent cartridge 120, wherein an area 111 is used to position the reagent cartridge 120 relative to portions of the heating and cooling subsystem 150 and the separation subsystem 160, as described in more detail below. In this regard, the area 111 may include one or more openings, recesses, and / or protrusions for providing an interface between complementary portions of the reagent cartridge 120 and associated portions of the heating and cooling subsystem 150 and the separation subsystem 160, and otherwise promoting and maintaining alignment between such portions.

[0056] The reagent kit 120 is used to contain materials for cell capture and / or sample processing in one or more compartments according to one or more workflows for a variety of applications. In this way, the reagent kit 120 can define a collection of storage volumes distributed across a collection of domains, wherein the collection of domains can be configured to provide a suitable environment for the material contents of each domain. The collection of storage volumes can directly accommodate sample processing materials, and / or can be optionally configured to receive and hold positions of separate containers (e.g., tubes, etc.) containing sample processing materials. The storage volumes of each domain can be distributed in an array or arranged in other ways. Depending on the application of use (e.g., refrigeration, heat transfer, magnetic separation, etc.), the storage volume can have a circular cross-section, a rectangular cross-section, or other morphology (e.g., cross-section, width, depth, etc.).

[0057] The collection of domains can additionally or alternatively be configured to provide modularity, where one or more domains can be prepackaged with materials that are stable over a longer shelf life, while other domains can be configured to receive materials with a short shelf life (e.g., immediately prior to use). The collection of domains can additionally or alternatively be configured to improve the operational efficiency of equipment that interfaces with materials of the reagent kit 120 (e.g., with respect to grouping similar materials, etc.). The collection of domains can additionally or alternatively define areas for receiving and / or processing materials (e.g., nucleic acid materials) extracted from the sample processing cartridge 130, which is described in more detail below.

[0058] Additionally or alternatively, the domains in a set of domains can be separate (e.g., domains for receiving heat are separate from domains for other storage temperatures or applications requiring different temperatures), overlapping, or otherwise arranged. The domains in a set of domains can additionally or alternatively be distinguished from each other by morphology (e.g., the length of the storage volume of each domain, the depth of the storage volume for accessing or interfacing with other elements of the platform, the width or depth of the domain configured for effective heat transfer, etc.). In some variations, the set of domains can also include at least one domain supporting an absorbent or porous material pad that can be used to receive droplets during processing (e.g., from the tip of a pipette, as described below). The internal surface properties of certain domains (e.g., for PCR reactions, for magnetic separation, etc.) can be configured to have a high surface finish to enable low binding or retention of biological molecules (e.g., nucleic acids or proteins). Multiple domains can also be mixed and matched to provide customers with a large number of available assays.

[0059] The independent storage volume in the set of the storage volume of test kit 120 can also include one or more seals, which are used to isolate the material in the test kit 120, prevent cross contamination between the material in the independent storage volume, prevent pollutants from entering the independent storage volume and / or prevent evaporation losses during storage and transportation. The seal can be a pierceable seal (for example, consisting of paper, consisting of metal foil and / or consisting of any other suitable material). However, the seal can be optionally configured to be non-pierceable (for example, the seal can be configured to be peeled off from test kit 120). In embodiments, some reagent containers can also be sealed by a hinged lid, which can be opened or closed by an instrument (for example, as described in more detail below), as required for processing in the appropriate steps of the scheme.

[0060] In a variation, a collection of domains may include a first domain for storing reagents that require a refrigerated environment (e.g., at a temperature of from 1°C to 15°C), a second domain for storing materials that can be stored in ambient conditions, a third domain for storing tubes with materials for performing a polymerase chain reaction (PCR) operation and engaging a heating element described below, a fourth domain for storing functionalized particles (e.g., beads with probes having barcode regions and other functional regions, as described in U.S. Application No. 16 / 115,370, etc.), and a fifth domain for performing a separation operation (e.g., separating a target from a non-target material by magnetic force). In a variation, domains that provide different environments for storage volumes may be configured differently. For example, a first domain (i.e., for refrigeration) may contain an insulating material and / or may include an insulating material about the storage volume of the domain (e.g., individually, about the entire domain). Additionally or alternatively, a domain for separation may include a magnetically conductive material configured to provide appropriate magnetic field characteristics for separation. Additionally or alternatively, domains used for thermal cycling applications or other heat transfer applications can be configured with thermally conductive materials to facilitate efficient heat transfer to and from the test kit. In an embodiment, the various domains can be optimally positioned so that there is minimal crosstalk between certain operations. For example, a domain used for frozen reagent storage volume can be maintained at a temperature (e.g., 4° C.) during operation, while a domain used for a PCR reaction may require heating (e.g., up to 95° C. during denaturation). Therefore, in order to minimize the effect of PCR thermal cycling on frozen reagents, a domain containing reagents stored at ambient temperature can be configured between the PCR thermal cycling domain and the freezing domain. To further prevent thermal crosstalk, additional buffer tubes containing only air can be used between key domains that require independent temperature control.

[0061] In variations, the process materials supported by the domains of the kit 120 may include one or more of the following: buffers (e.g., ethanol, priming buffers, lysis buffers, custom lysis buffers, sample wash buffers, saline with RNase inhibitors, bead wash buffers, RT buffers, buffers, etc.), oils (e.g., perfluorinated inert oils), PCR master mixes, cells, beads (e.g., functionalized beads), or any other suitable material for cell capture and / or sample processing. Additionally or alternatively, one or more of the collection of storage volumes may be empty (e.g., initially empty, empty during one or more processes, empty prior to being filled by an operator, etc.). Different storage areas in different domains of the kit may have initial reagent volumes ranging from a few microliters (e.g., 5 microliters) to 50 milliliters. Further details of the application of process materials and uses are described below with respect to the workflow of Section 3.

[0062] In a specific example, Figure 3A As shown, the test kit 120' includes: a first domain 121', which is located in a first peripheral area of ​​the test kit 120' and is used to store reagents that require a refrigerated environment; a second domain 122', which is located in a central area of ​​the test kit 120' and is used to store materials that can be stored under ambient conditions; a third domain 123', which is located in a peripheral area of ​​the test kit, close to the second domain 122, and is used to store tubes with materials for performing polymerase chain reaction (PCR) operations; a fourth domain 124', which is located in a peripheral area of ​​the test kit 120' and is used to store functionalized particles (e.g., beads with probes having barcode regions and other functional regions, as described in U.S. application No. 16 / 115,370, etc.); and a fifth domain 125', which is located in a peripheral area of ​​the test kit 120' and is used to perform separation operations (e.g., separating targets from non-target materials by magnetic force). In certain examples, the fourth domain 124' can be a modular component, whereby the fourth domain 124' can be stored separately from the rest of the reagent kit 120' until the functionalized particles are ready for use, at which time the fourth domain 124' is put into place and coupled to the reagent kit 120'.

[0063] In a specific example, the first domain 121' and the second domain 122' are covered by a first seal composed of metal foil, the third domain 123' and the fifth domain 125' are covered by a second seal composed of paper, and the fourth domain 124' is covered by a third seal composed of metal foil. However, variations of the example of the reagent kit 120' may be configured in another suitable manner.

[0064] exist Figure 3BIn another specific example of the 3' RNA processing scheme shown (e.g., corresponding to the workflow of Section 3.1 below), the kit 120" can include: a first storage volume 1201 for 100% molecular grade ethanol (e.g., having a volume of 4.46 mL); a second storage volume 1202 for a first wash buffer (e.g., having a volume of 6.6 mL); a third storage volume 1203 for a particle binding buffer (e.g., having a volume of 1.1 mL); a fourth storage volume 1204 for a lysis buffer (e.g., having a volume of 1.1 mL); a fifth storage volume 1205 for a perfluorinated inert oil (e.g., having a volume of 1.1 mL); a sixth storage volume 1206 for a particle binding wash solution (e.g., having a volume of 6.6 3 mL); a seventh storage volume 1207 for RT pre-reaction wash buffer (e.g., having a volume of 1.1 mL); an eighth storage volume 1208 for 0.1 M sodium hydroxide solution (e.g., having a volume of 1 mL); a ninth storage volume 1209 for a second wash buffer (e.g., having a volume of 2.5 mL); a tenth storage volume 1210 for mineral oil (e.g., having a volume of 1 mL); an eleventh storage volume 1211 for 80% molecular weight ethanol (e.g., having a volume of 1.1 mL); a twelfth storage volume 1212 for nuclease-free water (e.g., having a volume of 2.2 mL); a thirteenth storage volume 1213 for waste (e.g., having a volume of 12.36 mL); a 0.1 M 14th storage volume 1214 for DTT (e.g., having a volume of 0.15 mL); 15th storage volume 1215 for RT mix without SuperScript IV: 16th storage volume 1216 for SuperScript IV enzyme (e.g., having a volume of 0.011 mL); 17th storage volume 1217 for exonuclease buffer (e.g., having a volume of 0.22 mL); 18th storage volume 1218 for exonuclease (e.g., having a volume of 0.022 mL); 19th storage volume 1219 for second strand synthesis mix (e.g., having a volume of 0.128 mL); 20th storage volume 1220 for second strand synthesis primer (e.g., having a volume of 0.072 mL); 21st storage volume 1221 for PCR master mix for mRNA amplification (e.g., having a volume of 0.22 mL); 22nd storage volume 1222 for mixture (e.g., Kapa Biosystems TMa 22nd storage volume 1222 for HiFi Hot Start Ready Mix (2x) (e.g., having a volume of 0.33 mL); a 23rd storage volume 1223 for mRNA product (e.g., having a volume of 0.025 mL); a 24th storage volume 1224 for functionalized particles (e.g., having a volume of 0.11 mL); a 25th storage volume 1225 for magnetic recovery particles (e.g., having a volume of 0.03 mL); a 26th storage volume 1226 for AMPure XP particles (e.g., having a volume of 0.66 mL); a 27th storage volume 1227 for magnetic particle recovery collection; a 28th storage volume 1228 for magnetic particle preparation; a 29th storage volume 1229 for magnetic particle second strand synthesis; a 30th storage volume 1230 for magnetic particle recovery for cDNA amplification after PCR; a 31st storage volume 1231 for AMPure XP particle purification; a 32nd storage volume 1232 for first exonuclease treatment; a 33rd storage volume 1233 for magnetic particle recovery for cDNA amplification after PCR; a 34th storage volume 1234 for AMPure XP particle purification; a 35th storage volume 1235 for magnetic particle recovery collection; a 36th storage volume 1236 for magnetic particle preparation; a 37th storage volume 1237 for magnetic particle second strand synthesis; a 38th storage volume 1238 for magnetic particle recovery for cDNA amplification after PCR; a 39th storage volume 1240 for AMPure XP particle purification; a 39th storage volume 1241 for first exonuclease treatment 2 storage volume 1232 for the second exonuclease treatment; the 33rd storage volume 1233 for the second nuclease treatment; the 34th storage volume 1234 for the second chain synthesis; the 35th storage volume 1235 for the first cDNA amplification operation; the 36th storage volume 1236 for the second cDNA amplification operation; the 37th storage volume 1237 for the third cDNA amplification operation; the 38th storage volume 1238 for the fourth cDNA amplification operation; and the 39th storage volume 1239 for the cell suspension.

[0065] exist Figure 3CIn another specific example of the CITE-Seq processing protocol shown (e.g., corresponding to the workflow in Section 3.3 below), the reagent kit 120"' can include: a first storage volume 1201' for 100% molecular grade ethanol (e.g., having a volume of 4.46 mL); a second storage volume 1202' for a first wash buffer (e.g., having a volume of 6.6 mL); a third storage volume 1203' for a particle binding buffer (e.g., having a volume of 1.1 mL); a fourth storage volume 1204' for a lysis buffer (e.g., having a volume of 1.1 mL); a fifth storage volume 1205' for a perfluorinated inert oil (e.g., having a volume of 1.1 mL); a sixth storage volume 1206' for a particle binding wash solution (e.g., having a volume of 6 .63mL); a seventh storage volume 1207' for RT pre-reaction wash buffer (e.g., having a volume of 1.1mL); an eighth storage volume 1208' for 0.1M sodium hydroxide solution (e.g., having a volume of 1mL); a ninth storage volume 1209' for a second wash buffer (e.g., having a volume of 2.5mL); a tenth storage volume 1210' for mineral oil (e.g., having a volume of 1mL); an eleventh storage volume 1211' for 80% molecular weight ethanol (e.g., having a volume of 1.1mL); a twelfth storage volume 1212' for nuclease-free water (e.g., having a volume of 2.2mL); a thirteenth storage volume 1213' for waste (e.g., having a volume of 12.36mL); a 0.1M 14th storage volume 1214' for DTT (e.g., having a volume of 0.15 mL); 15th storage volume 1215' for RT mix without SuperScript IV: 16th storage volume 1216' for SuperScript IV enzyme (e.g., having a volume of 0.011 mL); 17th storage volume 1217' for exonuclease buffer (e.g., having a volume of 0.22 mL); 18th storage volume 1218' for exonuclease (e.g., having a volume of 0.022 mL); 19th storage volume 1219' for second strand synthesis mix (e.g., having a volume of 0.128 mL); 20th storage volume 1220' for second strand synthesis primer (e.g., having a volume of 0.072 mL); 21st storage volume 1221' for PCR master mix for cDNA amplification (e.g., having a volume of 0.22 mL); 22nd storage volume 1221' for mixture (e.g., Kapa Biosystems TMHiFi Hot Start Ready Mix (2x)); 23rd storage volume 1223' for index primers; 24th storage volume 1224' for PCR master mix for mRNA amplification (e.g., having a volume of 0.11 mL); 25th storage volume 1225' for ADT product (e.g., having a volume of 0.2 mL); 26th storage volume 1226' for mRNA product (e.g., having a volume of 0.025 mL); 27th storage volume 1227' for functionalized particles (e.g., having a volume of 0.11 mL); 28th storage volume 1228' for magnetic recovery particles (e.g., having a volume of 0.03 mL); 29th storage volume 1230' for AMPure 37 storage volume 1237' for AMPure XP particle ADT after PCR purification; 38 storage volume 1239' for mRNA AMPure XP particle purification; 39 storage volume 1240' for mRNA AMPure XP particle purification; 40 storage volume 1241' for mRNA AMPure XP particle purification; 41 storage volume 1242' for mRNA AMPure XP particle purification; 42 storage volume 1243' for mRNA AMPure XP particle purification; 43 storage volume 1244' for mRNA AMPure XP particle purification; 44 storage volume 1245' for mRNA AMPure XP particle purification; 45 storage volume 1246' for mRNA AMPure XP particle purification; 46 storage volume 1247' for mRNA AMPure XP particle purification; 47 storage volume 1248' for mRNA AMPure XP particle purification; 48 storage volume 1249' for mRNA AMPure XP particle purification; 49 storage volume 1250' for mRNA AMPure XP particle purification; 50 storage volume 1251' for mRNA AMPure XP particle purification; 51 storage volume 1252' for mRNA AMPure XP particle purification The 39th storage volume 1239' for AMPureXP particles; the 40th storage volume 1240' for the first exonuclease treatment; the 41st storage volume 1241' for the second exonuclease treatment; the 42nd storage volume 1242' for second chain synthesis; the 43rd storage volume 1243' for the first cDNA amplification operation; the 44th storage volume 1244' for the second cDNA amplification operation; the 45th storage volume 1245' for the third cDNA amplification operation; the 46th storage volume 1246' for the fourth cDNA amplification operation; the 47th storage volume 1247' for antibody-derived tag fraction amplification; the 48th storage volume 1248' for mRNA amplification; and the 49th storage volume 1249' for cell suspension.

[0066] 2.1.2 Components supported by the platform: sample box

[0067] like Figure 2A and Figure 2BAs shown, platform 110 also includes at least one region 112 for supporting a unit of sample processing cartridge 130, wherein region 112 is used to position sample processing cartridge 130 relative to portions of heating and cooling subsystem 150, pumping subsystem 157, and liquid level detection subsystem 159, which are described in more detail below. In this regard, region 112 may include one or more openings, recesses, and / or protrusions for providing an interface between complementary portions of sample processing cartridge 130 and associated portions of heating and cooling subsystem 150, pumping subsystem 157, and liquid level detection subsystem 159, and otherwise promoting and maintaining alignment between such portions.

[0068] The sample processing cartridge 130 is used to provide one or more sample processing areas in which cells are captured and optionally sorted, processed or otherwise treated for downstream applications, wherein the downstream applications can be performed on the sample processing cartridge 130 (e.g., on-chip) and / or remote from the sample processing cartridge 130 (e.g., off-chip). Portions of the sample processing cartridge 130 can be configured within a single substrate, but can additionally or alternatively include multiple portions across multiple substrates (e.g., connected by fluid pathways).

[0069] like Figure 4A-4C As shown, an example of a sample processing box 130' can include a base substrate 131, to which other elements are coupled and / or in which other elements are defined. In addition, with respect to sample processing involving microfluidic elements, the base substrate 131 can be used as a manifold for transferring fluids to the microfluidic elements, entering the sample processing volume at different stages of processing, and transferring waste materials generated during sample processing. In a variation, the base substrate 131 supports one or more of the following: a sample processing chip 132; an inlet reservoir 133, the inlet reservoir 133 being used to receive sample material (e.g., containing cells, containing particles, etc.) and deliver the sample material to the sample processing chip 132; an entry area 134, the entry area 134 being used to enter one or more areas of the sample processing chip 132; a cover 135, which covers the entry area and includes a gasket 136 that provides a sealing function; and a waste holding area 137, which is used to receive waste materials from the sample processing chip 132. The box can have additional gasket ports to also connect to an off-cartridge pumping system present in the instrument. However, variations of the base substrate 131 can include other elements. For example, as described in more detail below, the base substrate can include one or more openings, recesses and / or protrusions that provide additional coupling with the sample processing chip 132 so as to jointly define a valve area for opening and closing the flow through the sample processing chip 132.

[0070] like Figure 4A and Figure 4C As shown in (bottom view), the sample processing chip 132 (equivalently referred to herein as a microwell device or slide) defines a collection of wells (e.g., microwells). Each well in the collection of wells can be configured to capture a single cell and / or one or more particles (e.g., probes, beads, etc.), any suitable reagent, multiple cells, or any other material. In a variation, the microwells of the sample processing chip 132 can be configured for co-capture of a single cell with a single functional particle so that a single cell and / or material from a single cell can be analyzed without cross-well contamination. Embodiments, variations, and examples of the sample processing chip 132 are described in one or more of the following: U.S. Application No. 16 / 048,104 filed on July 27, 2018; U.S. Application No. 16 / 049,057 filed on July 30, 2018; U.S. Application No. 15 / 720,194 filed on September 29, 2017; U.S. Application No. 15 / 430,833 filed on February 13, 2017; U.S. Application No. 15 / 821,329 filed on November 22, 2017 ... No. 15 / 782,270, filed on July 2, 2018; No. 16 / 049,240, filed on July 30, 2018; No. 15 / 815,532, filed on November 16, 2017; No. 16 / 115,370, filed on August 28, 2018, No. 16 / 564,375, filed on September 9, 2019, and No. 16 / 816,817, filed on March 12, 2020, each of which is incorporated by reference in its entirety above.

[0071] In material composition, the sample processing chip 132 can be composed of microfabricated silicon or glass fused silica material, which is used to achieve higher resolution of a collection of holes, for example, by defining sharper edges in the collection of holes (for example, thinner hole walls, hole walls arranged at angles close to 90 degrees, etc.). Compared with conventional chip designs, the described materials and manufacturing processes can further achieve one or more smaller feature sizes (for example, length, width, total footprint, etc.). Additionally or alternatively, the substrate includes any other suitable material, such as but not limited to polymers, metals, biomaterials, or any other material or combination of materials. The sample processing chip 132 can be manufactured by a variety of processes such as precision injection molding, precision embossing, microlithography etching, LIGA-based etching, or by other suitable techniques.

[0072] In some modifications, one or more surfaces (for example, bottom surface, side surface, bottom surface and side surface, all surfaces, etc.) of the set in a hole can react with oligonucleotide molecules, for capturing biomarkers from independent cells into independent micropores. The oligonucleotide molecules present in each micropore and independent micropores can be barcoded to allow the biomarkers processed in each micropore to be connected back to specific holes, and therefore connected back to specific single cells. In a modification, the set in a hole includes the set of micropores, and the micropores have a hexagonal cross section intercepted transverse to the longitudinal axis of the hole, as described in one or more applications incorporated by reference above.

[0073] In one variation, Figure 4C As shown, the sample processing chip 132 may include an inlet opening 32; a first fluid distribution network 33, which is downstream of the inlet opening and is used to distribute fluid to a collection of microwells 34; a second fluid distribution network 35, which is downstream of the collection of microwells 34; and an outlet opening 36, which is coupled to the terminal portion of the second fluid distribution network 35, for transferring waste fluid from the sample processing chip 132. In this variation, the sample processing chip 132 is coupled to a first side (e.g., the lower side) of the base substrate 131 (e.g., by laser welding, adhesive bonding, solvent bonding, ultrasonic welding, or another technique). Coupling the sample processing chip 132 to the side of the base substrate 131 can transfer heat from the heating and cooling subsystem 150 to the collection of microwells 34 and / or other areas of the sample processing chip 132, wherein the heating and cooling subsystem 150 will be described in more detail below.

[0074] As described above, the base substrate 131 may also include an inlet reservoir 133 (e.g., defined on a second side of the base substrate 131 opposite to the first side to which the sample processing chip 132 is coupled). The inlet reservoir is used to receive sample materials (e.g., samples containing cells, samples containing barcoded cells, samples containing encapsulation materials, samples containing particles, etc.) and / or sample processing materials from the reagent kit 120 described above for delivery to the inlet opening 32 of the sample processing chip 132. In a variation, the inlet reservoir 133 may be defined as a recessed region within the surface of the base substrate 131, wherein the recessed region includes a pore that is aligned with and / or sealed with the inlet opening 32 of the sample processing chip 132. The inlet reservoir 133 of the base substrate 131 can interface with an upstream fluid containing a component and / or a bubble mitigation component, as described in one or more of the following: U.S. Application No. 16 / 048,104 filed on July 27, 2018; U.S. Application No. 16 / 049,057 filed on July 30, 2018; U.S. Application No. 15 / 720,194 filed on September 29, 2017; U.S. Application No. 15 / 430,833 filed on February 13, 2017; U.S. Application No. 15 / 821,329 filed on November 22, 2017 No. 15 / 782,270, filed on October 12, 2017; No. 16 / 049,240, filed on July 30, 2018; No. 15 / 815,532, filed on November 16, 2017; No. 16 / 115,370, filed on August 28, 2018, No. 16 / 564,375, filed on September 9, 2019, and No. 16 / 816,817, filed on March 12, 2020, each of which is incorporated by reference in its entirety above.

[0075] The inlet reservoir 133 may also be configured to interface with a level detection subsystem 159 supported by or otherwise interfaced with the platform 110, as described in more detail below. In particular, portions of the inlet reservoir 133 may be constructed of a material capable of sensing a level of liquid within the inlet reservoir 133 (e.g., by optical interrogation, by pressure sensing, by weight sensing, etc.). For example, the inlet reservoir 133 may be constructed of a material that is optically transparent or translucent to visible spectrum electromagnetic radiation and / or non-visible spectrum electromagnetic radiation (e.g., by fabricating with different materials, by fabricating to create a thin region of material at the inlet reservoir 133, etc.), wherein a sensing element of the level detection subsystem 159 may be configured to query the level of liquid within the inlet reservoir 133 accordingly.

[0076] In variations, one or more of the inlet reservoir 133 of the base substrate 131 and the inlet 32 ​​of the sample processing chip 132 may include a valve component that can be opened or closed by one or more components of the system 100. In a first variation, the inlet reservoir 132 includes a pore that can be accessed by a pipette tip or any other suitable attachment of a fluid handling subsystem coupled to the stage 170 (described in more detail below). In some embodiments, the pore can be closed and thereby prevent fluid from traveling from the inlet reservoir 132 to the sample processing chip 132. However, the inlet reservoir 132 can be configured in another suitable manner. The opening associated with the inlet reservoir 133 can have a conical surface that opens toward the top, allowing a pipette tip to be connected and sealed so that fluid (aqueous solution or oil or air) can be pumped directly into Figure 4C In the microchannel defined by 33.

[0077] like Figure 4A and Figure 4B As shown, base substrate 131 may also define an access area 134 for accessing one or more areas of sample processing chip 132, wherein the access area may allow observation of areas of sample processing chip 132 at different stages of sample processing and / or extraction of areas of sample processing chip 132 from sample processing chip 132. Figure 4A and Figure 4B As shown, the access region 134 can be defined as a recessed area within the base substrate 131 and includes an opening 37 that is aligned with an area of ​​the sample processing chip 132 that includes a collection of microwells. The sample processing chip 132 can have as few as 100 microwells and as many as 100 million microwells. Therefore, in variations where the microwell region is open to the environment (e.g., there is no cover to seal the holes), the openings 37 of the access region 134 can be used as microwells to provide access to the contents of the microwells for observation and / or material extraction (e.g., by magnetic separation, as described in further detail below). The openings 37 can match the morphology and footprint of the microwell region, and in a first variation, as shown in FIG. Figure 4B As shown, it can be a square opening. However, in other variations, the opening 37 can have another suitable shape.

[0078] like Figure 4A-4CAs shown, the base substrate 131 may include or otherwise be coupled to a cover 135 covering the access area 134, wherein the cover 135 may include a gasket 136 that provides a sealing function, and wherein the cover 135 is used to convert the access area 134 between an open mode and a closed mode to prevent sample loss due to evaporation during operation and / or contamination of the contents of the sample processing chip 132. The cover 135 can additionally or alternatively be used to protect the contents of the microwells or other processing areas of the sample processing chip 132 from debris, enable processing of the contents of the sample processing chip 132 (e.g., by isolating the area from the surrounding environment), initiate the start of a protocol (e.g., by opening to accept reagents from a pipette), prevent a user from manipulating the sample processing chip 132 (e.g., by closing after all necessary reagents have been added), define (e.g., with the cover 135) part or all of a fluid path, cavity, or reservoir (e.g., serving as the top surface of a fluid path between an inlet and a collection of microwells, serving as a boundary for a fluid path adjacent to a microwell area, serving as the top surface of a fluid path between a collection of microwells and a waste chamber, etc.), or perform any other suitable function.

[0079] like Figure 4B As shown, in at least one variation, the cover 135 can be complementary in form to the features of the entry region 134, so that the cover 135 matches the entry region 134 while providing a gap with the sample processing chip 132. Figure 4B and Figure 4C ), when the cover 135 is in the closed position, the cover 135 may be substantially flush with the base substrate 131 at the top surface. However, the cover 135 may be configured in another suitable manner in terms of morphology.

[0080] In a variation, the protrusion 38 of the cover 135 may interface with the opening 37 of the access area 134 so that access to the opening 37 is substantially prevented when the cover is in the closed position. Figure 4B As shown, in some variations, protrusion 38 may have a base (or other area) surrounded by a gasket 136 that is used to seal opening 37 of access area 134 when lid 135 is in the closed position. However, variations of lid 135 may omit the gasket and facilitate sealing of access area 134 in another suitable manner.

[0081] In some variations, the cover 135 may include a locking or latching mechanism that allows the cover 135 to be held in a closed position with the base substrate 131 until the locking / latching mechanism is released. Figure 5A-5CIn the variation shown, the peripheral portion of the cover 135 may include one or more tabs 39 that interface with corresponding tab receiving portions of the base substrate 131, wherein the tabs 39 are configured to flex when pushed into the base 131 until they interface with the tab receiving portions of the base substrate 131 and return from the flexed configuration to the latched state. Additionally or alternatively, in Figure 5A-5C In the variation shown, the locking / latching mechanism can include a release body 41 (e.g., a rod, a recess, a hook, etc.) that can interface therewith to release the tab 39 from the tab receiving portion and convert the cover 135 from a closed mode to an open mode relative to the base substrate 131. In this way, the cover 135 provides an open mode for the cover, in which the access area 134 is not covered, and a closed mode, in which the access area 134 is covered. Figure 5A-5C In the variation shown, the release element 41 includes a rod recessed away from the access area 134 of the base substrate 131, wherein the rod can be reversibly coupled to the decapping tool 145. In the variation, the decapping tool 145 can include a first region (e.g., a first end) that interfaces with an actuator (e.g., an actuation tip, a pipette coupled to a fluid handling subsystem of the gantry 170 described below, etc.); and a second region (e.g., a second end) including a connecting element 42 that is configured to interface with the release element 41 of the cover 135. Then, with the movement of the pipette / pipette interface, the decapping tool 145 can be configured to pull the release element 41 and / or push the cover 135 to convert the cover between an open mode and / or a closed mode. Thus, with respect to a fluid handling element coupled to a gantry 170 described below, the system 100 can provide an operating mode for: coupling a decapping tool 145 to an actuator (e.g., which is coupled to the gantry 170), the decapping tool including a linking element 42; moving the decapping tool to align with a release element 41 of the cover 135, reversibly coupling the linking element 42 to the release element 41; and applying a force to the release element 41, thereby releasing the cover 135 from a latched state and converting the cover 135 from a closed mode to an open mode. In order to effectively apply an unlocking force (e.g., by an actuator (e.g., which is coupled to the gantry 170), the base substrate 131 can be held in place (e.g., by a holding element described in Section 2.1.4, by a holding element of a heating and cooling subsystem, by a holding element of a liquid level detection subsystem, by a holding element of a platform, etc.), which passively or actively applies a reaction force against the unlocking force applied by the decapping tool 145).

[0082] However, in variations, the locking / latch mechanism may additionally or alternatively include or operate by: a lock and key mechanism, a magnetic element, or another suitable mechanism. In addition, in alternative variations, cover 135 may include another cover actuator, for example, including a motor that rotates the cover about an inlet parallel to the wide surface of sample processing box 130. The actuator may additionally or alternatively be configured to translate cover 135 (e.g., slide cover 135 parallel to the wide surface of sample processing box 130, translate cover 135 perpendicular to the wide surface, etc.) or otherwise move cover 135 to selectively cover and expose one or more predetermined areas (e.g., a collection of microwells). Thus, the cover 135 can be configured to operate in an automatic or semi-automatic manner such that the cover 135 automatically closes upon one or more triggers (e.g., a cell capture protocol is initiated by a user, a cell processing protocol is initiated by a user, all reagents for a selected protocol have been added from the reagent kit 120, etc.) and opens upon one or more triggers (e.g., a cell capture protocol has been completed, upon user request, it has been determined that cells are viable, it has been determined that single cells have been captured, etc.). Additionally or alternatively, operation of the cover 135 can be initiated and / or completed by a user, operated according to a schedule or other time pattern, or otherwise operated.

[0083] like Figure 4A-4C As shown, the base substrate 131 may also include a waste holding area 137 for receiving waste materials from the sample processing chip 132. The waste holding area 137 may also be used to maintain a desired pressure (e.g., vacuum pressure, etc.) within the sample processing chip 132 so that liquid can flow from the inlet reservoir 133 through the sample processing chip 132 and reach the waste holding area 137. The waste holding area 137 may be defined as a volume for receiving waste or other materials from the sample processing chip 132 (e.g., recessed into the base substrate 131, extending from the base substrate 132, coupled to an outlet of the base substrate 131, etc.). Figure 4A-4C In the illustrated variation, waste holding area 137 is defined on a side of base substrate 131 opposite to the side to which sample processing chip 132 is coupled, so that waste from sample processing chip 132 is pushed or pulled upward into waste holding area 137 by the force of pumping subsystem 157, which is described in more detail below. However, waste holding area 137 may additionally or alternatively be configured at another suitable location relative to base substrate 131 and sample processing chip 132 to receive waste.

[0084] The waste holding area 137 may have a volume capacity of 10 mL-100 mL or another suitable volume capacity.

[0085] like Figure 4A-4CAs shown, the waste containment area 137 may include a cover 48 (e.g., a cover that is substantially coplanar with the lid 135), which helps contain the waste within the waste containment area 137. Alternatively, the waste containment area 137 may not include a cover. Figure 4C As shown, an example of a waste containment area 137 can include a pump outlet 51 that is distinct from the cover, wherein the pump outlet 51 can allow remaining air in the waste chamber to be pressurized by a non-cartridge pump (e.g., via a pumping mechanism, etc.); however, variations of the waste containment area 137 can optionally omit the waste outlet.

[0086] With respect to the waste holding area 137, the system 100 may further include a valve 43 configured to allow and / or prevent flow from the sample processing chip 132 to the waste holding area 137. The valve 43 may interface with the outlet opening 36 of the sample processing chip 132 described above so as to enable and / or prevent flow out of the outlet opening 36 and into the waste holding area 137. The valve 43 may have a normally open state and be switched to a closed state when interfaced with a valve actuation mechanism. Alternatively, the valve 43 may have a normally closed state and be switched to an open state when interfaced with a valve actuation mechanism.

[0087] exist Figure 4A and Figure 6A-6B In the variation shown, the valve 43 includes an elastomeric body and is configured to couple the sample processing chip 132 to the base substrate 131 through an opening 44 of the sample processing chip 132, which is aligned with a corresponding valve receiving portion of the base substrate 131. In this variation, the convertible portion of the valve 43 is configured to be positioned along a flow path from the outlet opening 36 of the sample processing chip 132 to the entrance of the waste holding area 137 of the base substrate 132 (e.g., along a flow path from the microwell area to the outlet of the sample processing chip into the waste holding area of ​​the sample processing box). In the example, the opening 44 of the sample processing chip 132 is adjacent to the outlet opening 37 of the sample processing chip 132; however, in other variations, the outlet opening 37 and the opening 44 can be separated from each other and connected by another microfluidic channel. In this way, the closure of the valve 43 can prevent the flow from the outlet opening 37 to the waste holding area 137, and the valve 43 can be opened to allow the flow from the outlet opening 37 to the waste holding area 137.

[0088] exist Figure 6B In the variation shown in the cross-sectional image of the base substrate 131 shown, the valve actuator 45 can enter the base substrate 131 from below (e.g., from below the platform) and pass through the channel or other recess / opening of the base substrate 132 to interface with the valve 43. Specifically, as shown in Figure 6BAs shown in the top, when the tip 46 of the valve actuator 45 (aligned with the opening in the base substrate) pushes against the valve (e.g., the elastomeric membrane of the valve 43), the valve 43 can be switched to a closed state to fluidly separate the outlet opening 37 of the sample processing chip 132 from the waste holding area 137. Additionally or alternatively, as Figure 6B (bottom), removing the force by the valve actuator 45 can remove the pressure from the valve 43 and convert it to an open state to fluidically couple the outlet opening 36 of the sample processing chip 132 with the waste holding area 137. In this way, the valve actuation subsystem includes an engagement mode and a disengagement mode, in which the tip is extended into the valve opening to deform the elastomeric valve, thereby closing the flow path, and in the disengagement mode, the tip is retracted, thereby opening the flow path. However, the valve 43 can be configured in another suitable manner in addition or alternatively.

[0089] In other variations, the system may include similar mechanisms for coupling valves to other flow paths of the sample processing chip 132 and / or the base substrate 131 .

[0090] However, variations of base substrate 131 may include other elements. For example, as described in more detail below, base substrate 131 may include one or more openings, recesses, and / or protrusions that provide additional coupling to sample processing chip 132 to facilitate or inhibit flow through sample processing chip 132. For example, Fig. 6A As shown, the base substrate may include pump openings 46 that couple the base substrate 131 to pumping elements of a pumping subsystem 157 (eg, via the platform 110 ) to drive and / or stop fluid flow through the sample processing chip 132 .

[0091] However, base substrate 131 of sample processing cartridge 130 may include other suitable components.

[0092] 2.1.3 Platform Support Components: Tool Container

[0093] like Figure 2A and Figure 2B As shown, the platform 110 includes an area 113 of at least one unit for supporting a tool container 140, wherein the area 113 is used to position the tool container 140 relative to the fluid handling equipment of the stand 170 described below. The area 113 can also position the tool container 140 near the reagent kit 120 and sample processing cartridge 130 used to provide a more compact system and improve the efficiency of automated operations involving the contents of one or more of the reagent kit 120, the sample processing cartridge 130, and the tool container 140. The area 113 can also include an opening that allows the tool container 140 to be at least partially recessed below the surface of the platform 110.

[0094] The tool container 140 is used to contain one or more units of various tools in one or more compartments, the sample processing box decapping tool 145 and / or other tools, according to one or more workflows for various applications, the one or more units for fluid aspiration, fluid delivery, separation of target materials from non-target materials of the sample. In this way, the tool container 140 can facilitate the transfer and / or mixing of reagents and samples, fluidly couple and / or separate elements at various areas of the platform 110, or otherwise interface with one or more components of the system 100.

[0095] In the variant, one of the variants is Fig. 7A As shown in the figure, the tool container 140' may include a collection 141 of tips for fluid aspiration and / or fluid delivery (e.g., to and from the reagent cartridge 120, to and from the sample processing cartridge 130, etc.). The collection 141 of tips may include any or all of the following: standard pipette tips (e.g., P20 tips, P200 tips, P1000 tips, etc.); additionally or alternatively, the collection of tips may include piercing tools (e.g., to gain access to a reagent tube through a seal), blunt tips (e.g., for facilitating fluid flow, for blocking pores, for defining fluid paths, etc.), or any other suitable tips. The tips may be configured or otherwise used for any or all of the following: piercing (e.g., piercing a reagent strip foil), transferring and / or mixing a collection of reagents (e.g., a tip for mixing ethanol and priming buffer, a tip for transferring SPRI ethanol, a tip for transferring SPRI supernatant, a tip for transferring SPRI elution buffer, etc.), dispensing cells into cells of a sample processing cartridge 130, dispensing functionalized particles into cells of a sample processing cartridge, facilitating execution of a collection of predetermined processes (e.g., particle washing, cell lysis, oil dispensing, pre-reverse transcription washing, other workflows described below, etc.), or may have any other suitable function.

[0096] like Fig. 7A As shown, the collection 141 of tips can be arranged in an array (e.g., by type, by size, etc.) within the tool container 140, wherein the internal base of the tool container 140 may include a droplet capture portion for capturing droplets that leave the aspiration / delivery end of the tip from various fluid handling operations. In addition, the tool container 140 may include spacing elements to prevent the individual tips of the collection 141 of tips from contacting each other, thereby preventing cross contamination. In addition, the area of ​​one or more tips for interfacing with the pipette can be conductive (e.g., thermally conductive, electrically conductive, composed of metal, composed of conductive polymers, composed of semiconductor materials, etc.).

[0097] exist Figure 7BIn the specific example of the 3' processing scheme shown, the tool container 140" can include: a first tip 1401 for transferring ethanol, washing buffer and / or priming solution (e.g., having a tip volume of 200 mL, having a tip volume of 200 μL); a second tip 1402 for transferring a cell suspension (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a third tip 1403 for transferring functionalized particles (e.g., having a tip volume of 200 mL, having a tip volume of 200 μL); a fourth tip 1404 for transferring a washing buffer (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a fifth tip 1405 for transferring a particle binding buffer (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a sixth tip 1405 for transferring DTT and / or a lysis buffer (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a seventh tip 1407 for transferring a perfluorinated inert oil (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); an eighth tip 1408 for transferring DTT, a particle binding solution and / or a washing solution (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a seventh tip 1408 for transferring DTT and / or a pre-RT reaction washing solution (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 9th tip 1409 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); one or more magnetic sleeves 1410 for magnetic recovery; an 11th tip 1411 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for transferring a DTT and / or RT mixture solution; a 12th tip 1412 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for transferring an exonuclease treatment; a 13th tip 1413 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for delivering mineral oil; a 1440 tip 1441 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for transferring an exonuclease treatment; a 4th tip 1414 for transferring a sodium hydroxide solution; a 15th tip 1415 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 16th tip 1416 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for transferring a second washing buffer; a 17th tip 1417 for transferring a second strand synthesis solution; an 18th tip 1418 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for transferring a particle solution; a 19th tip 1419 (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL) for transferring a second washing buffer;a 20th tip 1420 for transferring a PCR master mix and / or a particle solution (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 21st tip 1421 for transferring mineral oil (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 22nd tip 1422 for transferring a PCR product (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 23rd tip 1423 for transferring an AMPure and / or a supernatant (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); 00 μL tip volume); a 24th tip 1424 for transferring ethanol (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 25th tip 1425 for transferring ethanol (e.g., having a tip volume of 1000 mL, having a tip volume of 1000 μL); a 26th tip 1426 for transferring ethanol (e.g., having a tip volume of 50 mL, having a tip volume of 50 μL); and a 27th tip 1427 for transferring nuclease-free water and / or a derivative product (e.g., having a tip volume of 50 mL, having a tip volume of 50 μL). ;

[0098] In variations, the tool container 140 may additionally or alternatively include other sample processing tools. In one variation, the tool container 140 may include one or more units of separation tool tips 142 for magnetically separating target material from non-target material (described in more detail below). Additionally or alternatively, in variations, the tool container 140 may additionally or alternatively include units of a decapping tool 145 for converting the lid 135 described above to an open configuration. However, with respect to the disposability of the tool container 140 and / or its contents, the tool container 140 may be configured to contain only disposable elements and omit reusable elements (e.g., units of a decapping tool 145, such as Figure 2B shown).

[0099] In addition, the unit of the contents of the tool container 140 may be additionally included in one or more of the reagent kit 120, the sample processing box 130, arranged at the platform 110 in other ways, separated from the platform 110, separated from the system 100, or arranged in other ways. In other variations, the reusable tools used in the tool container 140 may include other tools that use electrical, electromagnetic, optical, or a combination of different modalities to interface with the stage 170 and be moved to a specific position on the reagent kit 120 and / or the sample processing box 130 to provide specific energy (e.g., heat, optical signals, electromagnetic waves, etc.) and / or sense specific signals (e.g., light, heat, electromagnetic, etc.). These tools may be wired to the control electronics, or may be wirelessly charged and controlled / communicated.

[0100] 2.1.4 Platform Support Components - Registration and Retention Functions

[0101] like FIG. 8A to FIG. 8I As shown, the platform 110 may include a collection of retaining elements, which are positioned relative to an area 111 for a reagent box 120, an area 112 for a sample processing box 130, and an area 113 for a tool container 140, wherein the collection of retaining elements is used to register and retain the reagent box 120, the sample processing box 130, and the tool container 140 at the platform 110 during operation to process the sample, and to be able to release the reagent box 120, the sample processing box 130, and the tool container 140 from the platform 110 when appropriate. The collection of retaining elements may include mechanical retaining elements (e.g., recesses, protrusions, etc.) configured to provide retention by snap fit, press fit, ratchet, or other suitable mechanisms. Additionally or alternatively, the collection of retaining elements may include magnetic retaining elements or other suitable retaining elements. In a variation including a magnetic retaining element, the associated magnet may be positioned away from the magnetic separation area of ​​the reagent box 120 and the platform 110 (described below with respect to 2.1.8) so as not to interfere with the associated magnetic separation mechanism. Alternatively, associated magnets may be positioned proximate to the reagent cartridge 120 and the magnetic separation region of the platform 110 to facilitate magnetic separation operations performed at the reagent cartridge 120 (and / or other suitable portion of the system).

[0102] The holding element can provide a unified mechanism for each of the reagent box 120, the sample processing box 130 and the tool container 140. Alternatively, the reagent box 120, the sample processing box 130 and the tool container 140 can each include different holding elements that are appropriately operated by different mechanisms. The holding mechanism supported by the collection of holding elements can be manually operated (e.g., a user engages with the holding element to disengage and / or engage the holding component with the platform 110). Additionally or alternatively, the holding mechanism supported by the collection of holding elements can be non-manually operated (e.g., having an actuator coupled to the holding element to convert them between an engagement mode and a disengagement mode). The holding mechanism is configured to operate with an appropriate form (e.g., to facilitate engagement by a manual operator or device), forces of loading and unloading, and / or forces transmitted (e.g., transmitted to other sensitive elements of the platform 110).

[0103] exist Fig. 8AIn the variation shown, platform 110 may include a first subset of retaining elements 21 corresponding to region 111 for reagent cartridge 120 , a second subset of retaining elements 22 corresponding to region 112 for sample processing cartridge 130 , and a third subset of retaining elements 23 corresponding to region 113 for tool container 140 .

[0104] about Fig. 8A , a first subset of the retaining elements 21 at the region 111 may include a collection of snap-fit ​​flanges positioned around the region 111 (e.g., on opposite sides of the region 111) in a manner complementary to the corresponding retaining interface 31 of the reagent cartridge 120. In more detail, as Figure 8B and Figure 8C As shown, the holding interface 31 of the reagent box 120' may include a collection of connecting pieces (e.g., flexible connecting pieces) that can be compressed from a baseline position to be positioned at the area 111, wherein the release from the compressed configuration allows the collection of connecting pieces to latch with the buckle flange. The collection of connecting pieces of the holding interface 31 may also include a gripping feature (e.g., a protrusion, etc.) that allows the user to easily compress the collection of connecting pieces. In a related variation, the first subset of the holding element 21 of the reagent box 120' and the corresponding holding interface may be positioned in another manner. For example, the collection of connecting pieces of the reagent box may be positioned in a non-peripheral area of ​​the reagent box 120 (e.g., with a finger and / or thumb hole cutout), aligned with the corresponding holding element 21 of the platform 110. With regard to providing a stable contact, the holding element 21 may be configured to provide a biasing force against a platform element (e.g., a heating and cooling subsystem element, a magnetic separation subsystem element, etc.) that interfaces with the reagent box 120, so as to provide a stable contact between the surfaces. The biasing force used against the platform element may be at least 0.5 lbs or at least 1 lbs or at least 2 lbs or at least 3 lbs.

[0105] about Fig. 8A , a second subset of retaining elements 22 at region 112 may include a collection of snap-fit ​​flanges positioned around region 112 (e.g., on opposite sides of region 112 and on a third peripheral side of region 112) in a manner complementary to corresponding retaining interfaces 32 of sample processing cartridge 130. In more detail, as FIG. 8D to FIG. 8GAs shown, the holding interface 32 of the sample processing box 130 may include a collection of connecting pieces (e.g., flexible connecting pieces) that can be compressed from a baseline position to be positioned at the area 112, wherein release from the compressed configuration allows the collection of connecting pieces to latch with the snap flange. In this variation, the sample processing box 130 can be configured to be inserted into / interfaced with the first holding element 22a (e.g., on the side of the sample processing box near the inlet reservoir, on the side of the sample processing box near the waste holding area, etc.) before another holding interface 22b is locked with the opposite snap flange. Similar to the collection of connecting pieces of the reagent box 120, the collection of connecting pieces of the holding interface 32 of the sample processing box 130 can also include a gripping feature (e.g., a protrusion, etc.) that allows the user to easily compress the collection of connecting pieces. With respect to providing secure contact, retaining elements 22 may be configured to provide a biasing force against platform elements (e.g., heating and cooling subsystem elements, pumping subsystem elements, etc.) that interface with sample processing cartridge 130 to provide secure contact between the surfaces. In addition, the collection of retaining elements 22 of platform 110 may be configured to reversibly engage sample processing cartridge 120 and provide a reaction force against decapping tool 145 as described with respect to the decapping mode of operation.

[0106] about Fig. 8A , a third subset of the retaining elements 23 at region 113 may include a collection of snap-fit ​​flanges positioned around region 113 (e.g., on opposite sides of region 113) in a manner complementary to the corresponding retaining interface 33 of the tool container. In more detail, as Figure 8H and Figure 8I As shown, the holding interface 33 of the tool container 140 may include a collection of connecting pieces (e.g., flexible connecting pieces) that can be compressed from a baseline position to be positioned at the area 113, wherein release from the compressed configuration allows the collection of connecting pieces to latch with the snap flange. The collection of connecting pieces of the holding interface 33 may also include a gripping feature (e.g., a protrusion, etc.) that allows a user to easily compress the collection of connecting pieces.

[0107] However, in variations of the retaining elements of the platform 110 described above, other suitable features may be included or may be configured in another suitable manner relative to related elements.

[0108] 2.1.5 Platform Support Elements: Heating and Cooling Subsystems

[0109] like Figure 1A-Figure 1D and Figure 2BAs shown, the system 100 may include a heating and cooling subsystem 150 for transferring heat to and / or from desired areas of the reagent cartridge 120 and / or the sample processing cartridge 130. The heating and cooling subsystem 150 may additionally or alternatively be used to maintain a desired temperature within the internal volume of the system 100. In a variation, the heating and cooling subsystem 150 may include one or more of the following: a heating element (e.g., a Peltier heating element, a resistive heating element, other heating elements), a cooling element (e.g., a Peltier cooling element, a cooling aluminum block, a fluid path system that circulates a coolant, etc.), a thermal contact or non-contact body for transferring heat to the heating and cooling element or transferring heat from the heating and cooling element to other objects, a heat sink, a fan, a temperature sensor, and a thermal control circuit (e.g., having a processing element electrically coupled to a base 180 described in more detail below). In a variation, the cooling element may maintain the storage volume and / or the sample at between 2 degrees Celsius and 8 degrees Celsius, and further preferably maintained at 4 degrees Celsius. Additionally or alternatively, the cooling element may maintain one or more storage volumes / samples at any suitable temperature (eg, below 2 degrees Celsius, above 8 degrees Celsius, etc.).

[0110] One or more portions of the heating and cooling subsystem 150 may enter the opening of the platform 110 to thermally engage or otherwise couple with desired portions of other system elements (e.g., reagent cartridges, sample processing cartridges, tool containers, etc.) supported by the platform 110 to provide heat transfer functionality for a variety of applications. Alternatively, the platform 110 may be constructed of thermally conductive material in desired areas for heat transfer applications, and portions of the heating and cooling subsystem 150 may be configured to contact the thermally conductive material areas of the platform 110 for heat transfer.

[0111] exist Figure 2B In the particular example shown, the heating and cooling subsystem 150 includes a collection of thermal bodies that thermally engage heating elements with desired portions of the reagent cartridge 120 and sample processing cartridge 130 supported by the platform 110. The collection of thermal bodies includes a first thermal body 156' (e.g., a hot plate) that passes through an opening in the platform 110' and is configured to interface with the first domain 121' of the reagent cartridge 130' described above, wherein the first thermal body 156' includes an array of recesses for surrounding a storage volume of the first domain 121' (e.g., to provide a cooling environment for material stored in the storage space of the first domain 121'). Figure 2BAs shown, the collection of thermal bodies may also include a second thermal body 157' (e.g., a thermal plate) that passes through the opening of the platform 110' and is configured to interface with the third domain 123' of the reagent kit 130' described above, wherein the second thermal body 157' includes an array of recesses for surrounding the storage volume of the third domain 123' (e.g., to transfer heat to PCR and / or other heating operations of a process performed within the storage volume of the third domain 123'). Figure 2B and Fig. 6A As shown, the collection of thermal bodies may also include a third thermal body 158' (e.g., a thermal plate) that passes through the opening of the platform 110' and is configured to interface with the collection 34 of microwells of the sample processing chip 132 described above, wherein the third thermal body 158' provides a substantially flat substrate for uniformly transferring heat to and from the microwells of the sample processing box 130.

[0112] In a variation, a collection of thermal bodies may be coupled to a heat sink element 155 (e.g., on the side of the thermal body away from the interface with the reagent cartridge / sample processing cartridge) to provide a larger surface area for heat transfer. Figure 2D-2F As shown, the area between the platform 110 and the base 180 described in more detail below can include one or more fans 154 and / or ducts 153 to provide a thermal mechanism for convective heat transfer away from the collection of the thermal body as needed. In addition, in the variations described above, one or more portions of the heating and cooling subsystem 150 (e.g., the thermal body, etc.) can include features that help keep the corresponding box (e.g., reagent box, sample processing box, etc.) in place.

[0113] In variations, the thermal body and / or one or more of the other portions of the heating and cooling subsystem 150 may be coupled to an actuator that moves the thermal body into and out of thermal communication with an element supported by the platform 110; however, variations of the system 100 may omit the actuator of the heating and cooling subsystem 150.

[0114] 2.1.6 Platform support elements: pumping subsystem

[0115] like Figure 1A , Figure 2B and Fig. 6AAs shown, system 100 may include a pumping subsystem 157 (e.g., coupled to platform 110 and / or base 180) for providing positive and / or negative pressure to desired portions of sample processing cartridge 130 described above. In more detail, pumping subsystem 157 may be used to drive fluid from inlet reservoir 133 of sample processing cartridge 130 and into sample processing chip 132 of sample processing cartridge 130. Additionally or alternatively, pumping subsystem 157 may be used to remove fluid from waste holding area 137 of sample processing cartridge 130 and into an external waste container. In variations, the pumping subsystem 157 may include: one or more ports 58 (e.g., vacuum ports) configured to interface with the sample processing cartridge 130 through an opening in the platform 110; one or more pumps (e.g., vacuum pumps, peristaltic pumps, etc.) coupled to the ports 58; one or more manifolds providing a pressure drive path coupled to the pumps; one or more pressure sensors configured to detect pressure levels along the pressure paths; and / or one or more control circuit elements configured to control the operation of the pumping subsystem 157 (e.g., by electrically coupling to a processing element of the base 180 described in more detail below). Thus, in variations, portions of the pumping subsystem 157 that are not directly coupled to the sample processing cartridge 130 may be located between the platform 157 and the base.

[0116] In a variation, port 58 of pumping subsystem 157 can be coupled (e.g., physically connected, fluidically connected, etc.) to a suitable area (e.g., an inlet, a hole, etc.) of sample processing box 130, and can be additionally or alternatively coupled to reagent box 120, another fluid path of system 100, or any other suitable component of system 100.

[0117] In a first specific example, if Figure 2F and Fig. 6A As shown, the pumping subsystem 157 includes a pump 59 (e.g., a peristaltic pump) disposed between the platform 110 and the base 180 and coupled to the waste holding area at the waste outlet 51 through a coupler disposed at the bottom wide surface of the base substrate 131 of the sample processing cartridge 130, wherein the pump 59 can draw fluid through the sample processing chip 132 in the forward direction and the reverse direction by applying negative pressure and / or positive pressure accordingly. Additionally or alternatively, the pumping subsystem 157 can draw fluid from the inlet reservoir 133 and through the sample processing chip 132 at a predetermined pressure (e.g., -0.25 psi or -1 psi or -2.5 psi) according to a trigger event associated with the liquid level detection subsystem 159 described in more detail below.

[0118] With respect to the sample processing cartridge 130 and the force applied by the decapping tool 145 to convert the lid 135 between the closed state and the open state, the retaining element described above in Section 2.1.4 can provide a retaining force that balances / offsets the force applied by the decapping tool 145 to open the lid 135, as described above. Additionally or alternatively, one or more portions of the pumping subsystem 157 can hold the sample processing cartridge 130 in place and provide a retaining force that balances / offsets the force applied by the decapping tool 145 to open the lid 135, as described above. In a specific example, the port 58 can be coupled to the sample processing cartridge 130 to provide a reaction force to the decapping tool 145. However, other portions of the system 110 can additionally or alternatively provide a reaction force.

[0119] 2.1.7 Platform Support Components: Liquid Level Detection Subsystem

[0120] like Figure 1A , Figure 2B and Fig. 9 As shown, the system 100 may include a fluid level detection subsystem 159 that is at least partially supported by the platform 110 and is configured to interface with the sample processing cartridge 130. The fluid level detection subsystem 159 is used to detect and / or measure fluid parameters (e.g., binary presence of fluid, volume of fluid, fluid flow rate, fluid type, etc.) associated with fluid at the sample processing cartridge 130 and / or other fluid processing elements of the system 100. In variations, the fluid level detection subsystem 159 may include a fluid level sensor 63 coupled to a fluid level control circuit (e.g., by being electrically coupled to a processing element of the base 180 described in more detail below).

[0121] The cells of level sensor 63 may determine a fluid parameter associated with inlet reservoir 133 of sample processing cartridge 130 (e.g., fluid flowing from the inlet reservoir to the microwell). The cells of level sensor 63 may additionally or alternatively determine a fluid parameter associated with the outlet of sample processing cartridge 130 (e.g., fluid flowing from the microwell to the outlet, fluid flowing from the outlet to the waste holding area, etc.). The cells of level sensor 63 may additionally or alternatively determine a fluid parameter associated with the waste holding area of ​​sample processing cartridge 130 (e.g., the volume of fluid in the waste holding area), or any other suitable fluid parameter. Level sensor 63 may include any or all of the following: an optical sensor, a pressure sensor, a temperature sensor (e.g., for detecting a fluid at a certain temperature in a fluid path), or any other suitable sensor configured to detect the presence of a fluid in a sample processing cartridge and optionally determine a value associated with the fluid (e.g., volume flow rate, etc.).

[0122] In a first variation, the system includes an optical sensor 63 configured to detect the presence of fluid transferred from the inlet to the collection of wells. In an example, an infrared (IR) emitter / detector pair is used to determine the presence of fluid at the inlet reservoir 133 of the sample processing cartridge 130 and the volume of fluid transferred (e.g., further based on the duration of the fluid presence).

[0123] exist Figure 2B and Fig. 9 In the particular example shown, liquid level detection subsystem 159 includes IR emitter 63a, which is displaced across and opposite to IR detector 63b of inlet reservoir 133 of sample processing cartridge 130, wherein IR emitter 63a and IR detector 63b are configured to be positioned in the lower interior portion of base substrate 131 of sample processing cartridge 130 so that IR radiation passes through the aperture and interior volume of inlet reservoir 133 to enable liquid level detection. However, variations of liquid level sensor 63 may be configured in another suitable manner.

[0124] 2.1.8 Elements of platform support: separation subsystems and operation modes

[0125] like Figure 1A , Figure 2B and Figure 10A-10C As shown, the system 100 may include a separation subsystem 160 for facilitating separation of target material from non-target material (e.g., using magnetic force, using other forces). In a variation, the separation subsystem 160 may include embodiments, variations, and examples of components described in U.S. application 62 / 866,726, entitled “System and Method for Target Material Retrieval from Microwells” and filed on June 26, 2019, which is incorporated herein by reference in its entirety. However, variations of the separation subsystem 160 may additionally or alternatively include other components.

[0126] In one variation, Figure 10A-10CAs shown, the separation subsystem 160 can include a first body 161 and a magnetic distal region 163, the first body 161 including an interface 162 to a fluid processing subsystem (e.g., a pipette interface) of a stand 170 described below, the magnetic distal region 163 being configured to provide a magnetic force for target material separation. In this variation, the magnetic distal region 163 can be configured to couple with one or more units of the magnetic sleeve 1410 described above (e.g., of the tool container 140), wherein the magnetic sleeve 1410 can be a disposable element. In addition, the interface 162 can be configured to couple to a pipetting head, which is coupled to the stand 170 described in more detail below, so as to facilitate recovery of target material or non-target material by magnetic force, fluid aspiration, and / or fluid delivery operations provided by the pipetting head. Thus, the system 100 can include a separation mode in which the stage 170 conveys the first body 161 coupled to the magnetic sleeve 1410 between the sample processing box 130 and the reagent box 120 for magnetically separating the target material from the sample. In addition, embodiments of the method implemented using the separation subsystem 160 can produce rapid recovery of target material, wherein the recovery efficiency is >90%, wherein only magnetic particles coupled to the target material (or non-target material) of the sample are recovered. The separation subsystem 160 can therefore be used to produce increased selective recovery efficiency, and thus can reduce downstream costs and processing burdens with respect to processing reagents and other material costs (due to the reduced volume required, due to reduced fragmentation in the biochemical reaction).

[0127] like Fig. 10A As shown, the first body 161 can include an interface 162 to a fluid handling subsystem of a gantry 170 described below, wherein the interface includes a coupling region that is complementary to a corresponding coupling region of the fluid handling subsystem. The coupling region of the interface 162 can be operated by: a magnetic coupling mechanism; a press fit; a snap fit, a threaded mechanism; a male-female connector; or another suitable mechanism for providing reversible coupling to a fluid handling subsystem.

[0128] The magnetic distal region 163 of the first body 161 can include or be composed of a material for providing a permanent magnet, or can be optionally configured as an electromagnet (e.g., by coupling with suitable electronics of the system 100). In variations, the magnetic distal region 163 can be composed of one or more of: aluminum nickel cobalt, neodymium, neodymium iron boron, samarium cobalt, ferrite, and any other suitable magnetic material. In terms of morphology, the magnetic distal portion 163 can complement the morphology of the magnetic sleeve 1410 so that the cells of the magnetic sleeve 1410 can be coupled (e.g., reversibly coupled) to the magnetic distal portion. In addition, the morphology and magnetic pole configuration of the magnetic distal portion 163 are such that a nearly normal magnetic force is applied to most of the target micropores, removing trapped particles from these micropores.

[0129] The magnetic sleeve 1410 may include a first region 1410a configured to interface with the sample processing chip 132, for example, by entering the region 134, so as to enable material to be transferred from the sample processing chip 132. The magnetic sleeve 1410 may also include a second region 1410b for coupling with the magnetic distal portion 163 of the first body 161, and an internal cavity 1410c passing from the first region to the second region. The magnetic sleeve 1410 is used to provide a structure that separates the first body 161 from the physical contact hole or other sensitive materials of the sample processing chip 132, and supports the application of a magnetic field to a desired area for recovering target materials (or non-target materials). The magnetic sleeve 1410 can also prevent sample cross-contamination by serving as a disposable component that can be discarded between uses of the system 100.

[0130] The magnetic sleeve 1410 may be a prismatic shape having an interior cavity 1410c, wherein a cross section of the magnetic sleeve 1410 along its longitudinal axis is defined by a polygonal perimeter, an elliptical perimeter, an amorphous perimeter, or a boundary of any other suitable shape (e.g., a closed shape, an open shape). The cross section of the magnetic sleeve 1410 may be complementary to the shape of the footprint of the microwell region of the sample processing chip 132, but may not be complementary to the shape corresponding to the sample processing chip 132. The magnetic sleeve 1410 preferably has a wall thickness that supports the application of a magnetic force from the magnetic distal portion 163 to the sample processing chip 132 that interfaces with the first region 1410a of the magnetic sleeve 1410. The wall thickness may be constant or non-constant along the length of the adapter 210. In an example, the wall thickness may be in the range of from 0.2 to 3 mm thick; however, in other examples, the wall thickness may have any other suitable thickness. The surface of the magnetic sleeve 1410 that receives the magnetic particles is made smooth (say, the surface finish is better than SPIB1) so that small magnetic particles (1-3 microns) are not trapped in the surface during capture of beads onto its surface and subsequent release into another container.

[0131] The magnetic sleeve 1410 may additionally or alternatively include structural features that enable a separation mode of operation of the separation subsystem 160. For example, with respect to the release of the magnetic sleeve 1410 from the pipetting head (described in more detail below), the magnetic sleeve 1410 may include a protrusion 1410d configured to allow another object (e.g., a sleeve stripping tool 165) to provide a force against the protrusion 1410d to release the magnetic sleeve 1410 from the first body 161.

[0132] As described above, the magnetic sleeve 1410 is coupled to the area of ​​the sample processing chip 132 exposed by the access area 134 at the first area 1410a to facilitate applying a magnetic force to the area and enabling the material (e.g., target material or non-target material coupled to the magnetic particles) to be pulled into the magnetic sleeve 1410 for further downstream processing. The magnetic sleeve 1410 can therefore include a seal at the first area 1410a to facilitate the mechanism for pulling the target material from the sample processing chip 132 into the magnetic sleeve 1410. The seal can be a separate element or an element integrated with the magnetic sleeve 1410. However, the magnetic sleeve 1410 can omit the seal at the first area 1410a.

[0133] The magnetic sleeve 1410 can be made of a polymer material (e.g., plastic) that does not adversely affect the magnetic field applied by the magnetic distal portion 163 during operation. The magnetic sleeve 1410 can additionally or alternatively include a material (e.g., a metallic material) that is magnetic (e.g., including particles of a material (e.g., a metallic material) that is magnetic) or be made of a material (e.g., a metallic material) that is magnetic, or can generate an induced magnetic field to support an application using the system 100. The magnetic sleeve 1410 can additionally or alternatively be made of any other suitable material. The distribution of the material of the magnetic sleeve 1410 can be uniform or non-uniform in the body of the adapter relative to the desired magnetic effect at the capture area of ​​the sample processing chip 132. The internal cavity 1410c of the magnetic sleeve 1410 can include a medium (e.g., a magnetic medium, etc.), or can optionally not include any medium.

[0134] like Figure 1A , Figure 2B and Figure 11A-11B As shown, in a variation, the separation subsystem 160 can include a magnet subsystem 166 that includes a collection of magnets 167 within a housing 168, wherein the magnet subsystem 166 also includes a magnet actuator 169 that is configured to move the collection of magnets 167 relative to the platform 110 (e.g., through an opening in the platform 110) and to align / non-align with one or more separation reservoirs 129 of the reagent kit 120 described above. The magnet actuator 169 can also be coupled to a control circuit (e.g., at the base 180) described in more detail below. In addition, the magnet actuator 169 can be configured to convert the collection of magnets between a retracted state and an extended state, wherein in the extended state, the collection of magnets enters a first area of ​​the platform (e.g., as shown in FIG. 1 ). Fig.11A and Fig. 11B As such, the separation subsystem 160 may also include elements supported by the platform 110 and / or the base 180 to enable operations for separating target materials from non-target materials.

[0135] In variations, the collection of magnets 167 may include one or more permanent magnets and / or electromagnets (e.g., via suitable electronics coupled to the system 100). The permanent magnets may be composed of one or more of: Alnico, Neodymium, Neodymium Iron Boron, Samarium Cobalt, Ferrite, and any other suitable magnetic material.

[0136] exist Figure 11A-11B In the example shown, the set of magnets 167 may include a first subset 167a of magnets arranged in a linear array (e.g., for performing a purification operation at the reagent cartridge 120), wherein the positions of the first subset 167a of magnets correspond to the positions of the volume of the fifth domain 125' for particle separation / purification, as described above with respect to the reagent cartridge 120' and below with respect to the workflow in Section 3. Figure 11A-11B In the example shown, the set of magnets 167 also includes a second subset of magnets 167b (e.g., one or more magnets) that is displaced or otherwise offset from an axis associated with the first subset of magnets 167a to interface with the separation reservoir 129a of the reagent cartridge 120 (e.g., for initial bead recovery). However, the set of magnets 167 can be arranged in another suitable manner (e.g., in a distributed array, in quantity, etc.) with respect to providing an appropriate interface with the separation reservoir 129 of the reagent cartridge 120 or other cartridge.

[0137] The housing 168 is used to enclose the collection of magnets 167 and provide smooth operation associated with transitioning the collection of magnets 167 into / out of alignment with corresponding portions of the reagent cartridge 120. Fig. 11B As shown, with respect to a configuration in which there is a first subset of magnets 167a and a second subset of magnets 167b, the housing 168 can include a first surface (e.g., a first planar surface) of the first subset of tracking magnets 167a and a second surface (e.g., a second planar surface) of the second subset of tracking magnets 167b, wherein the first surface 168a and the second surface 168b are angled away from each other. In this variation, a pair of opposing walls can extend from the first surface and the second surface to facilitate smooth operation (e.g., sliding operation) of the housing 168 and the magnets through the platform 110 to interface with the reagent cartridge 120.

[0138] Regarding the kit 120', Figure 3A and Fig. 11BAs shown, the volumes of the reagent box 120 configured for magnetic separation can each include a flat surface 128a, or other surfaces that are configured to be complementary to the housing 168 at the side closest to the housing 168 during operation (e.g., in the extended magnet state). In addition, the volumes of the reagent box 120 configured for magnetic separation can each include a second surface 128b (e.g., a curved surface) that is displaced away from the housing 168 for aspiration and / or delivery of fluids by a pipette coupled to the stand 170 described below. A cross-section taken longitudinally through the separation volume / reservoir 129 of the reagent box 120 can further taper toward the base of the reagent box 120, such that a lower volume of fluid is required for the separation operation and / or to provide more efficient aspiration and separation of target and non-target materials.

[0139] like FIG. 12A to FIG. 12J As shown, the separation subsystem 160 can provide a series of operating modes for material separation, wherein, for example, Fig. 12A As shown, the operating mode involves the following specific system structural configuration: a first body 161 coupled to a pipette head or other actuatable component (e.g., as described with respect to the stand 170 below), the first body having a magnetic distal region 163, a magnetic sleeve 1410, a sleeve stripping tool 165, a separation reservoir 129, and a magnet 167b from the collection 167 of magnets described above.

[0140] In more detail, Fig. 12B As shown, the separation subsystem 160 can provide a first operating mode 164a, wherein the first operating mode 164a is a baseline operating mode in which the first body 161 is disengaged from the pipette interface or other actuatable component (e.g., as described with respect to the stand 170 below), and the magnetic distal region 163 of the first body 161 is disengaged from the magnetic sleeve 1410. The magnetic sleeve 1410 is further held above the separation reservoir 129 (or in a variation, in another position) by the sleeve stripping tool 165, and the magnet 167b is moved away from the separation reservoir 129 (e.g., by the magnet actuator 169 described above).

[0141] like Fig. 12C As shown, the separation subsystem 160 can provide a second operating mode 164b, wherein the second operating mode 164b is an initialization operating mode in which the first body 161 is coupled to a pipette interface or other actuator interface 6 (e.g., as described with respect to the stand 170 below), and the magnetic distal region 163 of the first body 161 is disengaged from the magnetic sleeve 1410. The magnetic sleeve 1410 is further held above the separation reservoir 129 by the sleeve stripping tool 165, and the magnet 167b is moved away from the separation reservoir 129 (e.g., by the magnet actuator 169 described above).

[0142] like Fig.12D As shown, the separation subsystem 160 can provide a third operating mode 164c, wherein, in the third operating mode 164c, the first body 161 is coupled to a pipette interface or other actuator interface 6 (e.g., described with respect to the stand 170 below) and moved to align with the separation reservoir 129. In the third operating mode 164c, the magnetic distal region 163 of the first body 161 is coupled to the magnetic sleeve 1410 above the separation reservoir 129 in a holding position of the magnetic sleeve 1410. In the third operating mode 164c, the magnet 167b is moved away from the separation reservoir 129 (e.g., by the magnet actuator 169 described above).

[0143] like Fig.12E As shown, the separation subsystem 160 can provide a fourth operating mode 164d, wherein, in the fourth operating mode 164d, the first body 161 is coupled to a pipette interface or actuator interface 6 (e.g., described with respect to the stand 170 below), and the magnetic distal region 163 of the first body 161 is coupled to a magnetic sleeve 1410 above the separation reservoir 129. In the fourth operating mode 164d, the pipetting head (or other actuatable component) moves the first body 161 and the magnetic sleeve 1410 out of the holding position provided by the sleeve stripping tool 165 to prepare for extracting material (e.g., functionalized particles from a sample processing cartridge) and / or delivering material from the pipetting interface coupled to the first body and the magnetic sleeve to the separation reservoir 129. In the fourth operating mode 164d, the magnet 167b is moved away from the separation reservoir 129 (e.g., by the magnet actuator 169 described above).

[0144] like Fig.12F As shown, the separation subsystem 160 can provide a fifth operating mode 164e, wherein, in the fifth operating mode 164e, the first body 161 is coupled to a pipette interface or actuator interface 6 (e.g., described with respect to the stand 170 below), and the magnetic distal region 163 of the first body 161 is coupled to the magnetic sleeve 1410 above the separation reservoir 129. In the fifth operating mode 164d, the pipette interface (or other actuatable component) delivers fluid from the pipette head to the separation reservoir 129, and the magnetic sleeve 1410, which is still coupled to the first body 161 (and coupled to the functionalized particles), is immersed in the fluid in the separation reservoir 129. In the fifth operating mode 164e, the magnet 167b is moved away from the separation reservoir 129 (e.g., by the magnet actuator 169 described above).

[0145] like Figure 12GAs shown, the separation subsystem 160 can provide a sixth operating mode 164f, wherein, in the sixth operating mode 164f, the first body 161 is coupled to a pipette interface or actuator interface 6 (e.g., described with respect to the stand 170 below), and the magnetic distal region 163 of the first body 161 is coupled to the magnetic sleeve 1410 above the separation reservoir 129. In the sixth operating mode 164f, the pipette interface (or other actuatable component) moves the magnetic sleeve 1410, which is still coupled to the first body 161, back to a holding position at the sleeve stripping tool 165, and immerses the magnetic sleeve 1410 (still coupled to the functionalized particles) in the fluid in the separation reservoir 129. In the sixth operating mode 164f, the magnet 167b is moved away from the separation reservoir 129 (e.g., by the magnet actuator 169 described above).

[0146] like Fig.12H As shown, the separation subsystem 160 can provide a seventh mode of operation 164g, wherein, in the seventh mode of operation 164g, the first body 161 is coupled to a pipette interface or actuator interface 6 (e.g., described with respect to the stage 170 below), and the magnetic distal region 163 of the first body 161 is coupled to the magnetic sleeve 1410 above the separation reservoir 129. In the seventh mode of operation 164g, the magnetic sleeve 1410, still coupled to the first body 161, is in a holding position at the sleeve stripping tool 165, and the magnetic sleeve 1410 (with the functionalized particles) is immersed in the fluid in the separation reservoir 129. In the seventh mode of operation 164g, the magnet 167b is moved toward the separation reservoir 129 (e.g., by the magnet actuator 169 described above) to prepare to attract and hold the target material or non-target material of the functionalized particles coupled to the fluid against the wall 128a of the separation reservoir 129.

[0147] like Fig.12IAs shown, the separation subsystem 160 can provide an eighth operating mode 164h, wherein, in the eighth operating mode 164h, the first body 161 is coupled to a pipette interface or an actuator interface 6 (e.g., described with respect to the stand 170 below) and is moved away from the separation reservoir 129 to be removed from the tool container described above and replaced with a suitable tip. In the eighth operating mode 164h, the magnetic distal region 163 of the first body 161 is disengaged from the magnetic sleeve 1410 above the separation reservoir 129 by moving the first body 161 away from the magnetic sleeve 1410 while keeping the magnetic sleeve 1410 in a proper position at the sleeve stripping tool 165. In the eighth operating mode 164h, the magnetic sleeve 1410 is immersed in the fluid in the separation reservoir 129. In the eighth operating mode 164h, the magnet 167b is still positioned adjacent to the separation reservoir 129 (e.g., by the magnet actuator 169 described above) for holding the target material or non-target material of the functionalized particles coupled to the fluid against the wall 128a of the separation reservoir 129. In the eighth operating mode 164h, the magnet 167b draws the target material toward the bottom of the separation reservoir 129 (e.g., for later extraction from the bottom by a pipette, for holding while the pipette draws material not bound by the magnet 167b).

[0148] like Fig.12J As shown, the separation subsystem 160 can provide a ninth mode of operation 164i, wherein, in the ninth mode of operation 164i, the pipetting head / actuator interface 6 is coupled with a suitable tip and moved into the separation reservoir 129 to aspirate material from the separation reservoir 129. In the ninth mode of operation 164i, the magnetic sleeve 1410 is still held in position above the separation reservoir 129 at the sleeve stripping tool 165 and is immersed in the fluid in the separation reservoir 129. In the ninth mode of operation 164i, the magnet 167b (e.g., by the magnet actuator 169 described above) is moved away from the separation reservoir 129 in coordination with aspiration of material by the pipetting head.

[0149] FIG. 13A to FIG. 13D Additional views of the configuration of the magnetic sleeve 1410 relative to the sleeve stripping tool 165 of the separation reservoir 129 of the reagent cartridge 120 are depicted with respect to the above described modes of operation.

[0150] However, variations of the separation subsystem 160 may include elements and modes of operation that provide target material recovery based on one or more of: gravity, buoyancy, centrifugal force, chemical separation, and / or any other suitable separation method. In yet another embodiment, the target material recovery operation by the separation subsystem 160 may be used to transfer target particles from a microwell chip to another substrate or another new, empty microwell chip while maintaining the relative spatial positions of the different particles being transferred.

[0151] 2.2 System-Stand

[0152] like Figure 1A-1B , Figure 2A and Figure 2C-2F As shown, the system 100 may include a stage 170 coupled to the platform 110, the stage 170 being used to support and / or enable actuation of one or more tools for various engagements with elements of the platform 110 along a set of axes. In a variation, the stage 170 provides one or more rails / tracks for moving tools, such as a pipette 174 having a pipette interface described below, in a three-dimensional space (e.g., a three-dimensional volume defined by a first side of the platform). In a variation, tools actuated using the stage 170 can be moved relative to the sample processing box 130, the reagent box 120, the tool container 140, or other elements for transfer of materials (e.g., cells, reagents, particles, etc.) across different components supported by the platform 110. Additionally or alternatively, the tools supported by the stage 170 can be used to read bar codes associated with various disposable items supported by the platform 110 (e.g., for identifying the correct settings for a run, for inventory management, etc.). The stage 170 is preferably capable of moving one or more tools along one or more axes parallel to the broad surfaces of the reagent cartridge 120, the sample processing cartridge 130, and the tool container 140 (eg, Figure 2A X and Y axes shown), and also along an axis perpendicular to the broad surface (e.g., Figure 2A The gantry 170 may be movable in a subset of these directions or in any other suitable direction. To enable movement, the gantry 170 includes or is otherwise coupled to one or more motors (e.g., a motor for each axis or direction of movement), one or more encoders for position recognition in each axis or direction of movement, and / or one or more switches (e.g., optical switches for each axis) for controlling the gantry 170 (e.g., where the switches are electrically coupled to the control circuitry described below with respect to the base 180).

[0153] In a first variant (for example, Figure 2A-2B and Figure 2D-2F As shown in FIG. 1 , the stage 170 includes a three-dimensional track assembly (XYZ track assembly), which includes an X-rail 171 for moving the tool along the X-axis, a Y-rail 172 for moving the tool along the Y-axis, and a Z-rail 173 for moving the tool along the Z-axis. Figure 2AIn the illustrated embodiment, the X-rail 171 is coupled to the Y-rail 172 and slides along the Y-rail 172, and the Z-rail 173 is coupled to the X-rail 171 and slides along the X-rail 171; however, in other embodiments, the X-rail 171, the Y-rail 172, and the Z-rail 173 may be coupled and engaged with each other in another suitable manner. Figure 2A In the variation shown, the stage 170 is configured to move tools using X-rails 171 and Y-rails 172 to align them with elements of the platform 110, and is configured to move tools using Z-rails 173 for access to elements of the platform (e.g., portions of the reagent box 120, elements of the sample processing box 130, tools of the tool container 140, etc.). In some variations, the stage 170 can be configured to return to a parked position (e.g., once a protocol is completed, when the system is shut down, before the cover is opened, etc.), wherein the parked position allows the user to access appropriate areas of the platform (e.g., for refilling tips, filling tubes with reagents, removing microwell boxes, etc.). However, the stage 170 can have another suitable baseline position. The various operating modes of the stage 170 will be further described in conjunction with the workflow of Section 3 below.

[0154] 2.2.1 Stand-Pipette

[0155] like Figure 2A As shown, the stand 170 can include and / or can be configured to interface with a pipette 174 for holding, moving, and / or otherwise interfacing with any number of tips or other tools, such as the tips or other tools of the tool container 140 described above. In variations, the pipette 174 assembly can include one or more of: a pump (e.g., a displacement pump) for providing a pressure differential for delivery and aspiration of fluids, a pressure sensor for sensing aspiration pressure, a level sensor for sensing a liquid level within the pipette 174, a tip detector (e.g., for enabling determination of whether a tip coupled to the pipette 174 is present), and a tip ejection motor coupled to a tip ejector for removing a tip from the pipette 174. As shown in FIG. Figure 2A As shown, the pipette 174 may be coupled to the Z-rail 173 of the stage 170 ; however, in other variations, the pipette 174 may additionally or alternatively be coupled to other portions of the stage 170 .

[0156] The pipette 174 is preferably operable in an automated manner (e.g., motorized, mechanized, automatically operated, etc.) and may be configured to control any or all of the following predetermined parameters: volume (e.g., dispensing a precise volume, aspirating a precise volume), the height above the well to which each material is dispensed (e.g., the priming buffer is dispensed between 0.25 mm and 0.3 mm above the top of each well, the cell suspension is dispensed at a height of 0.25 mm above the top of each well, etc.), or any other suitable attribute may be controlled according to any suitable parameter. Additionally or alternatively, the pipette 174 may be configured to operate in a manual manner (e.g., according to a user, with user intervention, held and used by a user, etc.) or in any suitable manner. In yet another embodiment, the pipette 174 may be used to pick up one or more tools associated with a tool container, such as any or all of the following tools: mechanical tools, magnetic tools, optical tools, and any other suitable tools. The tool may be moved to the reagent cartridge and / or microwell cartridge by the pipette 174 so that the tool may perform a specific mechanical / magnetic and / or optical function with respect to specific contents of the reagent cartridge or microwell cartridge.

[0157] 2.2.2 Bench - Other tools

[0158] In addition, the stand 170 may include or support one or more of the tools described above with respect to other elements. For example, the stand 170 may be coupled to a unit of the decapping tool 145 (in Figure 2B 1) is coupled to the cover 135 of the sample processing box 130 for switching between an open state and a closed state. The stage 170 may additionally or alternatively be coupled to a head actuator 175 (which is shown in FIG. Figure 2B ), the head actuator 175 is associated with the separation subsystem 160 described above (eg, with respect to actuation of the first body 161 having the magnetic distal region 163) for separating target material from non-target material.

[0159] In some variations, the stage 170 may be coupled directly or indirectly to a camera 176 (e.g., a camera coupled to a light) for enabling reading of labels (e.g., bar codes) associated with various disposable items supported by the platform 110 (e.g., for identifying the correct settings for a run, for inventory management, etc.). Additionally or alternatively, the camera 176 may include functionality for transmitting image data that captures the configuration of elements at the platform 110 without reading specific labels. Figure 2A and Figure 2FAs shown, the camera 176 can be coupled to the Z rail 173 via the pipette 174 described above, so that the camera 176 has a field of view associated with the object aligned with the pipette 174. However, the movement of the camera 176 can be selectively disengaged from the pipette 174, so that the camera 176 can move independently of the pipette 174. In a variation, the camera 176 can include components described in the applications incorporated by reference above. The camera image can also be used as an additional feedback mechanism for the precise movement of the stage 170 and / or its components (e.g., pipettes, tools, etc.) to a desired position, thereby significantly improving the accuracy and precision of the motion system (e.g., to less than 100 microns, less than 50 microns, less than 25 microns, less than 10 microns, less than 5 microns, less than 1 micron, etc.).

[0160] Additionally or alternatively, such as Figure 1A and Figure 1B As shown, the gantry 170 may include a variety of sensors for monitoring the environment or other parameters of the system 100. For example, in a variation, the sensor may include one or more of the following: a temperature sensor, a humidity sensor, a pressure sensor, an optical sensor, a vibration sensor, and other suitable sensors. However, the sensor may be positioned relative to the system 100 in another suitable manner (e.g., detached from the gantry 170, positioned at the platform 110, positioned at the base 180, etc.).

[0161] 2.3 System Base

[0162] like Figure 1AAs shown, the system 100 may include a base 180 for supporting control and processing architecture associated with the elements of the platform 110 and the stage 170 described above. In variations, the base 180 may support control and processing architecture for one or more system functions, including: pressure modification for fluid delivery throughout the sample processing cartridge 130 and / or the pipette 174; fluid level sensing (e.g., at the sample processing cartridge 130, at the pipette 174, at various storage volumes of the reagent cartridge 120, etc.); actuation of the lid opening mechanism of the sample processing cartridge 130; thermal cycling and / or other heating functions of the reagent cartridge 120 and / or the sample processing cartridge 130; cooling functions of the reagent cartridge 120 and / or the sample processing cartridge 130; separation functions (e.g., elution, magnetic separation, other separations, etc.); functions for controlling the stage 170; functions involving receiving sensor signals and Functions that return outputs; functions involving receiving sensor signals and performing various actions; functions for converting system doors between various states (e.g., open state, closed state, locked state, unlocked state, etc.); functions associated with system power management; functions associated with system status indicator elements (e.g., lights, audio output devices, visual output devices, etc.); functions associated with system input devices (e.g., buttons, keyboards, keypads, mice, joysticks, switches, touch screens, etc.); functions associated with display devices; functions associated with system data storage devices; functions associated with system transmission devices (e.g., wired transmission devices, wireless transmission switches, etc.); and other suitable functions.

[0163] In variations, the base 180 can thus support an electronic subsystem (e.g., a PCB, a power source, a communication module, an encoder, etc.) associated with a processing architecture (e.g., on the system, separate from the system, etc.), or any other suitable component, wherein the processing architecture can include any or all of the following: a processor (e.g., a microprocessor), a controller (e.g., a microcontroller), a memory, a storage device, software, firmware, or any other suitable component. In addition, the processing subsystem can include a machine vision module for reading labels, verifying protocols, performing error detection (e.g., detecting that the reagent does not match the assigned protocol), or performing any other function.

[0164] For example, in an exemplary operation flow, an operator can start the execution of a scheme (e.g., by pressing a button of the system, by selecting through a touch-sensitive display interface with the system, etc.). A barcode reader performs an error detection scheme by scanning the label of a platform element (e.g., a test kit, a sample processing box, a tool container, etc.) and comparing it with the scheme selected by the user; if the label does not match the selected scheme, a notification can be sent to the user, and if the label is correct, the scheme can be started. At this point, an operator may no longer be needed. According to one or more workflows, some of which are described in Section 3 below, the type and volume of the correct material (e.g., reagent / sample) are added to or removed from the sample processing box in an automated manner at the right time. Once the scheme is completed, the operator can continue to collect and / or process the contents of the microwell box as needed, and / or continue to establish a new run. Variations of the method and workflow implemented by system 100 are further described below.

[0165] Embodiments, variations, and examples of the control and processing architecture are further described in the following applications: U.S. Application No. 16 / 048,104, filed on July 27, 2018; U.S. Application No. 16 / 049,057, filed on July 30, 2018; U.S. Application No. 15 / 720,194, filed on September 29, 2017; U.S. Application No. 15 / 430,833, filed on February 13, 2017; U.S. Application No. 15 / 821,329, filed on November 22, 2017; U.S. Application No. 15 / 720,194, filed on September 29, 2017; U.S. Application No. 15 / 430,833, filed on February 13, 2017; U.S. Application No. 15 / 821,329, filed on November 22, 2017; U.S. Application No. 15 / 782,270, filed October 12; U.S. Application No. 16 / 049,240, filed July 30, 2018; U.S. Application No. 15 / 815,532, filed November 16, 2017; U.S. Application No. 16 / 115,370, filed August 28, 2018, U.S. Application No. 16 / 564,375, filed September 9, 2019, and U.S. Application No. 16 / 816,817, filed March 12, 2020, as incorporated by reference above.

[0166] 2.4 System – Functional coupling between platform, gantry and base

[0167] In variations, the platform 110, gantry 170 and base 180 of the system may be functionally coupled to provide multiple functions. The functional coupling may be electromechanical, mechanical, electrical, magnetic, pneumatic, fluidic, optical and / or other couplings according to various modes of operation. Figure 14A-Figure 14C Depicted are functional couplings for variations of the systems described below.

[0168] Fig.14AExamples of mechanical, electrical, pneumatic, optical, and magnetic communications between the various elements of the platform 110, the gantry 170, and the base 180 are depicted. In more detail, as Fig.14A As shown, the stage 170 supports elements associated with actuation, which include a Z-axis arm 801 associated with the Z-rail 173 described above, a 3-axis stage motor 802, a 3-axis encoder 803, a 3-axis optical switch 804, a magnetic actuator 805, and a decapping tool 806. The stage 170 also supports pipetting elements, which include: a pipette 174, a displacement pump 808, a pipette tip detector 809, a pipette tip ejection motor 810, a level sensor 811, and a pressure sensor 812. The stage 170 also supports a camera and light 176 and a collection of sensors 814 including a temperature sensor and a humidity sensor. As shown in FIG. Fig.14A As shown, the platform 110 supports the sample processing box 130, the reagent box 120 and the tool container 140 (including the collection of tools 865). Fig.14A As shown, the base 180 supports a first computing subsystem 815, one or more interface boards 816, and a gantry control circuit 817. Fig.14A , mechanical, electrical, pneumatic, optical, and magnetic connections between various elements of the platform 110, the gantry 170, and the base 180 are described using various line types.

[0169] Fig. 14B Examples of mechanical, fluid, magnetic, and pneumatic communication between various elements of the platform 110 and the base 180 are depicted. In more detail, as Fig. 14B As shown, the platform 110 supports a reagent kit 120, which includes: a storage volume for environmental reagents and functional beads 818, a sample storage volume 819 for receiving an original sample 820, a storage volume 821 for storing frozen reagents and / or sample processing results, a collection of storage volumes for separation particles and associated separation reservoirs 822, and a collection of storage volumes for PCR 823, and associated PCR covers 824; a tool container 140, which includes a collection of tools (e.g., pipette tips) 825; a sample processing box 130; and a collection of platform tools 826, which include a heater head 827, a suction head 828, and a magnetic head 829. Fig. 14BAs shown, a collection of elements enables the platform 110 to interface with the base 180, wherein the collection of elements may include one or more of the following: a vacuum port 830, a squeeze actuator 831, a sample processing box thermal body 832 (configured to match with the microwell area of ​​the sample processing box 130 or other areas of the sample processing box 130), a first reagent box thermal body 833 (configured to match with the domain of the reagent box 120 at the first area 111 for freezing reagents), a second reagent box thermal body 834 (configured to match with the domain of the reagent box 120 at the first area 111 for PCR thermal cycling), a liquid level sensor 836, and a magnet 835 for a separation process. Fig. 14B , mechanical, fluid, magnetic, and pneumatic connections between various elements of the platform 110 and the base 180 are depicted using various line types.

[0170] Fig. 14C An example of electrical communication between various elements of the platform 110 and the base 180 is depicted. In more detail, as Fig. 14C As shown, the platform 110 supports a sample processing cartridge 130; a reagent cartridge 120, which includes: a storage volume 821 for storing frozen reagents and / or sample processing results, a collection 822 of storage volumes for separation particles and associated separation reservoirs, and a collection 823 of storage volumes for PCR and associated PCR covers; and a collection of platform tools 826, which includes a heater head 827. Fig. 14C As shown, a collection of elements enables the platform 110 to interface with the base 180, wherein the collection of elements may include one or more of the following: a vacuum port 830, an extrusion actuator 831, a sample processing box thermal body 832, a first reagent box thermal body 833, a second reagent box thermal body 834, a magnet 835 for the separation process, and a liquid level sensor 836.

[0171] In more detail, the vacuum port 830 is coupled to a pumping subsystem 837, which includes an exhaust valve manifold 838 and a pressure sensor 839. The sample processing cartridge thermal body 832 is coupled to a thermal cycler 840 of a heating and cooling subsystem, which includes a heater 841 (e.g., a Peltier heater, a thermocouple, a heat sink, and a fan) and a thermal control circuit 842. The first reagent cartridge thermal body 833 is coupled to a cooling device 843 of the heating and cooling subsystem, which includes a cooling element 844 (e.g., a Peltier cooler, a thermocouple, a heat sink, and a fan) and a thermal control circuit 845. The second reagent cartridge thermal body 834 is coupled to a heating device of the heating and cooling subsystem (e.g., for on-board PCR), which includes a first heater 846 (e.g., a Peltier heater, a thermocouple, a heat sink, and a fan), a second heater 847 coupled to the heat sink, and a thermal control circuit 848. The magnet 835 is coupled to a magnet actuator 849. Finally, the liquid level sensor 836 is coupled to the liquid level controller 850.

[0172] like Fig. 14C As shown, the base 180 also supports a first computing subsystem 815, one or more interface boards 816, a speaker 851, a door lock element 852 (engaged with a door magnet 853 to provide an operating mode of the door 90, as described above), a power inlet 854 coupled to a power source 855 coupled to an AC mains 856, a power switch 857, an indicator light 858 (e.g., for power / grounding effects), an exhaust fan 859, a collection of ports (e.g., including an Ethernet port 860 and a USB port 861), and a display 862 (e.g., a touch screen display). Elements of the base can also engage with external system accessories (e.g., input devices 863, storage devices 864). In Fig. 14C , electrical connections between various elements of platform 110 and base 180 are depicted using various line types.

[0173] 3. Methods

[0174] like Fig.15As shown, an embodiment of a method 300 for sample processing includes: positioning a reagent kit, a tool container, and a sample processing box on a platform of a sample processing system S310; transferring the contents of the reagent kit between the sample processing boxes by using a tool of the tool container in an operating sequence S320; and processing the sample at the sample processing box and / or the reagent kit S330. Additionally or alternatively, the method 300 may include any one or all of the processes described in the following applications: U.S. Application No. 16 / 048,104 filed on July 27, 2018; U.S. Application No. 16 / 049,057 filed on July 30, 2018; U.S. Application No. 15 / 720,194 filed on September 29, 2017; U.S. Application No. 15 / 430,833 filed on February 13, 2017; U.S. Application No. 15 / 821,329 filed on November 22, 2017; U.S. Application No. 15 / 821,329 filed on November 22, 2017; U.S. Application No. 15 / 782,270, filed October 12; U.S. Application No. 16 / 049,240, filed July 30, 2018; U.S. Application No. 15 / 815,532, filed November 16, 2017; U.S. Application No. 16 / 115,370, filed August 28, 2018, U.S. Application No. 16 / 564,375, filed September 9, 2019, and U.S. Application No. 16 / 816,817, filed March 12, 2020, each of which is incorporated by reference in its entirety.

[0175] The method is preferably performed by embodiments, variations or examples of the system described above (e.g., about the transmission of content between multiple elements and / or sample processing), but may be performed additionally or alternatively by any other suitable system. The method is also preferably at least partially automated (e.g., requiring a user to load reagents and select a protocol, without requiring user intervention, etc.), but one or more parts may be additionally or alternatively performed manually (e.g., for quality control steps, for all protocols, for rare protocols, etc.).

[0176] Specific workflows associated with method 300 and system elements described above are described in further detail below, wherein samples (e.g., samples comprising cell-derived material, proteins, mRNA, proteins and mRNA; samples comprising multiple samples, each sample being labeled with a multiplexed barcode; samples comprising encapsulated particles from cell- or non-cell-derived biomarkers, etc.) can be processed according to a workflow (e.g., the workflows in Sections 3.1 to 3.3 below), and then through a library preparation workflow (e.g., the workflow in Section 3.4 below), followed by next generation sequencing (NGS).

[0177] 3.1 Methods – Exemplary Workflow of the 3' Protocol for mRNA Synthesis / cDNA Amplification

[0178] like Fig.16 As shown, a variation 300' of a method configured for mRNA synthesis / cDNA amplification may include: performing a run preparation operation, wherein the run preparation operation configures the system for performing an mRNA synthesis / cDNA amplification protocol S305'; upon completion of the run preparation operation, initiating a sample processing cartridge of the system S310'; co-capturing a collection of cells and a collection of functionalized particles of the sample in a single cell form at the sample processing cartridge S315'; lysing the collection of cells, and subsequently binding mRNA released from the lysed cells with functionalized particles (e.g., barcoded microspheres) (e.g., with associated washing of any unbound mRNA from the functionalized particles); )S320'; perform a reverse transcription operation; recover a collection of functionalized particles with associated bound target contents from the sample processing box S325'; perform a nuclease exonuclease treatment operation S330' on a certain volume of cell-derived contents associated with the collection of functionalized particles; perform a chain denaturation and second chain synthesis operation S335' on a certain volume of cell-derived contents; perform a cDNA amplification operation S340' on a certain volume of cell-derived contents; perform an mRNA particle purification operation S345' on the PCR products of the cDNA amplification operation; and perform a run completion operation S360' upon completion of the mRNA particle purification operation.

[0179] In more detail, performing the run preparation operation S305' may include sub-steps associated with one or more of the following: preparing a cell suspension; initializing and performing operational checks of a system subsystem (e.g., associated with a platform, associated with a gantry, associated with a base, etc.); returning the gantry to an initial position; removing one or more seals from a reagent kit and / or loading reagents onto a reagent kit; positioning a sample processing cartridge unit; removing one or more seals from a tool container positioned at the platform; dispensing a cell suspension into a storage container prior to use; verifying the correct positioning and status (e.g., regarding expiration dates) of the disposable items of the protocol when scanning the labels of the disposable items with a camera (e.g., a machine vision camera); receiving sample identification information (e.g., from an operator); and starting to run a sample. The steps of S305" may be automatically implemented by the system and / or by an operator. In addition, a variety of sub-steps may be performed once, or may be repeated as recommended.

[0180] In more detail, triggering the system's sample processing box S310' upon completion of the run preparation operation, and jointly capturing a collection of cells of the sample and a collection of functionalized particles in the form of single cells at the sample processing box S315' may include one or more of the following: dispensing a triggering solution (e.g., in a manner that prevents bubbles from being trapped within the sample processing box) into an inlet reservoir of the sample processing box; incubating the triggering solution within the sample processing box; dispensing one or more cleaning solutions into the inlet reservoir of the sample processing box; transferring the solutions to a waste holding area of ​​the sample processing box; dispensing a cell suspension into the inlet reservoir of the sample processing box and capturing cells in the form of single cells within the wells of the sample processing box; dispensing a collection of functionalized particles into the inlet reservoir of the sample processing box and jointly capturing the collection of functionalized particles and the collection of cells; incubating the contents of the wells of the sample processing box; and a variety of tools involved in the pick / release sub-step (e.g., by a stand coupled to a pipette interface). Step S310' and step S315' are preferably automatically performed by the system, but may alternatively be performed in another suitable manner. In addition, various sub-steps may be performed once, or may be repeatedly performed as suggested.

[0181] In more detail, at the sample processing box, in the presence of functionalized particles (e.g., barcoded microspheres), performing a reverse transcription operation S320' on a collection of lysates of cells may include one or more of the following: dispensing one or more wash solutions into the inlet reservoir of the sample processing box; transferring the solution to a waste holding area of ​​the sample processing box; dispensing a particle binding buffer into the inlet reservoir of the sample processing box; dispensing a DTT solution into the inlet reservoir of the sample processing box; dispensing a lysis solution into the inlet reservoir of the sample processing box; replacing the fluid above the wells of the sample processing box with oil, thereby isolating the contents of the wells and preventing unwanted material from being transferred across the wells (e.g., as in U.S. application Ser. No. 16 / 564,375, filed on Sept. 9, 2019, which is incorporated herein by reference in its entirety); replacing the oil with air from the inlet reservoir; dispensing a particle binding wash buffer into the inlet reservoir of the sample processing box; dispensing a pre-RT reaction wash buffer into the inlet reservoir of the sample processing box; dispensing a cDNA synthesis solution (e.g., SuperScript IV TM ) into the inlet reservoir of the sample processing box; dispensing the RT mixture into the inlet reservoir of the sample processing box; incubating the contents of the sample processing box; performing the incubation step; and picking up / releasing the various tools involved in the sub-steps. Step S320' is preferably performed automatically by the system, but can be optionally performed in another suitable manner. In addition, the various sub-steps can be performed once, or can be repeatedly performed as suggested.

[0182] In more detail, recovering the set of functionalized particles with associated bound target content from the sample processing cartridge S325' may include performing a magnetic separation operation (eg, as described above) using manual or automated operations. In addition, multiple sub-steps may be performed once, or repeated as suggested.

[0183] In more detail, the exonuclease treatment operation S330' of a volume of cell-derived contents associated with the collection of functionalized particles may include one or more of the following: mixing water and exonuclease buffer to produce an exonuclease solution having a desired concentration; dispensing the exonuclease solution containing the functionalized particles into a first PCR container; dispensing oil (e.g., mineral oil) into the first PCR container; thermal cycling and incubating the contents of the first PCR container; extracting the product of the first PCR container; performing a separation operation on the product of the first PCR container; discarding waste from the separation operation; performing an incubation step; and a plurality of tools involved in the pick / release sub-steps. Step S330' is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or may be performed repeatedly as suggested.

[0184] In more detail, performing the strand denaturation and second strand synthesis operation S335' may include one or more of the following: transferring a hydroxide solution (e.g., a sodium hydroxide solution) to a second magnetic separation container; mixing the contents of the second magnetic separation container; actuating a magnetic separation subsystem (e.g., a collection of magnets coupled to an actuator, as described above) near the second magnetic separation container to separate functionalized magnetic particles toward the magnet; discarding waste material from the second magnetic separation container; dispensing a wash solution into the second magnetic separation container; mixing a second strand synthesis primer enzyme in a processing container near the second magnetic separation container; mixing the second strand synthesis primer enzyme with the contents of the second magnetic separation container; dispensing the product of the second magnetic separation container into a second PCR container; thermally cycling the contents of the second PCR container while mixing; transferring the product of the second PCR container to a third magnetic separation container; magnetically separating the product of the third magnetic separation container from waste and discarding the waste; transferring the wash solution to the third magnetic separation container; and a variety of tools involved in the pick-up / release substep. Step S335' is preferably performed automatically by the system, but may be optionally performed in another suitable manner. Furthermore, multiple sub-steps may be performed once, or may be performed repeatedly as suggested.

[0185] In more detail, performing the cDNA synthesis operation S340′ may include one or more of the following: mixing a polymerase mix (eg, Kapa Biosystems HiFi Hot Start Ready Mix) in a cold storage volume; TM); mixing the PCR master mix from the cold storage volume with the contents of the third magnetic separation container; dividing the contents of the third magnetic separation container equally into the third PCR operation container, the fourth PCR operation container, the fifth PCR operation container, and the sixth PCR operation container; dividing oil (e.g., mineral oil) equally into the third PCR operation container, the fourth PCR operation container, the fifth PCR operation container, and the sixth PCR operation container; running the third PCR operation; and picking up / releasing the various tools involved in the sub-steps. Step S340' is preferably performed automatically by the system, but can optionally be performed in another suitable manner. In addition, multiple sub-steps can be performed once, or repeatedly as suggested.

[0186] In more detail, the mRNA particle purification operation S345′ using the PCR product of the cDNA amplification operation may include one or more of the following: distributing the products from the third PCR operation container, the fourth PCR operation container, the fifth PCR operation container, and the sixth PCR operation container into the fourth magnetic separation container; TM ) and transfer to a fourth magnetic separation container; diluting and mixing ethanol with nuclease-free water in a tenth magnetic separation container; removing waste from the tenth magnetic separation container after incubation; mixing nuclease-free water with the target contents of the tenth magnetic separation container; magnetically separating the target mRNA-cDNA product from the tenth magnetic separation container; transferring the target mRNA-cDNA product from the tenth magnetic separation container to a cold storage device; and a plurality of tools involved in the pick-up / release substeps. Step S345' is preferably performed automatically by the system, but can be optionally performed in another suitable manner. In addition, multiple substeps can be performed once, or repeatedly as suggested.

[0187] In more detail, performing the run completion operation S360' upon completion of the mRNA particle purification operation may include one or more of the following: returning the rack to an initial configuration; providing notification that sample processing is complete; releasing the reagent kit and / or sample processing cartridge from the system for off-board storage; discarding system waste; performing a system cleaning operation; and converting the system to an inactive (e.g., idle, off) state. The steps of S360' may be automatically performed by the system and / or by an operator. In addition, multiple sub-steps may be performed once, or may be repeated as suggested.

[0188] Exemplary details of the steps of method 300' are further described in Table 1 of Appendix A. With respect to the steps of method 300', a description of ambient temperature and frozen reagents and locations associated with the storage volume of the embodiments of the test kit described above is described in Table 2 of Appendix A. With respect to Table 2 of Appendix A, the volume of reagents can be scaled based on the size of the sample processing chip used and / or the number of reaction runs. With respect to magnetic separation operations, a description of the equipment used and associated reagents and locations associated with the storage volume of the embodiments of the test kit described above is described in Table 3 of Appendix A. With respect to amplification (e.g., PCR) operations, a description of the equipment used and associated reagents and locations associated with the storage volume of the embodiments of the test kit described above is described in Table 4 of Appendix A. With respect to fluid aspiration and delivery operations, a description of the equipment and locations associated with the embodiments of the tool containers described above is described in Table 5 of Appendix A. With respect to actuation of components for protocol-related automation, gantry arm and pipette operations (e.g., with respect to devices coupled to disposable devices, devices detached from disposable devices, fluid mixing, waste discarding, aspiration, delivery, aliquoting, etc.) are described in Table 6 of Appendix A. With respect to transitions between modes of protocol-related automation, sample processing cartridge operations are described in Table 7 of Appendix A. With respect to transitions between modes of protocol-related automation, heating and cooling subsystem modes of operation are described in Table 8 of Appendix A. With respect to transitions between modes of protocol-related automation, operations of the magnetic separation subsystem are described in Table 9 of Appendix A. With respect to amplification operations, PCR program details associated with method 300' are described in Table 10 of Appendix A.

[0189] 3.2 Methods – Exemplary workflow for single-cell cytometry

[0190] like Fig.17As shown, a variation 300" of a method configured for single-cell cytometry may include: performing a run preparation operation, wherein the run preparation operation configures the system for performing a single-cell cytometry protocol S305"; upon completion of the run preparation operation, initiating a sample processing cartridge of the system S310"; capturing a collection of cells and a collection of functionalized particles of the sample in single-cell form at the sample processing cartridge S315"; lysing the collection of cells, and subsequently binding antibody-labeled oligonucleotides released from the lysed cells to functionalized particles (e.g., barcoded microspheres) S320" (e.g., wherein unbound material is washed and a reverse transcription operation is then performed); recovering the collection of functionalized particles having associated bound target contents from the sample processing cartridge S321" 5"; performing exonuclease treatment operation S330" on a certain volume of cell-derived contents associated with the collection of functionalized particles; performing chain denaturation and second chain synthesis operation S335" on the volume of cell-derived contents; performing cDNA amplification operation S340" on the volume of cell-derived contents; performing particle purification operation S345" on the PCR products of the cDNA amplification operation; performing a collection of antibody-derived tag purification and amplification operations S350" on the output of the particle purification operation; performing a collection of mRNA purification and amplification operations S355" on the output of the collection of antibody-derived tag purification and amplification operations; and performing a run completion operation S360" after completing the collection of mRNA purification and amplification operations.

[0191] In more detail, performing the run preparation operation S305" may include sub-steps associated with one or more of the following: preparing a cell suspension; initializing and performing operational checks of a system subsystem (e.g., associated with a platform, associated with a gantry, associated with a base, etc.); returning the gantry to an initial position; removing one or more seals from a reagent kit and / or loading reagents onto a reagent kit; positioning a sample processing cartridge unit; removing one or more seals from a tool container positioned at the platform; dispensing a cell suspension into a storage container before use; verifying the correct positioning and status (e.g., relative to an expiration date) of disposable items used for the protocol when scanning the label of the disposable item with a camera (e.g., a machine vision camera); receiving sample identification information (e.g., from an operator); and starting to run the sample. The steps of S305" may be automatically implemented by the system and / or by an operator. In addition, multiple sub-steps may be performed once, or may be repeated as recommended.

[0192] In more detail, triggering the system's sample processing box S310" upon completion of the run preparation operation and capturing a collection of cells of the sample and a collection of functionalized particles together in the form of single cells at the sample processing box S315" may include one or more of the following: dispensing a triggering solution into the inlet reservoir of the sample processing box; incubating the triggering solution within the sample processing box; dispensing one or more cleaning solutions into the inlet reservoir of the sample processing box; transferring the solution to the waste holding area of ​​the sample processing box; dispensing a cell suspension into the inlet reservoir of the sample processing box and capturing cells in the form of single cells within the wells of the sample processing box; dispensing a collection of functionalized particles into the inlet reservoir of the sample processing box and capturing the collection of functionalized particles and the collection of cells together; incubating the contents of the wells of the sample processing box; and a variety of tools involved in the pick-up / release sub-steps. Steps S310" and S315" are preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0193] In more detail, performing a reverse transcription operation S320" by lysing a collection of cells at the sample processing box may include one or more of the following: dispensing one or more cleaning solutions into the inlet reservoir of the sample processing box; transferring the solution to the waste holding area of ​​the sample processing box; dispensing a particle binding buffer into the inlet reservoir of the sample processing box; dispensing a DTT solution into the inlet reservoir of the sample processing box; dispensing a lysis solution into the inlet reservoir of the sample processing box; replacing the fluid above the well of the sample processing box with oil, thereby isolating the contents of the well and preventing undesirable material from being transferred across the well (e.g., as in U.S. application No. 16 / 564,375 filed on September 9, 2019, which is incorporated herein by reference in its entirety); replacing the oil with air from the inlet reservoir; dispensing a particle binding cleaning buffer into the inlet reservoir of the sample processing box; dispensing a pre-RT reaction wash buffer into the inlet reservoir of the sample processing box; dispensing a cDNA synthesis solution (e.g., SuperScript IV TM ) into the inlet reservoir of the sample processing box; dispensing the RT mixture into the inlet reservoir of the sample processing box; incubating the contents of the sample processing box; performing an incubation step; and picking up / releasing various tools involved in the sub-steps. Step S320" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps can be performed once, or repeatedly as recommended.

[0194] In more detail, recovering a collection of functionalized particles having contents with associated binding targets from the sample processing cartridge S325" may include performing a magnetic separation operation (e.g., as described above) using manual or automated operation. Additionally, multiple sub-steps may be performed once, or may be repeated as suggested.

[0195] In more detail, performing exonuclease treatment operation S330" on a volume of cell-derived contents associated with the collection of functionalized particles may include one or more of the following: mixing water and exonuclease buffer to produce an exonuclease solution having a desired concentration; dispensing the exonuclease solution containing the functionalized particles into a first PCR container; dispensing oil (e.g., mineral oil) into the first PCR container; thermal cycling and incubating the contents of the first PCR container; extracting the product of the first PCR container; performing a separation operation on the product of the first PCR container; discarding waste from the separation operation; performing an incubation step; and a variety of tools involved in picking up / releasing sub-steps. Step S330" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0196] In more detail, performing chain denaturation and second chain synthesis operation S335" may include one or more of the following: transferring a hydroxide solution (e.g., sodium hydroxide solution) to a second magnetic separation container; mixing the contents of the second magnetic separation container; actuating a magnetic separation subsystem near the second magnetic separation container (e.g., a collection of magnets coupled to an actuator, as described above) to separate functionalized magnetic particles toward the magnet; discarding waste material from the second magnetic separation container; dispensing a wash solution into the magnetic separation container; mixing a second chain synthesis primer enzyme in a processing container near the second magnetic separation container; mixing the second chain synthesis primer enzyme with the contents of the second magnetic separation container; dispensing the product of the second magnetic separation container into a second PCR container; thermally cycling the contents of the second PCR container while mixing; transferring the product of the second PCR container to a third magnetic separation container; magnetically separating the product of the third magnetic separation container from waste and discarding the waste; transferring the wash solution to the third magnetic separation container; and a variety of tools involved in the pick-up / release sub-step. Step S335" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. Furthermore, multiple sub-steps may be performed once, or may be performed repeatedly as suggested.

[0197] In more detail, performing cDNA synthesis operation S340" may include one or more of the following: mixing a polymerase mixture (e.g., Kapa Biosystems HiFi Hot Start Ready Mix) in a cold storage volume; TM); mixing the PCR master mix from the cold storage volume with the contents of the third magnetic separation container; dividing the contents of the third magnetic separation container equally into a third PCR operation container, a fourth PCR operation container, a fifth PCR operation container, and a sixth PCR operation container; dividing oil (e.g., mineral oil) equally into a third PCR operation container, a fourth PCR operation container, a fifth PCR operation container, and a sixth PCR operation container; running a first PCR operation; and picking up / releasing multiple tools involved in sub-steps. Step S340" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0198] In more detail, the particle purification operation S345" using the PCR product of the cDNA amplification operation may include one or more of the following: distributing the products from the third PCR operation container, the fourth PCR operation container, the fifth PCR operation container, and the sixth PCR operation container into the fourth magnetic separation container; separating the target contents of the fourth magnetic separation container and transferring the target contents to the fifth magnetic separation container; placing PCR purification particles (e.g., AMPure beads XP TM ) mixed and transferred to a fifth magnetic separation container; transferring the product of the fifth magnetic separation container to a sixth separation container; and a variety of tools involved in the pick-up / release sub-steps. Step S345" is preferably performed automatically by the system, but can optionally be performed in another suitable manner. In addition, multiple sub-steps can be performed once, or repeatedly as suggested.

[0199] In more detail, the set of antibody-derived tag purification and amplification operations S350'' that are performed on the output of the particle purification operation may include one or more of the following: TM ) and transfer to the sixth magnetic separation container; discard the waste from the sixth magnetic separation container; dilute the ethanol with nuclease-free water in the processing container and mix; transfer the ethanol to the sixth magnetic separation container; discard the waste from the sixth magnetic separation container while performing magnetic separation; transfer the target contents of the sixth magnetic separation container to the seventh magnetic separation container; place PCR purification particles (e.g., AMPure beads XP TM) into a seventh magnetic separation container; transferring ethanol to the seventh magnetic separation container; discarding waste from the seventh magnetic separation container when performing magnetic separation; dispensing water into the seventh magnetic separation container; transferring the purified cDNA from the seventh magnetic separation container to the seventh PCR operation container; and a variety of tools involved in the picking / releasing sub-steps. Step S350" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0200] In more detail, performing a set of mRNA purification and amplification operations S355"' using the output of the set of antibody-derived tag purification and amplification operations can include one or more of the following: transferring a PCR index primer to a seventh PCR operation container; transferring a polymerase blend to the seventh PCR operation container; mixing the contents of the seventh PCR operation container; transferring oil (e.g., mineral oil) to the seventh PCR operation container; initiating a second PCR operation; transferring the PCR product from the seventh PCR operation container to an eighth magnetic separation container; transferring PCR purification particles (e.g., AMPure beads XP TM ) is transferred to an eighth magnetic separation container; ethanol is transferred to the eighth magnetic separation container; waste from the eighth magnetic separation container is discarded when performing magnetic separation; water is dispensed into the eighth magnetic separation container; the purified cDNA is transferred from the eighth magnetic separation container to a storage container; and a variety of tools involved in the pick-up / release sub-steps. Step S355" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0201] In more detail, performing a run completion operation S360" after completing a set of mRNA purification and amplification operations may include one or more of the following: returning the rack to an initial configuration; providing notification that sample processing is complete; releasing a reagent kit and / or sample processing cartridge from the system for off-board storage; discarding system waste; performing a system cleaning operation; and transitioning the system to an inactive (e.g., idle, off) state. The steps of S360" may be automatically implemented by the system and / or by an operator. In addition, multiple sub-steps may be performed once, or may be repeated as recommended.

[0202] Exemplary details of the steps of method 300" are further described in Table 1 of Appendix B. With respect to the steps of method 300", descriptions of ambient temperature and frozen reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 2 of Appendix B. With respect to Table 2 of Appendix B, the volume of reagents can be scaled based on the size of the sample processing chip used and / or the number of reaction runs. With respect to magnetic separation operations, descriptions of the equipment used and associated reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 3 of Appendix B. With respect to amplification (e.g., PCR) operations, descriptions of the equipment used and associated reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 4 of Appendix B. With respect to fluid aspiration and delivery operations, descriptions of the equipment and locations associated with the embodiments of the tool containers described above are described in Table 5 of Appendix B. With respect to actuation of components for automation of protocol aspects, gantry arm and pipette operations (e.g., with respect to devices connected to disposable devices, devices separated from disposable devices, fluid mixing, waste disposal, aspiration, delivery, aliquoting, etc.) are described in Table 6 of Appendix B. With respect to transitions between modes of protocol aspects automation, sample processing box operations are described in Table 7 of Appendix B. With respect to transitions between modes of protocol aspects automation, heating and cooling subsystem operating modes are described in Table 8 of Appendix B. With respect to transitions between modes of protocol aspects automation, operation of the magnetic separation subsystem is described in Table 9 of Appendix B. With respect to amplification operations, PCR program details associated with Method 300" are described in Table 10 of Appendix B.

[0203] 3.3 Methods – Exemplary workflow for the CITE-Seq protocol (single-cell multi-omics)

[0204] like Fig.18As shown, a variation 300'" of a method configured for single-cell multi-omics may include: performing a run preparation operation, wherein the run preparation operation configures the system for performing a single-cell multi-omics protocol S305'"; upon completion of the run preparation operation, initiating a sample processing box of the system S310'"; capturing a collection of cells and a collection of functionalized particles of the sample together in the form of a single cell at the sample processing box S315'"; at the sample processing box, following lysis of the collection of cells, binding mRNA and antibody-labeled oligomers from the lysed cells to the collection of functionalized particles, wherein unbound material is washed and a reverse transcription reaction is subsequently performed S320'"; recovering a collection of functionalized particles having associated bound target contents from the sample processing box S325'"; and performing a reverse transcription reaction on the functionalized particles. A certain volume of cell-derived contents associated with the collection of particles is subjected to exonuclease treatment operation S330"'; a chain denaturation and second chain synthesis operation S335"' is performed on the volume of cell-derived contents; a cDNA amplification operation S340"' is performed on the volume of cell-derived contents; a particle purification operation S345"' is performed on the PCR product of the cDNA amplification operation; a collection of antibody-derived tag purification and amplification operations S350"' is performed on the output of the particle purification operation; a collection of purification and amplification operations S355"' is performed on the output of the collection of antibody-derived tag purification and amplification operations; a collection of mRNA particle purification and amplification operations S360"' is performed and after completing the collection of mRNA purification and amplification operations, a run completion operation S365"' is performed.

[0205] In more detail, performing the run preparation operation S305'" may include sub-steps associated with one or more of the following: preparing a cell suspension; initializing and performing operational checks of a system subsystem (e.g., associated with a platform, associated with a gantry, associated with a base, etc.); returning the gantry to an initial position; removing one or more seals from a reagent kit and / or loading reagents onto a reagent kit; positioning a sample processing cartridge unit; removing one or more seals from a tool container positioned at the platform; dispensing a cell suspension into a storage container before use; verifying the correct positioning and status (e.g., relative to an expiration date) of disposable items used for the protocol when scanning the label of the disposable item with a camera (e.g., a machine vision camera); receiving sample identification information (e.g., from an operator); and starting to run a sample. The steps of S305'" may be automatically performed by the system and / or by an operator. In addition, multiple sub-steps may be performed once, or may be repeated as recommended.

[0206] In more detail, triggering the system's sample processing box S310"' upon completion of the run preparation operation, and capturing a collection of cells of the sample and a collection of functionalized particles together in the form of single cells at the sample processing box S315"' may include one or more of the following: dispensing a triggering solution into the inlet reservoir of the sample processing box; incubating the triggering solution within the sample processing box; dispensing one or more cleaning solutions into the inlet reservoir of the sample processing box; transferring the solution to the waste holding area of ​​the sample processing box; dispensing a cell suspension into the inlet reservoir of the sample processing box and capturing cells in the form of single cells in the wells of the sample processing box; dispensing a collection of functionalized particles into the inlet reservoir of the sample processing box and capturing the collection of functionalized particles together with the collection of cells; incubating the contents of the wells of the sample processing box; and a variety of tools involved in the pick-up / release sub-steps. Steps S310"' and S315"' are preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0207] In more detail, performing a reverse transcription operation at the sample processing box by lysing the cell collection S320"' may include one or more of the following: dispensing one or more wash solutions into the inlet reservoir of the sample processing box; transferring the solution to the waste holding area of ​​the sample processing box; dispensing a particle binding buffer into the inlet reservoir of the sample processing box; dispensing a DTT solution into the inlet reservoir of the sample processing box; dispensing a lysis solution into the inlet reservoir of the sample processing box; displacing the fluid above the well of the sample processing box with oil, thereby isolating the contents of the well and preventing undesirable material from being transferred across the well (e.g., as in U.S. application No. 16 / 564,375 filed on September 9, 2019, which is incorporated herein by reference in its entirety); displacing the oil with air from the inlet reservoir; dispensing a particle binding wash buffer into the inlet reservoir of the sample processing box; dispensing a pre-RT reaction wash buffer into the inlet reservoir of the sample processing box; dispensing a cDNA synthesis solution (e.g., SuperScript IV TM ) into the inlet reservoir of the sample processing box; dispensing the RT mixture into the inlet reservoir of the sample processing box; incubating the contents of the sample processing box; performing the incubation step; and picking up / releasing the various tools involved in the sub-steps. Step S320 is preferably performed automatically by the system, but can be optionally performed in another suitable manner. In addition, multiple sub-steps can be performed once, or can be repeatedly performed as suggested.

[0208] In more detail, recovering a collection of functionalized particles having associated bound target content from the sample processing cartridge S325'" may include performing a magnetic separation operation (e.g., as described above) using manual or automated operation. Additionally, multiple sub-steps may be performed once, or may be repeated as suggested.

[0209] In more detail, performing an exonuclease treatment operation S330'" on a volume of cell-derived contents associated with the collection of functionalized particles may include one or more of the following: mixing water and an exonuclease buffer to produce an exonuclease solution having a desired concentration; dispensing the exonuclease solution containing the functionalized particles into a first PCR container; dispensing oil (e.g., mineral oil) into the first PCR container; thermal cycling and incubating the contents of the first PCR container; extracting the product of the first PCR container; performing a separation operation on the product of the first PCR container; discarding waste from the separation operation; performing an incubation step; and a variety of tools involved in picking up / releasing sub-steps. Step S330'" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0210] In more detail, performing chain denaturation and second chain synthesis operations S335'" may include one or more of the following: transferring a hydroxide solution (e.g., sodium hydroxide solution) to a second magnetic separation container; mixing the contents of the second magnetic separation container; actuating a magnetic separation subsystem near the second magnetic separation container (e.g., a collection of magnets coupled to an actuator, as described above) to separate functionalized magnetic particles toward the magnet; discarding waste material from the second magnetic separation container; dispensing a wash solution into the magnetic separation container; mixing a second chain synthesis primer enzyme in a processing container near the second magnetic separation container; mixing the second chain synthesis primer enzyme with the contents of the second magnetic separation container; dispensing the product of the second magnetic separation container into a second PCR container; thermally cycling the contents of the second PCR container while mixing; transferring the product of the second PCR container to a third magnetic separation container; magnetically separating the product of the third magnetic separation container from waste and discarding the waste; transferring the wash solution to the third magnetic separation container; and a variety of tools involved in the pick-up / release sub-steps. Step S335'" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. Furthermore, multiple sub-steps may be performed once, or may be performed repeatedly as suggested.

[0211] In more detail, performing cDNA synthesis operation S340" may include one or more of the following: mixing a polymerase mixture (e.g., Kapa Biosystems HiFi Hot Start Ready Mix) in a cold storage volume; TM); mixing the PCR master mix from the cold storage volume with the contents of the third magnetic separation container; dividing the contents of the third magnetic separation container equally into a third PCR operation container, a fourth PCR operation container, a fifth PCR operation container, and a sixth PCR operation container; dividing oil (e.g., mineral oil) equally into a third PCR operation container, a fourth PCR operation container, a fifth PCR operation container, and a sixth PCR operation container; running a first PCR operation; and picking up / releasing multiple tools involved in sub-steps. Step S340'' is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0212] In more detail, the particle purification operation S345'' of the PCR product of the cDNA amplification operation may include one or more of the following: distributing the products from the third PCR operation container, the fourth PCR operation container, the fifth PCR operation container, and the sixth PCR operation container into the fourth magnetic separation container; separating the target contents of the fourth magnetic separation container and transferring the target contents to the fifth magnetic separation container; placing PCR purification particles (e.g., AMPure beads XP TM ) mixed and transferred to a fifth magnetic separation container; transferring the product of the fifth magnetic separation container to a sixth separation container; and picking up / releasing various tools involved in the sub-steps. Step S345"' is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or may be repeatedly performed as suggested.

[0213] In more detail, the set of antibody-derived tag purification and amplification operations S350" performed on the output of the particle purification operation may include one or more of the following: PCR purified particles (e.g., AMPure beads XP TM ) and transfer to the sixth magnetic separation container; discard the waste from the sixth magnetic separation container; dilute and mix the ethanol with nuclease-free water in the processing container; transfer the ethanol to the sixth magnetic separation container; discard the waste from the sixth magnetic separation container while performing magnetic separation; transfer the target contents of the sixth magnetic separation container to the seventh magnetic separation container; place PCR purification particles (e.g., AMPure beads XP TM) into a seventh magnetic separation container; transferring ethanol to the seventh magnetic separation container; discarding waste from the seventh magnetic separation container when performing magnetic separation; dispensing water into the seventh magnetic separation container; transferring the purified cDNA from the seventh magnetic separation container to the seventh PCR operation container; and a variety of tools involved in the picking / releasing sub-steps. Step S350"' is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0214] In more detail, performing a set of purification and amplification operations S355" on the output of the set of antibody-derived tag purification and amplification operations may include one or more of the following: transferring a PCR index primer to a seventh PCR operation container; transferring a polymerase mixture to the seventh PCR operation container; mixing the contents of the seventh PCR operation container; transferring oil (e.g., mineral oil) to the seventh PCR operation container; initiating a second PCR operation; transferring the PCR product from the seventh PCR operation container to an eighth magnetic separation container; transferring PCR purification particles (e.g., AMPure beads XP TM ) is transferred to an eighth magnetic separation container; ethanol is transferred to the eighth magnetic separation container; waste from the eighth magnetic separation container is discarded when performing magnetic separation; water is dispensed into the eighth magnetic separation container; the purified cDNA is transferred from the eighth magnetic separation container to a storage container; and a variety of tools involved in the pick-up / release sub-steps. Step S355" is preferably performed automatically by the system, but may optionally be performed in another suitable manner. In addition, multiple sub-steps may be performed once, or repeatedly as suggested.

[0215] In more detail, the set S360"' of performing the mRNA particle purification and amplification operations may include one or more of the following: transferring nuclease-free water to a fifth magnetic separation container for mixing and incubation; transferring the product of the fifth magnetic separation container to a ninth magnetic separation container; transferring PCR purification particles (e.g., AMPure beads XP TM) and transfer to a ninth magnetic separation container; transfer ethanol to a ninth magnetic separation container; after incubation, remove waste from the ninth magnetic separation container; mix nuclease-free water with the target contents of the ninth magnetic separation container; mix and incubate the contents of the ninth magnetic separation container; transfer the purified cDNA product from the ninth magnetic separation container to an eighth PCR operation container; transfer the PCR master mix for mRNA amplification to an eighth PCR operation container; mix the contents of the eighth PCR operation container; add a polymerase mixture (e.g., Kapa Biosystems HiFi hot start ready mixture) is transferred to the eighth PCR operation container; the contents of the eighth PCR operation container are mixed; oil (such as mineral oil) is transferred to the eighth PCR operation container; a third PCR operation is performed in the eighth PCR operation container; the product of the third PCR operation is transferred from the eighth PCR operation container to the tenth magnetic separation container; the PCR purification particles are transferred to the tenth magnetic separation container; the contents of the tenth magnetic separation container are mixed; ethanol is transferred to the tenth magnetic separation container; waste from the tenth magnetic separation container is discarded; nuclease-free water is transferred to the tenth magnetic separation container; the contents of the tenth magnetic separation container are mixed and incubated; magnetically separate the target mRNA-cDNA product from the tenth magnetic separation container; transfer the target mRNA-cDNA product from the tenth magnetic separation container to a cold storage device; and a variety of tools involved in the pick-up / release sub-steps. Step S360"' is preferably performed automatically by the system, but can optionally be performed in another suitable manner. In addition, multiple sub-steps can be performed once, or repeatedly as suggested.

[0216] In more detail, performing a run completion operation S365'" after completing a set of mRNA purification and amplification operations may include one or more of the following: returning the rack to an initial configuration; providing notification that sample processing is complete; releasing a reagent kit and / or sample processing cartridge from the system for off-board storage; discarding system waste; performing a system cleaning operation; and transitioning the system to an inactive (e.g., idle, off) state. Step S365'" may be performed automatically by the system and / or by an operator. In addition, multiple sub-steps may be performed once, or may be repeated as recommended.

[0217] Exemplary details of the steps of method 300' are further described in Table 1 of Appendix C. With respect to the steps of method 300', descriptions of ambient temperature and frozen reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 2 of Appendix C. With respect to Table 2 of Appendix C, the volume of reagents can be scaled based on the size of the sample processing chip used and / or the number of reactions run. With respect to magnetic separation operations, descriptions of the equipment and associated reagents used and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 3 of Appendix C. With respect to amplification (e.g., PCR) operations, descriptions of the equipment and associated reagents used and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 4 of Appendix C. With respect to fluid aspiration and delivery operations, descriptions of the equipment and locations associated with the embodiments of the tool containers described above are described in Table 5 of Appendix C. With respect to actuation of components for protocol-related automation, operation of the gantry arms and pipettes (e.g., with respect to devices coupled to disposable devices, devices separated from disposable devices, fluid mixing, waste discarding, aspiration, delivery, aliquoting, etc.) is described in Table 6 of Appendix C. With respect to transitions between modes of protocol-related automation, operation of the sample processing cartridge is described in Table 7 of Appendix C. With respect to transitions between modes of protocol-related automation, the heating and cooling subsystem operating modes are described in Table 8 of Appendix C. With respect to transitions between modes of protocol-related automation, operation of the magnetic separation subsystem is described in Table 9 of Appendix C. With respect to amplification operations, PCR program details associated with Method 300 are described in Table 10 of Appendix C.

[0218] 3.4 Methods – Exemplary workflow for library preparation

[0219] like Fig.19 As shown, a variation 300"" of a method configured for library preparation may include: performing a run preparation operation, wherein the run preparation operation configures the system for executing a library preparation protocol S305""; performing a library preparation operation S310"" upon completion of the run preparation operation; performing a library purification operation S315"" on the output of the library preparation operation; and performing a run completion operation S360"" upon completion of the library purification operation.

[0220] In more detail, performing the run preparation operation S305"" may include sub-steps associated with one or more of the following: quantifying the concentration of the product (e.g., the DNA concentration of the product generated from the previous protocol; thawing the reagent kit for library preparation from a frozen storage state; processing the storage volume of the reagent kit (e.g., by vortexing, by centrifugation, etc.); diluting the sequencing adapter (e.g., NEBNext Illumina TMadapter) solution; return the sequencing adapter solution and previously removed storage to the reagent box; perform an operational check on the system subsystem (e.g., associated with the platform, associated with the stand, associated with the base, etc.); return the stand to the initial position; position the reagent box at the platform of the system; remove one or more seals from the reagent box and / or load reagents onto the reagent box; position the sample processing box unit at the platform of the system; remove one or more seals from the tool container positioned at the platform; receive an operator-loaded container (e.g., at the storage volume of the reagent box) for performing library preparation operations; initialize the heating and cooling subsystems (e.g., with an initial temperature set point); verify the correct positioning and status (e.g., relative to an expiration date) of disposable items used for the protocol when scanning the label of the disposable item with a camera (e.g., a machine vision camera); receive sample identification information (e.g., from an operator); and start running samples. The step of S305"" can be automatically implemented by the system and / or by an operator. In addition, multiple substeps can be performed once, or repeatedly as recommended.

[0221] In more detail, performing the library preparation operation S310"" upon completion of the run preparation operation may include sub-steps associated with one or more of the following: transferring the diluted cDNA from the operator-loaded tube to a first cold storage container containing a buffer; transferring the contents of the first cold storage tube to a second cold storage container; incubating the contents of the second cold storage container; transferring the cDNA mixture from the second environmental storage container to a first PCR operation container; fragmenting the contents of the first PCR operation container while performing a thermal cycling operation at the first PCR operation container; transferring the fragmented DNA from the first PCR operation container to a fourth cold storage container while mixing; storage container; in the case of mixing, transferring the contents of the fourth cold storage container to the fifth cold storage container; in the case of mixing and incubation, transferring the diluted adapter from the third cold storage container to the fifth cold storage container; in the case of incubation, transferring the contents of the fifth cold storage container to the second PCR operation container; in the case of mixing, transferring the contents of the second PCR operation container to the sixth cold storage container; in the case of incubation, transferring the contents of the sixth cold storage container to the second PCR operation container; transferring the contents of the sixth cold storage container to the second magnetic separation container; in the case of mixing, transferring PCR purification particles (e.g., AMPure beads XP TM) to a second magnetic separation container; discard waste from the second magnetic separation container; transfer ethanol to the second magnetic separation container; discard waste from the second magnetic separation container; in the case of incubation and magnetic separation, transfer TE buffer to the second magnetic separation container; transfer purified cDNA from the second magnetic separation container to a third PCR operation container; in the case of mixing, transfer the index PCR master mix to the third PCR operation container; perform a fourth PCR operation; perform a mixing step; perform an incubation step; and pick up / release the various tools involved in the sub-steps. Step S310"" is preferably performed automatically by the system, but can optionally be performed in another suitable manner. In addition, multiple sub-steps can be performed once, or repeatedly as suggested.

[0222] In more detail, performing a library purification operation S315"" on the output of the library preparation operation may include sub-steps associated with one or more of the following: in the case of mixing, transferring the product of the fourth PCR operation from the third PCR operation container to the third magnetic separation container; in the case of mixing, incubation and magnetic separation, transferring PCR purification particles (e.g., AMPureBeads XP TM ) to a third magnetic separation container; in the case of incubation, transfer ethanol to the third magnetic separation container; remove waste from the third magnetic separation container; in the case of mixing, incubation, and magnetic separation, transfer nuclease-free water to the third magnetic separation container; in the case of mixing, transfer the contents of the third magnetic separation container to a fourth magnetic separation container; in the case of mixing, incubation, and magnetic separation, transfer PCR purification particles (e.g., AMPure Beads XP TM ) is transferred to a fourth magnetic separation container; waste is removed from the fourth magnetic separation container; in the case of incubation, ethanol is transferred to the fourth magnetic separation container; waste from the fourth magnetic separation container is discarded; the steps for additional purification are repeated, wherein the target material is transferred from the fourth magnetic separation container to a fifth magnetic separation container, to a sixth magnetic separation container; in the case of incubation and magnetic separation, nuclease-free water is transferred to the sixth magnetic separation container; the purified cDNA for library construction is transferred to an eighth cold storage container; a mixing step is performed; an incubation step is performed; and the various tools involved in the pick / release sub-steps. Step S315"" is preferably performed automatically by the system, but can optionally be performed in another suitable manner. In addition, multiple sub-steps can be performed once, or repeatedly as suggested.

[0223] In more detail, performing a run completion operation S360""' upon completion of a library cleanup operation may include one or more of the following: returning the rack to an initial configuration; providing notification that sample processing is complete; releasing a reagent cartridge and / or sample processing cartridge from the system for off-board storage; discarding system waste; performing a system cleaning operation; and transitioning the system to an inactive (e.g., idle, off) state. Step S360"" may be performed automatically by the system and / or by an operator. Additionally, multiple sub-steps may be performed once, or may be repeated as recommended.

[0224] Exemplary details of the steps of method 300"" are further described in Table 1 of Appendix D. With respect to the steps of method 300"", descriptions of ambient temperature and frozen reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 2 of Appendix D. With respect to Table 2 of Appendix D, the volumes of reagents can be scaled based on the size of the sample processing chip used and / or the number of reaction runs. With respect to magnetic separation operations, descriptions of the equipment used and associated reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 3 of Appendix D. With respect to amplification (e.g., PCR) operations, descriptions of the equipment used and associated reagents and locations associated with the storage volume of the embodiments of the test kit described above are described in Table 4 of Appendix D. With respect to fluid aspiration and delivery operations, descriptions of the equipment and locations associated with the embodiments of the tool containers described above are described in Table 5 of Appendix D. With respect to actuation of components used for protocol-related automation, operation of the gantry arms and pipettes (e.g., with respect to devices coupled to disposable devices, devices separated from disposable devices, fluid mixing, waste discarding, aspiration, delivery, aliquoting, etc.) is described in Table 6 of Appendix D. With respect to transitions between modes of protocol-related automation, operation of the sample processing cartridge is described in Table 7 of Appendix D. With respect to transitions between modes of protocol-related automation, the heating and cooling subsystem operating modes are described in Table 8 of Appendix D. With respect to transitions between modes of protocol-related automation, operation of the magnetic separation subsystem is described in Table 9 of Appendix D. With respect to amplification operations, PCR program details associated with Method 300" are described in Table 10 of Appendix D.

[0225] However, embodiments of the system may be configured to implement other workflows, including variations of those described, and / or other workflows.

[0226] 4. Conclusion

[0227] The accompanying drawings show the architecture, function and operation of possible implementations of the system, method and computer program product according to preferred embodiments, exemplary configurations and variations thereof. In this regard, each block in the flow chart or block diagram can represent a part of a module, segment or code, which includes one or more executable instructions for realizing the specified logical function. It should also be noted that in some optional embodiments, the functions mentioned in the block can occur in an order other than the order indicated in the accompanying drawings. For example, the two blocks shown continuously can in fact be executed substantially simultaneously, or the blocks can sometimes be executed in reverse order, depending on the functions involved. It should also be noted that each square block in the block diagram and / or flow chart description and the combination of the square blocks in the block diagram and / or flow chart description can be implemented by a combination of a dedicated hardware-based system or dedicated hardware and computer instructions that performs a specified function or action.

[0228] As those skilled in the art will recognize from the previous detailed description and from the accompanying drawings and claims, modifications and changes may be made to the preferred embodiments of the invention without departing from the scope of the invention defined in the appended claims.

Claims

1. A system for automatically processing a sample, the system comprising: A platform configured to support and position: a reagent cartridge at a first region of a first side of the platform; a sample processing box at a second area of ​​the first side, wherein the sample processing box includes a cover covering an entry area into a microwell area of ​​the sample processing chip, the microwell area including a collection of microwells, the cover including an open mode and a closed mode, in which the entry area is uncovered and in which the entry area is covered; as well as a tool container at a third region of the first side; a stage coupled to the platform and comprising: a collection of tracks defining a collection of movement paths along axes for a pipette interface within a three-dimensional volume positioned above the first side of the platform; a decapping tool configured to be coupled to the pipette interface, the system further comprising a decapping mode in which the stage conveys the decapping tool for engagement with a release body of the lid of the sample processing cartridge to convert the lid from the closed mode to the open mode; and a base positioned below the platform and opposite the first side of the platform and supporting: a heating and cooling subsystem including a collection of thermal bodies for transferring heat to the reagent cartridge and the sample processing cartridge; a separation subsystem comprising a collection of magnets coupled to a magnet actuator, the magnet actuator configured to transition the collection of magnets between a retracted state and an extended state, wherein in the extended state the collection of magnets enters the first region of the platform; A pumping subsystem includes a vacuum port connecting the second region of the first side with a side of the platform opposite the first side.

2. The system of claim 1 , wherein the kit comprises a set of domains, the set of domains comprising a first domain and a second domain, the first domain being configured to cooperate with a first thermal body in the set of thermal bodies delivered to the first region of the first side of the platform and configured for material cooling, and the second domain being configured to match a second thermal body in the set of thermal bodies delivered to the first region of the first side of the platform and configured for thermal cycling.

3. The system of claim 1, wherein the sample processing cartridge comprises a base substrate coupled to a sample processing chip.

4. The system of claim 3, wherein the collection of thermal bodies includes a third thermal body that opens into the second region of the first side of the platform for heating the material at the collection of microwells.

5. The system of claim 3, wherein the sample processing box further comprises an elastomeric valve coupling the sample processing chip to the base substrate along a flow path from the microwell region to an outlet of the sample processing chip into a waste holding region of the sample processing box.

6. The system according to claim 5 further includes a valve actuation subsystem, wherein the valve actuation subsystem includes a valve actuator and a tip coupled to the valve actuator, wherein the tip is aligned with the valve opening leading to the second area of ​​the platform, wherein the valve actuation subsystem includes an engagement mode and a disengagement mode, wherein the tip is extended into the valve opening to deform the elastomeric valve, thereby closing the flow path, and the tip is retracted in the disengagement mode to open the flow path.

7. A system according to claim 1, wherein the platform includes a collection of retaining elements at the second area, the retaining elements being configured to reversibly engage the sample processing box, provide a reaction force against the decapping tool, and provide thermal contact with the thermal body in the collection of thermal bodies with a retaining force of at least 0.5 lbs.

8. The system of claim 1, wherein at least one of the collection of thermal bodies and the vacuum port is coupled to the sample processing cartridge to provide a reaction force against the decapping tool.

9. The system of claim 1, wherein in the closed mode, the cover and the access region define a boundary of a fluid path adjacent to the microwell region of the sample processing chip, the boundary being sealed by a gasket coupled to the cover.

10. The system of claim 1, wherein the separation subsystem further comprises a magnetic body configured to couple to the pipette interface at a first end and to couple to a magnetic sleeve of the tool container at a second end, the system further comprising a separation mode, in which the stage transfers the magnetic body coupled to the magnetic sleeve between the sample processing box and the reagent cartridge for magnetically separating target material from the sample.

11. The system of claim 1, wherein the collection of magnets of the separation subsystem comprises a first subset of magnets arranged in a linear array along a first axis for performing a purification operation at the reagent cartridge.

12. The system of claim 11, wherein the set of magnets of the separation subsystem further comprises a second magnet offset from the first axis for performing a target material separation operation at a separation reservoir of the reagent cartridge.

13. The system of claim 12, wherein the separation reservoir comprises a flat surface and a curved surface, wherein the flat surface is configured to be adjacent to the second magnet in the extended state of the set of magnets, and the curved surface is away from the second magnet in the extended state of the set of magnets.

14. The system of claim 1 , further comprising a processing and control subsystem supported between the platform and the base, the processing and control subsystem comprising non-transitory instructions stored in a computer-readable medium for performing automated sample processing operations, the automated sample processing operations comprising at least one of: controlling heat transfer between the heating and cooling subsystem, the reagent cartridge, and the sample processing cartridge; switching the set of magnets between the retracted state and the extended state; The pressure applied to the sample processing cartridge by the pumping subsystem is controlled through the vacuum port.

15. A system for automatically processing a sample, the system comprising: A platform configured to support and position: a reagent cartridge at a first region of a first side of the platform; and a sample processing box at a second area of ​​the first side, wherein the sample processing box includes a cover covering an entry area into a microwell area of ​​the sample processing chip, the microwell area including a collection of microwells, the cover including an open mode and a closed mode, in which the entry area is uncovered and in which the entry area is covered; a stage coupled to the platform and configured to hold a pipette, the stage comprising: a collection of tracks defining a collection of movement paths along axes for a pipette interface within a three-dimensional volume positioned above the first side of the platform; a decapping tool configured to be coupled to the pipette interface, the system further comprising a decapping mode in which the stage conveys the decapping tool for engagement with a release body of the lid of the sample processing cartridge to convert the lid from the closed mode to the open mode; and a base positioned below the platform and opposite the first side of the platform and supporting: A separation subsystem includes a collection of magnets coupled to a magnet actuator configured to transition the collection of magnets between a retracted state and an extended state, wherein in the extended state the collection of magnets enters the first region of the platform.

16. A system according to claim 15, wherein the separation subsystem further includes a magnetic body, which is configured to be coupled to the pipette interface at a first end and to the magnetic sleeve at a second end, and the system further includes a separation mode, in which the stage transfers the magnetic body coupled to the magnetic sleeve between the sample processing box and the reagent box for magnetically separating the target material from the sample.

17. A system according to claim 15, wherein the sample processing box includes a base substrate coupled to a sample processing chip, the sample processing chip includes a micropore area, the micropore area includes a collection of micropores configured to capture cells in single-cell form, and the sample processing box also includes an elastomeric valve, which couples the sample processing chip to the base substrate along a flow path from the micropore area to the outlet of the sample processing chip into a waste holding area of ​​the sample processing box.

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