Systems and methods for leak control in particle capture systems

Abstract

The invention relates to a system and method for leak control in a particle capture system. A system and method for target substance capture, the method comprising: receiving a set of target cells into an array of apertures defined at a surface plane of a substrate; receiving a set of particles into the array of pores, thereby co-capturing the set of target cells and the set of particles; upon receiving the wash fluid into the cavity in communication with the array of apertures, effecting a desired state of the array of apertures; receiving a lysis buffer into the chamber; receiving a separation fluid into the cavity, thereby displacing the lysis buffer from the cavity, and separating each hole in the array of holes from an adjacent hole at a surface plane; and retaining intracellular substances of the set of target cells individually together with the set of particles in the array of pores.

Description

[0001] This application is a divisional application of an application with an application date of March 16, 2020, application number 202080028946.2, and invention name “System and method for leakage control in a particle capture system”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 16 / 564,375, filed September 9, 2019, and U.S. Provisional Application No. 62 / 834,824, filed April 16, 2019, both of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present invention relates generally to the field of cell capture and cell processing, and more particularly to new and useful methods for leakage control within the field of cell capture and cell processing. background

[0004] As interest in cell-specific drug testing, diagnostics, and other assays increases, systems and methods that allow for the isolation, barcoding, identification, and retrieval of single cells have become highly desirable. Single-cell capture systems and methods have been shown to be particularly advantageous for these applications. However, drifting of biological target material (e.g., material derived from cells after cell lysis, cell-free nucleic acid material, etc.) can be particularly problematic in these systems and methods, resulting in nonspecific capture of target material (e.g., messenger ribonucleic acid [mRNA], proteins, etc.), and / or leakage of target material between chambers or partitions of the capture device.

[0005] Therefore, there is a need in the art of cell capture and cell processing to create new and useful systems and methods for capturing and / or processing cells while simultaneously controlling leakage. Summary of the invention

[0006] This application provides the following items: 1. A method, comprising: receiving a group of target cells in single cell form into an array of wells defined at a surface plane of a substrate, wherein each well in the array of wells extends perpendicular to and into the surface plane and into the substrate; receiving a set of particles into the array of wells, thereby co-capturing the set of target cells and the set of particles across the array of wells; achieving a desired state of the well array after receiving a wash fluid into a chamber in communication with the well array and transferring excess particles in the set of particles out of the well array; providing a lysis environment for the group of cells after receiving a lysis buffer into the chamber; separating each well in the array of wells from adjacent wells in the array of wells at the surface plane after displacing the lysis buffer from the cavity with the gas volume; preventing evaporation of the contents of the array of wells after receiving the partitioning fluid into the chamber; and Intercellular matter of the population of target cells is retained within the array of wells, wherein heat is transferred into the array of wells.

[0007] 2. A method according to item 1, wherein the substrate comprises: a short axis, a long axis and a fluid channel network, wherein the fluid channel network defines one or more flow paths that are preferentially aligned with the short axis and coupled to the cavity, wherein receiving one or more of the lysis buffer and the separation fluid comprises receiving one or more of the lysis buffer and the separation fluid into the fluid channel network.

[0008] 3. A method according to item 1, wherein each of the set of holes includes a width configured to prevent a target cell in the set of target cells from being positioned laterally adjacent to a particle in the set of particles, wherein receiving the set of particles and the set of target cells includes maintaining the set of particles closer to the surface plane than the set of target cells.

[0009] 4. The method according to item 1, wherein receiving the lysis buffer comprises receiving the lysis buffer at a temperature between 1°C and 15°C.

[0010] 5. A method according to item 4, wherein receiving the lysis buffer includes retaining the lysis buffer in the cavity for a residence time of less than 40 seconds.

[0011] 6. The method according to item 1, wherein retaining intracellular substances comprises retaining mRNA substances of the group of target cells that are individually hybridized with the group of particles within the well array.

[0012] 7. The method of claim 1, wherein each of the plurality of particles comprises a polystyrene coating and a unique biopolymer composite barcode.

[0013] 8. A method according to item 1, wherein receiving the group of cells includes receiving the group of cells at a first concentration, the first concentration is configured to be less than 50% occupancy of the hole array, thereby facilitating capture of the group of cells in single cell form, and wherein receiving the group of particles includes receiving the group of particles at a second concentration higher than the first concentration.

[0014] 9. A method according to item 1, wherein the cross-section of each hole in the hole array defines a polygon, and wherein each hole in the hole array includes a depth greater than the sum of the particle diameter of the group of particles and the cell diameter of the group of target cells.

[0015] 10. A method according to claim 1, wherein the separation fluid comprises oil.

[0016] 11. A method, comprising: receiving a group of target cells in single cell form into an array of wells defined at a plane of a substrate surface; receiving a set of particles into the array of wells, thereby co-capturing the set of target cells and the set of particles across the array of wells; achieving a desired state of the well array after receiving a wash fluid into a chamber in communication with the well array and transferring excess particles in the set of particles out of the well array; providing a lysis environment for the group of cells after receiving a lysis buffer into the chamber; receiving a separation fluid into the cavity, thereby displacing the lysis buffer from the cavity and separating each well in the array of wells from adjacent wells in the array of wells at the surface plane; and The intercellular material of the group of target cells is retained within the well array separately with the group of particles, thereby mitigating leakage of intracellular material of a first well of the well array to a second well of the well array.

[0017] 12. A method according to item 11, wherein the substrate comprises: a fluid channel network, the fluid channel network defining one or more flow paths preferentially aligned with the short axis of the substrate and coupled to the cavity, wherein receiving one or more of the lysis buffer and the separation fluid comprises receiving one or more of the lysis buffer and the separation fluid into the fluid channel network.

[0018] 13. The method of claim 11, wherein receiving the group of particles and the group of target cells comprises maintaining the group of particles closer to the surface plane than the group of target cells.

[0019] 14. The method according to item 11, wherein receiving at least one of the group of target cells, the group of particles, the washing fluid, the lysis buffer and the separation fluid includes receiving through a laminar flow process.

[0020] 15. The method according to item 11, wherein the separation fluid comprises at least one of air and oil.

[0021] 16. The method according to item 11, wherein receiving the lysis buffer comprises receiving the lysis buffer at a temperature between 1°C and 15°C.

[0022] 17. The method according to item 11, wherein the lysis buffer is pre-cooled to below 15°C before entering the cavity above the well array.

[0023] 18. A method according to item 11, wherein receiving the lysis buffer includes retaining the lysis buffer in the cavity for a residence time of less than 40 seconds.

[0024] 19. The method of clause 11, wherein retaining intracellular material comprises transferring heat through the substrate and into the well array, generating a well temperature within the well array of between 15°C and 35°C.

[0025] 20. The method according to item 11, wherein retaining intracellular substances comprises retaining mRNA substances of the group of target cells that are individually hybridized with the oligonucleotide probes coupled to the group of particles within the well array.

[0026] 21. The method according to item 20 further comprises flowing a wash buffer over the hole array at a flow rate greater than 50 μL / sec after the mRNA substance is hybridized with the set of particles, wherein the wash buffer is configured to remove unbound RNA.

[0027] 22. The method according to item 20 further comprises performing a reverse transcription process using the mRNA material retained in the hole array. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a schematic representation of an embodiment of a system for single cell capture and processing with leakage control.

[0028] Figure 1B is a schematic representation of an embodiment of a system for single cell capture and processing with leakage control.

[0029] Figure 2A-2C Variations of components of a system for single cell capture and processing with leakage control are depicted.

[0030] Figure 3 Specific examples of components of a system for single cell capture and processing with leakage control are depicted.

[0031] Figure 4 is a schematic representation of an embodiment of a method for single cell capture and processing with leakage control.

[0032] Figure 5A-5HVariations of the method for single cell capture and processing with leak control are depicted.

[0033] Figure 6A-6G Variations of systems and methods for single cell capture and processing with leakage control are depicted. Fig.6D In the present invention, a cold (e.g., between 2°C and 10°C) lysis buffer (e.g., having a predetermined mixture and concentration) is pumped over the microwells for a predetermined residence time (e.g., less than 30 minutes). The residence time and the lysis concentration are important. The amount of lysis reagent delivered to the microwell is proportional to the concentration of the lysis reagent and the residence time. The residence time is optimized so that enough lysis reagent is delivered to the microwell, but not enough time to cause cell lysis, release mRNA, and cause it to diffuse out of the microwell. The diffusion time of the mRNA can optionally be slowed by lowering the temperature and / or deepening the wells. In the present invention, a cold (e.g., between 2°C and 10°C) lysis buffer (e.g., having a predetermined mixture and concentration) is pumped over the microwells for a predetermined residence time (e.g., less than 30 minutes). The residence time and the lysis concentration are important. The amount of lysis reagent delivered to the microwell is proportional to the concentration of the lysis reagent and the residence time. The residence time is optimized so that enough lysis reagent is delivered to the microwell, but not enough time to cause cell lysis, release mRNA, and cause it to diffuse out of the microwell. The diffusion time of the mRNA can be optionally slowed by lowering the temperature and / or deepening the wells. Fig. 6E In the present invention, after lysis, the buffer is retained above the microwells for a predetermined amount of time (e.g., between 5 seconds and 60 seconds), and air is pumped over the microwells to remove the lysis buffer and separate the microwells from each other. Fig. 6F In the process, oil is pumped over the micropores to separate them from each other. The oil helps prevent Fig. 6E During the incubation time (e.g., long incubation time) described in , the liquid evaporates from the microwells. Figure 6G In the process, the temperature of the microwell is raised (e.g., to 26°C) and maintained (e.g., between 15 and 30 minutes). The cells are lysed, thereby releasing the mRNA. The mRNA begins to bind to the oligonucleotide fragments of the beads through the interaction of poly A (present on the mRNA) and poly T (present on the beads). The oil partition prevents the mRNA from one well from escaping to the adjacent well.

[0034] Figure 7A-7B Variations of system and method configurations for single cell capture and processing with leakage control are depicted. DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] 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.

[0036] 1. System like Figure 1AAs shown, an embodiment of a system for single cell capture and processing includes a slide 10 defining a set of wells 20 configured to receive any or all of: a set of cells 31, a set of particles 32, and any other suitable substance (e.g., reagents). In addition or alternatively, the system may include any or all of the system components described in: 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 / 821,329, filed on November 22, 2017; U.S. application No. 15 / 782,270, filed on October 12, 2017; U.S. application No. 16 / 049,240, filed on July 30, 2018; and U.S. application No. 16 / 115,370, filed on August 28, 2018, each of which is incorporated by reference in its entirety.

[0037] In more detail, about Figure 1B In the illustrated embodiment, a system 100 for separating and analyzing a group of cells includes: a substrate 110 having a wide surface; a well array 120 defined on a first side 112 (e.g., an upper wide surface) of the substrate, each well 128 in the well array 120 including an open surface 122 defined at the first side 112, a bottom surface 124 defined within the substrate near a second side 114 (e.g., a lower wide surface) directly opposite the first side 112, and a set of walls 126 extending between the bottom surface 124 and the open surface 122 to form a well cavity of the well 128. The well array 120 can be arranged in an active region of the substrate or in any other suitable arrangement. To facilitate delivery of a sample or fluid to the well array 120, the system 100 can also include a fluid delivery module 140, which is configured to couple with the substrate 110 and transfer a sample containing a group of cells and / or another fluid to the well array 120. Additionally or alternatively, the system 100 may include a flow control subsystem (e.g., a pressure pump system) 180 configured to control the flow of a fluid (e.g., a biological sample, a treatment reagent, a solution containing non-cellular particles) through the system 100 (e.g., the direction, velocity, volume of the fluid), and any other suitable flow through the system that requires control and / or actuation. Additionally or alternatively, the system 100 may include a thermal control module 190 for controlling the temperature of portions of the system 100. Additionally or alternatively, system100 may include an imaging subsystem 194 configured to perform optical imaging, illumination, or irradiation of the contents of the well array 120, and to enable identification, localization, and quantification of cells retained by the wells of the well array 120. Additionally or alternatively, the system 100 may include an extraction module that may extract one or more of: target cells, particles, cell-particle pairs, genetic complexes, and / or genetic products from the well array. However, variations of system 100 may include any other suitable components in any suitable configuration and / or combination, as described in U.S. application Ser. No. 15 / 333,420, entitled “Cell Capture System and Method of Use,” filed on Oct. 25, 2016, U.S. application Ser. No. 14 / 208,298, entitled “System for capturing and analyzing cells,” filed on Mar. 13, 2014, and U.S. application Ser. No. 14 / 289,155, entitled “System and Method for Isolating and Analyzing Cells,” filed on May 28, 2014, each of which is incorporated by reference in its entirety.

[0038] The described system embodiments function to prevent non-specific capture of target material (e.g., messenger RNA [mRNA], protein, etc.), and / or leakage of target material between the pores of the system to facilitate the performance of downstream sample processing steps. The described system embodiments are configured to perform the steps of the method embodiments described in Section 2 below, and / or any other suitable method.

[0039] The described system embodiments also function to separate, capture, retain and analyze cells of a cell population in at least one of single cell form and single cluster form at known addressable locations, and further facilitate the performance of multiple single cell assays that can be performed on individual target cells (e.g., rare cells in a biological sample) or cell clusters (e.g., doublets, triplets). In a preferred embodiment, the system 100 functions to facilitate the preparation of a genetic library of captured single cells (e.g., cDNA generated from captured mRNA from lysed target cells, amplified cDNA, amplified DNA) for sequencing proximately following cell capture and within the same device. After the cells are captured at a defined location determined by the single cell capture well, the fluid delivery module of the system 100 can be used to simultaneously, sequentially and / or repeatedly provide and deliver reagents to enable various cellular, subcellular or molecular reactions to be performed in each single cell / cell cluster. Additionally or alternatively, the system 100 can function to capture and process non-cellular particles (e.g., nucleic acid materials, other biological materials, other non-biological materials, particles containing molecular probes or reagents, etc.) and combinations of single cells and single non-cellular particles (e.g., in co-captured cell-particle pairs within separate microwells). In addition, the system 100 can implement controlled and rapid thermal regulation of the well array and the fluids that are additionally or alternatively delivered to the well array (e.g., heating and cooling cycles from 95°C to 1°C) to maintain cell or biological material vitality, improve the efficiency of biological assays, and perform various biochemical processes within a set of wells. The system 100 can also allow optical interrogation and detection of events on each captured cell at the single cell / cluster level. The system 100 can additionally or alternatively implement the selective release and / or selective removal of one or more captured cells or non-cellular particles for further processing and analysis. For example, system 100 enables co-capture of a single cell and particle within a single well, release of intracellular contents (e.g., mRNA, etc.) from the cell via a lysis step, binding of the intracellular contents to barcoded probes of the particles, execution of a reverse transcription (RT) reaction within the well, and selective retrieval of target-amplified contents associated with the intracellular contents from the well.

[0040] In some embodiments, system 100 can confer the benefits of real-time cell tracking, live cell and / or other biological material retrieval, biochemical processes (e.g., cell lysis, cell fixation, polymerase chain reaction, reverse transcription, etc.) and selective molecular analysis (e.g., electrophoresis, sequencing, fluorescence imaging) in the same microfluidic chip or off-chip. In some embodiments, system 100 can be used to capture circulating tumor cells (CTCs) and subpopulations of CTCs, such as circulating stem cells (CSCs), but can be used in addition or alternatively to capture any other suitable cells (e.g., erythrocytes, monocytes, macrophages, osteoclasts, dendritic cells, microglia, T cells, B cells, megakaryocytes, germ cells, nurse cells, neural cells, stem cells, etc.) or biological materials that may be of interest. System 100 is preferably defined on substrate 110, more preferably on a microfluidic chip, but can be optionally located on or defined by any suitable substrate.

[0041] In a particular example, the system 100 can be used with one or more methods operable for single cell molecular reactions, wherein such a system can facilitate efficient capture of cells (e.g., 100 cells, 1000 cells, 10,000 cells, 100,000 cells, 1,000,000 cells, etc.) in a single cell form (or single cluster form) within a well and on-chip delivery of reagents to the well, culturing, and thermal cycling to provide a cell capture to molecular reaction workflow. In more detail, the microfluidics and other portions of the system can be operable to perform assays (e.g., assays associated with ARV7 mRNA) using polymerase chain reaction (PCR) with samples (e.g., prostate clinical samples) having single cell or single cell cluster resolution. In a particular example, system 100 can accommodate sample volumes on the order of as low as 10 μl up to 1 mL within fluid reservoir 160 associated with well array 120, where the sample can contain between 500 and 100,000 target cells, thereby providing the ability to process larger sample volumes containing large numbers of cells of interest.

[0042] The system can include and / or be configured to interface with any number or type of cells. The cell can include any or all mammalian cells (e.g., human cells, mouse cells, etc.), embryos, stem cells, plant cells, or any other suitable types of cells. The cell preferably includes a target substance (e.g., a target lysate), which derives from within the cell and is finally released and captured by the cell capture system, but can additionally or alternatively include any other substance in one or more cells or on the cell. In some variations, the target cell substance includes mRNA. Additionally or alternatively, the target cell substance can include any suitable RNA, DNA, protein, or other substances.

[0043] The system may additionally include and / or be configured to engage with a group of particles, each of which is preferably functionalized with a group of probes configured to bind to a target substance of a cell. The probe preferably includes nucleotides (e.g., polynucleotides, oligonucleotides, etc.); in addition or alternatively, the particle may be functionalized, unfunctionalized, or exist in any other suitable form in other ways. As described below, the diameter of the particle is preferably substantially similar to (e.g., equal to) the hole diameter and / or the cell diameter. The diameter may be between 10 microns and 30 microns (e.g., 20 microns) so that only a single particle can enter each hole containing a cell. Alternatively, the size of the particle diameter is set so that only a single particle can enter any hole, multiple particles can enter each hole, and the particle can have any suitable diameter (e.g., less than 10 microns, greater than 30 microns, variable, etc.). In a related embodiment, the hole is configured to capture a single cell and a single particle together along (or mainly along) the longitudinal axis of the hole; however, the hole can be configured in any other suitable manner.

[0044] The particles are preferably in the form of beads, and are also preferably in the form of microspheres, but the particles may additionally or alternatively include any other material in any suitable form. The particles may have any suitable set of properties, such as any suitable physical properties (e.g., non-swelling behavior, solubility, etc.), magnetic properties, optical properties, surface properties (e.g., porous, non-porous, pores of a specific size range, etc.), chemical properties (e.g., biocompatibility, surface functional groups, binding affinity, etc.), thermal properties, or other properties. In some variations, the particles are barcoded with unique barcodes (e.g., using biopolymer complexes, functionalized, or otherwise treated). The particles may include any one or all of the following: polystyrene, glass, PMMA, polymers with a magnetic core, polymers with a hydrogel surface, and / or a combination of one or more other suitable materials. In a specific example, the particles include monodisperse (+ / - 10% Cv) 20 micron high-density biobeads (HDBB).

[0045] The density of the particles is preferably greater than most or all of the buffer solution (e.g., an initial buffer containing a solution, a solvent, etc.) and / or the flow solution used during method 100, so that the particles are configured to enter and stay in separate micropores. Alternatively, the particles can have a density less than one or more solutions, a variable density, or any other suitable density. In one variation, the particles include a set of HDBB particles with an optimal joint density, which is configured to maximize the binding between HDBB and mRNA released from lysed cells. In another variation, the microspheres include glass beads with a polystyrene coating and a diameter of about 20 microns (e.g., between 15 and 30 microns). However, system 100 can additionally or alternatively operate with any other suitable particles.

[0046] Individual cell and / or particle capture is preferably achieved by flowing or dispensing a sample containing a group of single cells in a fluid layer on the well array 120 in a direction parallel to the wide surface of the substrate (e.g., substantially parallel to the wide surface of the substrate, within 0.1 degrees parallel to the wide surface of the substrate, within 1 degree parallel to the wide surface of the substrate, within 45 degrees parallel to the wide surface of the substrate, completely parallel to the wide surface of the substrate, etc.), and capturing the cells after the cells fall through the fluid layer toward the well array 120 under the influence of gravity. Alternatively, individual cell capture may be achieved by delivering a sample containing a group of single cells into a fluid layer provided by a fluid reservoir 160 on the well array 120 in a direction perpendicular to the wide surface of the substrate, and capturing the cells after the cells fall through the fluid layer toward the well array 120 under the influence of gravity. However, in some variations, individual cell capture may additionally or alternatively be achieved by any suitable mechanism to transfer a single cell into a well in a group of wells. In addition, the system 100 is preferably configured to prevent undesirable fluid flow, which can lift cells from the substrate or move cells / cell clusters out of the pores 128 in which the cells are captured and completely retained. However, in some variations, the system 100 can be configured to facilitate moving cells / cell clusters in any suitable manner. The flow path of the fluid (e.g., biological sample, treatment reagent) through the system 100 is preferably multi-directional and uniform, so that each cell / cell cluster in the system 100 experiences consistent conditions (e.g., the gradient length scale of the flow properties (such as pressure, density, temperature, solution composition and other suitable properties) along the flow path is large relative to the length scale of the system); however, the flow path can optionally be unidirectional, bidirectional or have any other suitable characteristics.

[0047] In a specific example variation, such as Figure 2A-2C As shown, the flow path 141 of the fluid through the system includes a set of fluid paths 146 (e.g., coupled to a manifold of an array of wells) of equal length (e.g., substantially equal length, equal length within a manufacturability tolerance, etc.), which are configured such that a reagent supplied to the set of fluid paths 146 at a manifold inlet 440 reaches each array of inlets 444 (e.g., a single well, an area along a first edge of a reservoir, an area along a first edge of an active area of ​​a substrate, etc.) at substantially the same point in time (e.g., simultaneously, within 1 second, within 1 minute, etc.), and passes through the active area 116 of the substrate (e.g., including the array of wells 120) through the array of outlets 445 to the manifold outlet 442. Cell transport, separation, sorting, and viability maintenance can also be accomplished by controlling the sample flow through the system (e.g., by adjusting the flow so that the characteristic length scale of the flow is of similar order of magnitude to the characteristic length scale of the wells, by dithering the flow between high and low flow conditions, etc.) or by any other suitable means. However, the flow characteristics of the fluid through the system 100 may be configured in other ways.

[0048] In operation, the system 100 preferably receives a biological sample including a target cell population and facilitates uniform distribution of the biological sample across the well array 120 (e.g., using uniform cross flow, smearing, a cytospin procedure, pipetting aliquots of the sample at different areas of the array, etc.). However, the system 100 may additionally or alternatively facilitate the use of positive pressure (e.g., positive pressure at the inlet of the array) and / or negative pressure (e.g., negative pressure at the outlet of the array) applied by the flow control subsystem 180 to distribute the fluid (e.g., biological sample, treatment reagents, non-cellular particles) across a set of wells. Additionally or alternatively, the actuation pressure that facilitates sample distribution may be cycled in a pulse width modulated or sinusoidal manner to provide a net actuation pressure that is net positive at the inlet or net negative at the outlet. Thus, as the biological sample flows across the well array 120, desired cells having defined characteristics (e.g., size-based characteristics, density-based characteristics, adhesion-based characteristics, etc.) may be trapped within the wells 128. For example, in a variation of the system 100 configured to capture CTCs, the holes are preferably configured based on the morphological features that define the CTC cells, so as to facilitate the capture and retention of CTCs in the form of single cells or single clusters. However, the system 100 may additionally or alternatively be configured to retain and facilitate the processing of any other suitable particles of interest in any other suitable form. The actuation pressure is preferably provided by a flow control subsystem 180 (e.g., a manually operated pipette, an automated fluid handling robot, a vacuum pressure system, an electromechanical micropump, etc.) that is fluidically connected to the system 100, but may alternatively or additionally be provided by any suitable mechanism.

[0049] In a variation, the system 100 receives a cell population at a first density and / or total number of cells, which facilitates capturing the cell population as a single cell at the well array 120 (e.g., the density / total number is less than the number of wells in the well array 120). In this variation, the system 100 also receives a particle population at a second density higher than the first density and / or total number of particles, which facilitates co-capture of the cell population with the particle population. However, in other variations, the well array 120 can receive cells and / or particles in any other suitable manner. System components are described in more detail in the following sections.

[0050] 1.1 System-Baseboard like Figure 1B As shown, the substrate 110 functions to provide a medium, and the hole array 120 (group of micropores, micropores, holes) can be defined at the medium. In a variation, the substrate 110 can have a first side (e.g., an upper wide surface) 112 and a second side (e.g., a lower wide surface) directly opposite the first side. The upper wide surface 112 of the substrate 110 is preferably a flat surface, so that the microfluidic elements of the system 100 (e.g., inlet, outlet, inlet manifold, outlet manifold, fluid channel, etc.) are at least partially defined at the flat surface. Optionally, the upper wide surface 112 of the substrate 110 can be a non-flat surface, so that the microfluidic elements of the system 100 are at least partially defined at the non-flat surface. In a variation, the non-flat surface can be a concave surface, a convex surface, or a surface having a concave, flat and / or convex surface. Such a variation can facilitate various methods of depositing and distributing samples at the hole array 120. In any variation of the substrate 110 including a non-planar upper wide surface 112, one or more non-planar portions are preferably shallow (e.g., having a small depth relative to the width of the wide surface) or short (e.g., having a small height relative to the width of the wide surface); however, one or more non-planar portions may additionally or alternatively include deep (e.g., having a large depth relative to the width of the wide surface) or tall (e.g., having a large height relative to the width of the wide surface) portions. However, the surface may alternatively have any other suitable axis or type of symmetry, or may be asymmetric.

[0051] The composition of the substrate 110 can provide desired characteristics related to any one or more of the following: mechanical characteristics (e.g., mechanical properties of the substrate as a mechanical stimulus), optical properties (e.g., transparency), electrical properties (e.g., conductivity), thermal properties (e.g., thermal conductivity, specific heat, etc.), physical characteristics (e.g., wettability, porosity, etc.), and any other suitable characteristics. The substrate 110 is preferably composed of a rigid material (e.g., transparent material, translucent material) with high transparency to facilitate imaging of the substrate 110 to analyze captured single cells / cell clusters. The high transparency material is preferably optically transparent, but may additionally or alternatively be transparent and / or translucent to other parts of the electromagnetic spectrum (e.g., microwave, near infrared, ultraviolet, etc.). In several such variations, substrate 110 may be composed of any one or more of glass, fused quartz, ceramics, silicone-based materials (e.g., polydimethylsiloxane (PDMS)), polymers (e.g., agarose, polyacrylamide, polystyrene, polycarbonate, polymethyl methacrylate (PMMA), polyethylene glycol, etc.), paper, porous materials, and any other suitable material with high transparency, including composites thereof. Alternatively, substrate 110 may be composed of any other suitable material having any other suitable optical properties. Additionally or alternatively, the substrate may be composed of any one or more of ceramic materials, semiconductor materials, polymers, and any other suitable materials.

[0052] The substrate 110 can be processed using any one or more of the following methods: etching methods, molding methods, printing methods (e.g., 3D printing processes), machining methods, and any other suitable manufacturing processes suitable for brittle, elastic, or ductile substrate materials. In addition, the features defined at the upper wide surface 112, including the hole array, can be produced by any one or more of the following processes: molding, by polishing, by spinning the material in a mobile phase and then solidifying the material, by machining, by printing (e.g., 3D printing), by etching, and by any other suitable process. In a specific example, a photolithography process and a deep reactive ion etching (DRIE) process are used to etch microfluidic elements into a silicon mold to define the hole array 120 in a silicon mold. In a specific example, the etched elements of the silicon mold are then transferred to a polymethyl methacrylate (PMMA) sheet as the substrate 110 using a hot embossing process. The substrate 110 in the specific example has a size of 3 inches x 1 inch to substantially match the size of a glass microscope slide. In variations of the particular example and / or for other variations of the well array 120, hot embossing of cyclic olefin polymer (COP) may replace PMMA to form the microfluidic structures of the well array 120. However, the substrate 110 may alternatively be any other suitable substrate 120 processed in any other suitable manner.

[0053] Preferably, the substrate includes features that allow for interaction (e.g., reversible or irreversible connection, coupling) with other subcomponents of the system 100. In one variation, the substrate 110 can be coupled to a component fluid delivery module 140, wherein the substrate includes one or more inlets and outlets (an example of which is shown in Figure 2A-2C ) to transfer fluids into and out of the active area of ​​the substrate. In another example, as described above, the substrate 110 can be coupled to an adapter 150 aligned over the hole array (in Figure 1B ), to cooperatively define a fluid reservoir 160 to transfer fluid across the array of holes during operation of the system 100.

[0054] The substrate 110 may additionally or alternatively be coupled to a substrate platform that functions to reversibly attach and align the substrate to a platform, heating element (e.g., thermal control module 190), and / or stage on which an assay is performed, wherein the stage may be used to physically adjust the position of the substrate within the system 100 to improve accessibility of the well array to other elements of the system, such as the imaging subsystem 194, thermal control module 190, and / or extraction module. Preferably, the substrate platform may be configured to accommodate, secure, and manipulate substrates having various array configurations (e.g., an array having 50,000 wells, an array having 1M wells, etc.) with high precision, and may include an optional substrate attachment mechanism.

[0055] 1.2 System-hole array The well array (set of microwells, microwells, wells) 120 functions to capture a group of cells at addressable known locations so that the group of cells can be individually identified, processed, and analyzed. Thus, the well array 120 is preferably configured to facilitate cell capture in at least one of a single cell form and a single cluster (e.g., a cell-particle pair) form. However, the well array 120 may additionally or alternatively be configured to receive any other suitable type of particles in any other suitable form. For example, the well array 120 may be configured (e.g., sized, shaped) to receive mammalian cells, embryos, microspheres, particles, cell-particle pairs, and cells conjugated to microspheres.

[0056] like Figure 1B As shown, the hole array 120 is preferably defined at the upper wide surface 112 of the substrate 110, and each hole 128 in the hole array 120 includes a bottom surface 124 defined within the substrate and close to the second side (e.g., the lower wide surface 114), an open surface 122 directly opposite to the bottom surface 124 and close to the upper wide surface, and a set of walls 126 extending between the bottom surface and the open surface of the hole cavity 128 defining the hole.

[0057] The hole array 120 is defined at an active region of the substrate 110, wherein the active region may be the substrate ( Figure 2A-2C) (e.g., 1 square inch, 10 centimeters, 2 square inches, 3 square inches, 4 square inches, etc.). Preferably, the active area of ​​the substrate (and the well array) is accessible to other components of the system 100 (including the imaging subsystem 194, the fluid delivery module 140, the thermal control module 190, and / or the extraction module) to perform separation, processing, and analysis of individual captured cells. Well array 120 may include any suitable number of wells (e.g., on a scale of 100, 1,000, 10,000 wells, 50,000 wells, 100,000 wells, 1 million wells, 2 million wells, 3 million wells, 4 million wells, 5 million wells, 6 million wells, 7 million wells, 9 million wells, 10 million wells, etc.). In a preferred variation, the well array includes at least 250,000 wells. In a particular example, the well array includes approximately 1 million wells. However, the array of holes may be configured in any other suitable way.

[0058] With respect to the bottom surface 124 and the open surface 122, each hole 128 preferably has at least one wall (e.g., a set of walls) 126 extending between the bottom surface 124 and the open surface 122. In one variation, as at least Figure 1BAs shown, the wall 126 of each well at least partially physically and fluidically separates the individual well 128 from at least one other adjacent well, defines the depth, width and / or cross-sectional dimensions of the well, and is preferably perpendicular to the plane defined by the horizontal axis of the open surface 122. Preferably, the wall thickness of the wall 126 is between 4 microns and 5 microns, but can be any dimension less than 20 microns. The wall 126 can extend perpendicularly from the plane defined by the open surface 122 to the bottom surface 124 to define the cavity 128; therefore, in some variations, the cavity 128 of each well in the array of wells can be prismatic (e.g., cylindrical prismatic, hexagonal prismatic, polygonal prismatic, non-polygonal prismatic, etc.). In a specific example, the cavity of each well defines a hexagonal prism with a depth greater than the sum of the particle diameter of a set of particles and the cell diameter of a set of target cells. However, the walls 126 may extend between the open surface 122 and the bottom surface 124 in any other suitable manner in other variations (e.g., curvilinear walls, straight walls, curved walls, etc.). For example, the walls 126 in the set of walls may gradually reduce the characteristic dimension (e.g., diameter, horizontal cross-section, vertical cross-section) of the hole from the open surface to the bottom surface (e.g., by forming discrete steps, by gradually adjusting the characteristic dimension in a linear or non-linear manner with any suitable slope, etc.). However, in some variations, the hole 128 may not have a well-defined wall 126 perpendicular to the plane defined by the open surface 122 (e.g., the bottom surface may extend directly to the open surface in some manner without forming a wall perpendicular to the open surface). In an example, the bottom surface 124 and the open surface 122 may be separated by a distance (e.g., the height of the cavity 128) between 0.5 microns and 50 microns (e.g., about 25 microns for applications involving capture of a single target cell and about 40 microns for applications involving capture of a single cell-particle pair) with or without the wall. However, the wells of the well array may be configured with any other physical features and / or dimensions to perform the separation, processing, and analysis steps described in method 200. Additionally or alternatively, a set of walls may include a set of channels that fluidically couple each well to at least one adjacent well in well array 120. In such variations, one or more of a set of channels may be defined within a region of substrate 110 between adjacent wells, or may be defined by overlapping portions of adjacent wells. In a particular example, a channel may have a characteristic dimension of 5 microns, and in variations of a particular example, a channel may have a characteristic dimension ranging from 0.5 microns to 75 microns.

[0059] In addition, the hole array 120 is preferably arranged in a compressed array, but may alternatively be arranged in any other suitable manner. In one example, the hole array 120 may be arranged in a hexagonal tightly compressed array. In another example, the hole array may be arranged in a rectangular array. In another example, the hole array 120 may be arranged in any suitable irregular or uneven manner, for example to facilitate fluid flow from one portion of the hole array 120 to another portion of the hole array 120. In a specific example, the shortest distance from the center of each hole to the center of an adjacent hole of the hole array is approximately 30 microns. However, the hole array 120 may alternatively be arranged with any suitable spacing between the holes (e.g., in a compressed or non-compressed configuration) and in any other suitable manner.

[0060] In such Figure 3 In a specific example configuration of the group of holes shown, the hole array is arranged in a hexagonal tightly packed configuration, wherein each hole of the hole array includes a hexagonal open surface aligned with a wide surface of the substrate (e.g., surface plane 118). In addition, each hole includes a hexagonal footprint at the bottom surface opposite the hexagonal open surface. Each hole of the hole array has a cavity 128 forming a hexagonal prism, including a wall set of approximately 5 microns in thickness, approximately 40 microns in height, and a characteristic width of approximately 25 microns. The substrate defines 267,000 such hexagonal holes within an active area of ​​the substrate of approximately 150 square millimeters. However, the hole array can be configured in any other suitable manner.

[0061] 1.3 System - Fluid Delivery Module, Thermal Control Module and Imaging Subsystem The system 100 may include a fluid delivery module 140 that functions to transfer a sample comprising a cell population, a particle population, and / or another fluid such as a treatment reagent and / or a distribution fluid to the well array 120, and may be coupled to the substrate 110. Thus, the fluid delivery module may include one or more inlets, outlets, fluid guides, and / or structures that enable fluids to be transferred into, out of, and through various portions of the system.

[0062] The system 100 may further include a thermal control module 190 that functions to heat and / or cool the substrate and its contents to control the temperature of the contents of the set of wells and / or fluid delivery modules during operation of the system 100. In variations, the thermal control module may heat and / or cool biological samples and / or fluids containing cells of interest to facilitate cell capture and analysis, and may further function to facilitate reactions that require cycling from low to high temperatures, such as for cell lysis, enzyme activation for probe hybridization, and thermal cycling of biological sample mixtures for molecular diagnostic protocols, such as polymerase chain reaction (PCR). The thermal control module 190 may include a heater, a heat sink, a sensor, a fan, and one or more processors; however, the thermal control module may include any suitable components to sense and adjust the temperature of the well array, and to sense and adjust the temperature of the well array according to any instructions (e.g., user input, automatically, a preset temperature schedule, according to a specific assay, etc.). In variations, the heater 192 of the thermal control module is preferably a thin heater (e.g., a Peltier device) configured to controllably heat and cool the biological sample and / or fluid. The thermal control module 190 may additionally and / or alternatively include a temperature sensor or any other suitable element configured to facilitate temperature control. For example, the temperature sensor may be coupled to a thermally conductive substrate, to a heating element, or to a plate heater. Temperature control may be achieved using pulse width modulation through fuzzy logic control, a proportional-integral-differential algorithm, or any other suitable means. Temperature control may be provided to a resolution of 1°C or any other suitable resolution for a given application.

[0063] The system 100 may additionally include an imaging subsystem 194 that functions to image the contents of a group of wells, and may further function to distinguish target objects (e.g., CTCs, labeled cells, microspheres) captured in the group of wells from other cells or objects in a sample introduced into the system 100. The imaging subsystem 194 preferably includes a fluorescence microscope, but may additionally or alternatively include any suitable imaging mechanism (e.g., an optical microscope, a CCD camera, a photodiode array, a light emitting diode, a reflector, one or more processors, etc.). The fluorescence microscope is preferably operable (e.g., in an identification mode, a detection mode, etc.) to detect a fluorescent signal emitted from a target object in one or more of a group of wells, and thereby identify that the well contains the target object. In a particular example, the imaging system (e.g., a fluorescence imaging system) may be operated in a mode for providing real-time or near real-time fluorescence imaging of a sample processed according to an assay. The imaging subsystem 194 is preferably located below the substrate and oriented to image the contents of a set of holes through the transparent (or translucent) material of the substrate; alternatively, the imaging subsystem 194 can be located above the substrate and oriented to image the contents of a set of holes that are not obstructed by the material of the substrate itself. However, the imaging subsystem 194 can be otherwise positioned in any suitable manner.

[0064] Other aspects of embodiments of components of system 100 are described in greater detail in U.S. Application No. 16 / 115,059, filed on August 28, 2018, each of which is incorporated by reference in its entirety.

[0065] 2. Methods like Figure 4 As shown, the method 200 for single cell capture and processing includes receiving a group of cells into a group of wells S210; receiving a group of particles into the group of wells S220; receiving a washing fluid into a cavity connected to the group of wells to wash away excess particles S230 and any floating biomarkers (mRNA, protein, etc.); applying a cold lysis buffer (temperature < 15°C) to the group of cells S250; distributing each well in the well array S260; providing a lysis environment for the group of cells S270; and washing away unbound substances S290. In addition, the method 100 may include any one or all of the following: soaking the contents of the wells S240; adjusting the temperature (e.g., slowly lowering the temperature) S280; treating the cell material (e.g., target cell material) S299; any process described above in the description of the system embodiment; and any other suitable process.

[0066] The method 200 functions to prevent non-specific capture of target material (eg, messenger RNA [mRNA], protein, etc.), and / or leakage of target material between wells of the system to facilitate performance of downstream sample processing steps. Fig. 6A An example of a desired specifically captured state is shown in (right, top), and Fig. 6A An example of a state of undesirable non-specific capture and leakage is shown in (right, bottom).The described method embodiments are preferably implemented by one or more system embodiments described in Section 1 above and / or any other suitable system components.

[0067] 2.1 Methods - Cell and particle loading like Figure 4 As shown, method 200 includes receiving a group of cells (including a target cell subpopulation) into a well array of a slide S210, wherein, in an embodiment, block S210 may include receiving the target cells in a single cell form into a well array defined at a surface plane of a substrate, wherein each well in the well array extends perpendicular to the surface plane of the substrate and extends to the surface plane and into the substrate. Block S210 can be implemented using the embodiments of the substrate, well array, and / or fluid delivery module described above. Block S210 can be implemented in addition or alternatively by other system components.

[0068] A group of cells may include the target cells of interest described above, and / or any other suitable cells, tissues, or other biological matter (e.g., non-cellular biological matter). Figure 5A As shown, in one variation, a group of cells can be derived from dissociated tissue, processed into a single cell suspension, with optional antibody tagging of surface proteins; however, a group of cells can additionally or alternatively be obtained from any other suitable source and / or processed in any other suitable manner. A group of cells can also include cells from the same species or different species (e.g. Figure 6B shown).

[0069] Receiving a group of cells preferably includes underloading the group of cells relative to the number of holes of the slide (e.g., target less than 10% hole occupancy, target less than 20% hole occupancy, target less than 30% hole occupancy, etc.) to facilitate the capture of cells in single cell form across the hole array. The cell loading process is configured to maintain the viability of the cells by using a gentle loading process (e.g., gravity loading, low-speed centrifugation, etc.), and maintain the viability of the cells after loading. Cell loading can include any one or all of the following: gravity-induced entry (e.g., pipetting), low-speed centrifugation, magnetic attraction, force (e.g., along an axis parallel to the wide surface of the slide, along an axis perpendicular to the wide surface of the slide, etc.), changing the direction of the slide (e.g., centrifugal smear, tilting, shaking, etc.), or any other suitable process. In a first example, loading a group of cells includes pipetting aliquots between 50 microliters and 500 microliters into the holes. Loading a group of cells can also include loading any suitable buffer or other fluid.

[0070] like Figure 5B As shown, receiving a group of cells can include performing a quality control operation using the imaging subsystem described above to observe the correct loading of the group of cells across the hole array. However, block S210 can additionally or alternatively include any other suitable quality control operation (e.g., using a sensor-based method) to determine the correct loading of the group of cells across the hole array.

[0071] like Figure 4 , Figure 5C and Figure 6C As shown, method 200 also includes receiving a group of particles into the wells of the slide S220, thereby co-capturing a group of target cells and a group of particles across the well array. As shown in the figure, the wells of the well array are of high aspect ratio, so that a group of cells are configured to be located in the well at a lower region, and a group of particles are configured to be located above a group of cells at an upper region of the well (for a well containing a single cell and a single particle in a desired state), thereby preventing the cells and particles from being laterally positioned in the same plane within the well; however, with respect to the co-capture of a group of cells and a group of particles across the well array, the wells can be configured in another manner.

[0072] A set of particles is preferably overloaded with respect to cells (e.g., the bead concentration is the particle suspension, higher than the cell concentration in the cell suspension), but may additionally or alternatively be underloaded with respect to cells, overloaded with respect to the number of wells, underloaded with respect to the number of wells, or loaded in another manner (e.g., batched). The ratio of bead concentration to cell concentration may be between 10:1 and 20:1. Alternatively, the ratio of bead concentration to cell concentration may be less than or equal to 10:1, greater than or equal to 20:1, or any other suitable ratio.

[0073] Receiving a set of particles is preferably performed concurrently (e.g., at the same time, at least partially overlapping in time, etc.) with receiving a set of cells at block S210, because reducing the time between cell loading and particle loading can function to prevent undesirable cell lysis. Alternatively, receiving a set of particles can be performed after block S210, before S210, with a time delay (e.g., less than 5 minutes, between 0 and 60 minutes, greater than 60 minutes, etc.) with respect to block S210, or at any other suitable time. Receiving a set of particles can include any or all of the loading processes described above with respect to cell loading (e.g., gravity loading) or any other suitable loading process.

[0074] In one variation, after a group of cells are loaded into the wells of a slide by a gentle loading process, a group of particles are loaded into the wells by the same gentle loading process (e.g., gravity loading). In a specific example, a group of cells is underloaded with respect to a group of wells, and a group of particles is overloaded with respect to the group of cells.

[0075] In another variation, a group of cells and a group of particles are loaded together (eg, from the same suspension). However, blocks S210 and S220 may additionally or alternatively be performed in any other suitable order or manner.

[0076] like Figure 5C As shown, receiving a set of particles can include performing a quality control operation using the imaging subsystem described above to observe the correct loading of the set of particles (e.g., with respect to a set of cells) across the hole array. However, block S220 can additionally or alternatively include any other suitable quality control operation (e.g., using a sensor-based method) to determine the correct loading of a set of cells across the hole array.

[0077] 2.2 Method - Excess Particle Removal and Hole Soaking like Figure 4 and Figure 5D As shown, method 200 may optionally include washing away excess particles S230, which functions to remove any floating excess particles above the wells (e.g., completely above the wells, partially exposed from the wells, etc.). Thus, box S230 includes achieving a desired state of the well array after receiving a wash fluid into a chamber connected to the well array and transferring excess particles in a group of particles out of the well array. Box S230 may additionally or alternatively function to remove any other excess material (e.g., floating cellular material, debris, etc. from prematurely lysed cells). The one or more wash fluids used in box S230 may include any suitable buffer, liquid (e.g., water, oil, etc.), air, or any other suitable substance. The wash flow is preferably configured to remove material above the well walls without removing material (e.g., cells, particles, buffers, liquids, etc.) from within the wells; additionally or alternatively, the wash flow may be configured to prevent one or more wash buffers from entering a group of wells. Thus, the wash fluid is preferably applied in a laminar flow process (e.g., Figure 5D ), but may additionally or alternatively include or cause turbulence. The flow rate is preferably less than 100 microliters / second, but may alternatively be equal to 100 microliters / second, greater than 100 microliters / second, or have any other suitable flow rate. Additionally or alternatively, any other suitable parameters, such as other flow parameters (e.g., flow duration), structural parameters (e.g., diameter of one or more fluid paths, arrangement of fluid paths, location of fluid paths on a set of holes, hole height, hole width, etc.) may be configured to prevent one or more substances from leaving the holes.

[0078] In a first variation, the characteristic length scale of the flow is of similar order to the characteristic length scale of the pore (eg, height, width, etc.).

[0079] Method 200 may optionally include soaking the contents of the well S240, which functions to prevent cells from lysing before a desired time point (e.g., before lysis buffer delivery, during lysis buffer delivery, etc.), allowing cells and / or particles to have time to settle in a group of wells, or performing any other suitable function. The wells can be soaked with a wash buffer (e.g., as used above to wash away excess particles), different buffers (e.g., for loading cells and / or particles with an initial buffer, a cell suspension buffer, a particle suspension buffer, etc.), water, or any other suitable fluid. Frame S240 can be performed after frame S230, before frame S230, during frame S230, at multiple times during the method, or at any suitable point during the method. Alternatively, the method can be performed without frame S240. In a first variation, after frame S230, the wells are soaked at 4 degrees Celsius for 11 minutes; however, the soaking process can be performed using another suitable temperature, duration, or other soaking parameters.

[0080] 2.3 Methods - Cell lysis and pore partitioning to prevent leakage like Figure 4 , Figure 5E and Fig.6D As shown, method 200 includes receiving a lysis buffer at a well array, thereby delivering the lysis buffer to the cells S250, which functions to initiate cell lysis (e.g., immediately, after a time delay, etc.), so that the release of target cell material can be captured by a group of particles. Therefore, after receiving the lysis buffer in a cavity (e.g., a reservoir described above) that is fluidly connected to the well array, frame S250 can help provide a lysis environment for a group of cells. The lysis buffer is preferably received with flow parameters (e.g., flow rate, flow duration, etc.) configured to allow the lysis buffer to enter the wells without removing the contents from the wells. Frame S250 preferably includes flowing the lysis buffer over a group of wells (e.g., at a well located Figure 1BIn the embodiment of the present invention, the lysis buffer is preferably applied by laminar flow, but turbulence can be included or induced in addition or alternatively. Lysis buffer is preferably applied by the same fluid manifold (e.g., fluid reservoir, fluid path, fluid reservoir connected to a group of fluid path fluids, etc.) used in frame S230, but alternatively, another fluid manifold connected to the hole can be used. In addition or alternatively, any or all of the fluid delivery subsystems (e.g., reservoirs, fluid paths, etc.) described in the following may be used: 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 / 821,329, filed on November 22, 2017; U.S. application No. 15 / 782,270, filed on October 12, 2017; U.S. application No. 16 / 049,240, filed on July 30, 2018; and U.S. application No. 16 / 115,059, filed on August 28, 2018, each of which is incorporated by reference in its entirety.

[0081] like Fig.6DAs shown in frame S250', the lysis buffer is preferably cooled (e.g., to 4 degrees Celsius, to less than 10 degrees Celsius, between 0 and 20 degrees Celsius, above 20 degrees Celsius, etc.), which functions to slow down the time for the target cell material (e.g., mRNA) to diffuse from the cell and / or a set of holes. The structure of the hole (e.g., depth, width, shape, shape, etc.) can be configured to slow down the diffusion time of the mRNA in addition or alternatively. The concentration of the lysis buffer is preferably configured to prevent cell lysis during the lysis buffer delivery time. The lysis buffer preferably includes one or more lysing agents, such as any one or all of the following: Triton X-100 (e.g., 1%, less than 1%, greater than 1%, etc.), sodium deoxycholate [DOC] (e.g., 0.1%, less than 0.1%, greater than 0.1%, etc.), sodium dodecyl sulfate [SDS] (e.g., 0.2%, less than 0.2%, greater than 0.2%, etc.), or any other suitable solubilizing agent. Additionally or alternatively, the lysis buffer may include any or all of the following: Tris (e.g., 100 mM, less than 100 mM, greater than 100 mM, pH 7.5, pH less than 7.5, pH greater than 7.5, etc.), lithium chloride [LiCl] (e.g., 500 mM, less than 500 mM, greater than 500 mM, etc.), ethylenediaminetetraacetic acid [EDTA] (e.g., 2 mM, less than 2 mM, greater than 2 mM, etc.), dithiothreitol [DTT] (e.g., 5 mM, less than 5 mM, greater than 5 mM, etc.), and / or any other suitable reagents or components.

[0082] In one variation, the lysis buffer includes 100 mM Tris, pH 7.5, 500 mM LiCl, 1% Triton X-100, 2 mM EDTA, 0.1% DOC, 0.2% SDS, and 5 mM DTT. However, variations of the lysis buffer may include altered components and / or component concentrations.

[0083] like Fig.6D As shown, the residence time of the lysis buffer (e.g., the time before S280, the time before S290, etc.) is preferably less than or equal to 60 seconds (e.g., between 5 seconds and 60 seconds), and further preferably less than or equal to 30 seconds (e.g., between 5 seconds and 30 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, etc.). Optionally, the residence time can be greater than 30 seconds, greater than 60 seconds, or have any other suitable value.

[0084] In About Fig.6DIn a variation described, the lysis buffer is optimized so that enough lysing agents are delivered to a group of holes during the lysis buffer delivery time, but not enough to start lysing cells. In addition or alternatively, the lysis buffer may include enough lysing agents delivered to the hole, but not enough to release the target cell material (e.g., mRNA), diffuse the target cell material out of the hole, or have any other suitable result. The amount of the lysis buffer applied (e.g., the volume delivered, the duration, etc.) is preferably determined based on the concentration of the lysis buffer, the diffusion rate of the key lysing agent and the residence time of the lysis buffer on the micropore, but may alternatively be variable or otherwise determined. In another embodiment, the lysis buffer is configured to be in an inactive state during its delivery, and becomes active afterwards by any one or all of the following: heat, light, the addition of a cleavage reaction initiator, or any other suitable process or agent. In addition, in some embodiments, the lysis buffer is precooled to less than 15°C before entering the cavity through the micropore.

[0085] like Figure 4 As shown, method 200 also includes a separation hole S260, which plays a role in isolating adjacent holes and their contents from each other, and preventing substances (e.g., target cell substances, cell debris, particles, etc.) from drifting out of the hole (e.g., drifting out of the hole upward) and entering another hole. Therefore, frame S260 may include separating each hole in the hole array from adjacent holes in the hole array after replacing the lysis buffer with a separation fluid (e.g., gas, immiscible liquid) from a cavity or reservoir connected to the hole array fluid. Frame S260 can also or alternatively function to remove excess lysis buffer (e.g., lysis buffer outside the outer hole), maintain the separation of adjacent holes during the lysis buffer culture, or perform any other suitable function. Frame S260 is preferably performed during the execution of frame S250 or in a predetermined time period (e.g., 5-60 seconds thereafter) after the execution of frame S250, but can also or alternatively be performed at any other suitable time during the execution of method 200.

[0086] Frame S260 can include introducing one or more barriers above the hole, for example, a cavity or reservoir in fluid communication with the hole array. The barrier can include a physical barrier, a secondary fluid (secondary fluids) (e.g., a separator fluid) or any suitable barrier. The barrier can be introduced into the same manifold associated with one or more of the frames S210, S220 and / or S230, or introduced in other ways. The secondary fluid can include any one or all of a gas, a liquid (e.g., non-polar, polar, viscous, non-viscous, etc.), a colloid or any other suitable fluid. The secondary fluid can include non-settling particles containing a target biomarker scavenger, which can be combined with any target biomarker flowing out of any micropore.

[0087] Block S260 preferably includes positioning each hole (e.g., above) in a group of holes. Fig. 6E About S260' and Fig. 6F In one embodiment, a partitioning fluid (e.g., air, oil, buffer, water, etc.) is dispensed (e.g., through a fluid path subsystem) in the direction shown in S260”, which functions to form a barrier (e.g., semi-permeable, non-permeable, selectively permeable, etc.) to prevent one or more contents of the well from drifting out of the well. In one variation, the partitioning fluid (e.g., air) achieves such a barrier based on the flow rate of the partitioning fluid (e.g., Fig. 6E '). In a particular instance, pumping a separation fluid over a set of holes at a flow rate above a predetermined threshold functions to prevent material within the holes from drifting upward and out of the holes. Additionally or alternatively, the flow rate can function to remove material that may drift above the wall (e.g., in a horizontal direction) before the material may drift into another hole. In a second variation, the separation fluid implements a barrier by preventing liquid from evaporating from the holes (e.g., during a long incubation step). This can be achieved by any or all of the following: the density of the separation fluid (e.g., where the density of the separation fluid is less than the lysis buffer), the weight of the separation fluid, the pressure achieved by the separation fluid, the temperature of the separation fluid and / or the slide (e.g., a cold slide), or any other suitable feature.

[0088] Block S260 preferably separates the material along a vertical axis (e.g., is a horizontal separation), wherein the separation is at the height of one or more walls defining a set of holes. Additionally or alternatively, the separation can be established at a height below one or more walls (e.g., within the hole), above the height of one or more walls, at a variable height, or in any other suitable location. Further, additionally or alternatively, block S260 can include separating the material along a horizontal axis, along another axis, or in any other suitable manner. In a first variation (e.g., as Figure 6E-6F as shown), substances (e.g. Fig. 6E The air and Fig. 6F The oil in the pores provides a pore separation along the vertical axis, wherein the separation is established at a height corresponding to the terminal portion of the wall of the pores.

[0089] Block S260 can be achieved at least in part by a fluid path subsystem engaged with the slide (e.g., the location and arrangement of the fluid paths, the number of fluid paths, the length of the fluid paths, the diameter of the fluid paths, the fluid path subsystem within the slide, the fluid path subsystem outside the slide, etc.) and / or flow parameters (e.g., flow rate, flow duration, etc.) of one or more fluids (e.g., buffer, air, oil, etc.) through the fluid path subsystem. In a first variation, the fluid path subsystem is configured to transfer the fluid from the fluid manifold to an area above a set of holes. In a specific example, the fluid path configured to distribute the separation fluid (e.g., air, oil, buffer, water, etc.) has a diameter similar to the size of the space above the wall of the set of holes. In addition, the fluid path can define an inlet above the set of holes so that the separation fluid is distributed in laminar flow above the set of holes. In a second variation, the flow rate of the separation fluid (e.g., air, oil, etc.) is configured to fill the space above the set of holes (e.g., during at least a majority of the duration of dispensing the separation fluid, greater than 80% of the time of dispensing the separation fluid, etc.). Additionally or alternatively, S600 may be implemented by any one or all of the following: separation fluid properties (eg, density, viscosity, etc.), temperature, or any other suitable properties.

[0090] In variations (e.g., Figure 6E-6G As shown, Figure 6G Aspects of cell lysis within the compartment wells are also described and depicted), block S260 includes dispensing air (e.g. Fig. 6E ), which functions to remove excess lysis buffer (e.g., remaining from block S250) and separate the wells from each other, and then distributes the oil (e.g., Fig. 6F ), which functions to prevent liquid or other contents from evaporating from a group of holes or to prevent molecules from diffusing from one micropore to another micropore. In optional variations, only air is dispensed, only oil is dispensed, or any other fluid or combination of fluids is dispensed.

[0091] like Figure 4 and Figure 5E As shown, method 200 may include providing a lysis environment S270 for a group of cells, which functions to release target cell material (e.g., mRNA) from the cells. With regard to frame S270, for each hole, at least a subset of target cell material is then combined with a probe (e.g., an oligonucleotide fragment) of a particle initially captured in the hole with the cell. The combination of target cell material and probe is preferably achieved by multiple (A) present on the target cell material (e.g., mRNA) and multiple (T) present on the particle. However, the combination of target content and particle probe can be achieved in addition or alternatively by any other suitable reaction or process.

[0092] Frame S270 is preferably performed while maintaining the barrier (e.g., oil) as described about frame S260, which prevents cell material from moving or leaking into adjacent holes from the source hole. In some variations, separation fluid speed and / or separation fluid barrier are essential for achieving this. In addition or alternatively, the predetermined mixture of lysis buffer can be essential for maintaining binding time or other binding conditions. In a particular instance, the method includes waiting for a predetermined duration (e.g., between 15 and 30 minutes, less than 15 minutes, greater than 30 minutes, etc.) for the cracking of target cell material (e.g., mRNA) and the combination with a group of particles.

[0093] The temperature for cell lysis is preferably between 20 degrees Celsius and 30 degrees Celsius (e.g., room temperature, between 20 and 25 degrees Celsius, 26 degrees Celsius, etc.). Figure 6G , block S270' may include transferring heat to the well array to promote lysis and preserve the contents of the well array. Optionally, the cells may be lysed at a temperature below 20 degrees Celsius, above 30 degrees Celsius, or any other suitable temperature.

[0094] In a first variation, cells are lysed at room temperature for between 15 and 30 minutes. In a specific example, cells are lysed at 26 degrees Celsius for 25 minutes. In another variation, cells can be lysed at a temperature of 40 degrees Celsius or 50 degrees Celsius.

[0095] 2.4 Methods - Additional Steps like Figure 4 As shown, method 200 may optionally include slowing down the temperature and soaking S280, which may function to prevent any unbound target cell material from migrating (e.g., by evaporation, by diffusion, etc.) away from a group of holes. The temperature is preferably between 0 degrees Celsius and 5 degrees Celsius, and the soaking time is preferably between 5 minutes and 20 minutes. In a particular instance, frame S280 includes slowing down the temperature to 2 degrees Celsius and keeping for 11 minutes. In addition or alternatively, S280 may include any other suitable process at any suitable parameter.

[0096] like Figure 4As shown, method 200 may optionally include washing away unbound material S290, which functions to remove unbound target cell material (e.g., unbound mRNA) after lysis. The slide temperature during S290 is preferably maintained at room temperature, but can be maintained at another suitable temperature, variable, room temperature or any other suitable temperature in addition or alternatively. S290 preferably includes flowing in wash buffer (e.g., by any or all fluid paths used in the previous steps). Wash buffer may include detergent, preferably, PBS containing RNAse inhibitors (e.g., PBS containing 1% IGEPAL). In addition or alternatively, any other liquid or material may be used to wash away unbound material. Washing flow is preferably configured so that the floating target cell material from hole is pumped to a waste reservoir, and there is no chance to diffuse into adjacent holes. In a variation, the flow is between 50 microliters / second and 1000 microliters / second. In embodiments, washing unbound material may include flowing a wash buffer configured to remove unbound RNA over the well array at a flow rate greater than 50 μL / sec after the mRNA material is hybridized to a set of particles.

[0097] Frame S290 can optionally include removing washing buffer, which is preferably performed by inflowing air (e.g., air washing).Frame S290 can additionally or alternatively include washing holes with washing beads, wherein the washing beads are bound to the target cell material of migration (e.g., floating above a group of holes, in a group of holes, etc.).One group of washing beads is preferably magnetic (e.g., oligo d (T) magnetic beads, oligo (dT) magnetic beads, etc.), but can additionally or alternatively be non-magnetic, configured to combine any suitable probe, or otherwise configured.Therefore, washing beads can be a composition that is convenient to retrieve the washing beads with the undesirable or leaked material of combination from the system.

[0098] The washing process is preferably performed more than once (eg, twice, three times, between 1 and 10 times, etc.), but may alternatively be performed once, or the method may be performed without washing.

[0099] In a first variation, frame S290 includes washing the slide glass with a wash buffer solution without washing beads. In a second variation, S290 includes washing the slide glass with a group of magnetic washing beads. In a first specific example, 1 micron of magnetic oligo d (T) beads were used. In a second specific example, eight micron of magnetic oligo d (T) beads were used. In another variation, the wash buffer solution can include oligo dT fragments containing non-extendable bases. These oligo dT fragments are combined with any unbound target mRNA molecules, and these unbound target mRNA molecules leave or remain in the micropores, but after combining, due to the presence of non-extendable bases around the oligo dT sequence, they cannot be converted into cDNA during downstream reverse transcription.

[0100] like Figure 4 As shown, the method can optionally include any treatment of the cells S299. The cells can be treated within the well, outside the well (e.g., after extracting a single cell, after extracting all cells, etc.), or at any other suitable location or combination of locations.

[0101] In one variation, block S299 includes a first operation that includes an on-chip reverse transcriptase process, such as Fig. 5F In the first example, this was performed at a temperature of 58°C and for a duration of 16 minutes. In another variation, the on-chip reverse transcriptase process was performed immediately after cell lysis while compartmentalized in its microwells. For this process, the reverse transcription reagents were optimized and co-delivered with the lysis buffer mixture.

[0102] Block S299 may also include removing a set of particles bound to the target cell material and continuing with a set of steps to prepare the material for DNA sequencing, such as Figure 5G In an example, this includes extracting a set of particles from the well array (e.g., using an aspiration process, using a pressure-driven process, using a magnetic retrieval process, using a gravity or centrifugal force-related process, etc.) and separating them from any remaining wash beads (e.g., before removing the first set of beads, after removing the first set of beads, etc.).

[0103] Block S299 may additionally or alternatively include any other suitable processing and analysis steps. This may include any or all future processing of the cells (e.g., by reverse transcription), polymerase chain reaction sequencing (e.g., single cell genomics, DNA / RNA sequencing, library preparation, such as Figure 5H S299c in, etc.), data analysis or any other suitable process.

[0104] Regarding the system configuration for further preventing leakage, Figure 7A-7BDepicted are exemplary fluidic network configurations and flow directions across a substrate associated with preventing the contents of a well from leaking into adjacent wells. Fig. 7A A first configuration is depicted in which a substrate for a well array includes a short axis, a long axis, and a fluid channel network defining one or more flow paths that are preferentially aligned with the long axis and coupled to a cavity in fluid communication with the microwell region. Due to the configuration in which the fluid travels a longer distance across the well array, Fig. 7A The configuration shown may contribute to a higher risk of leakage of the hole contents. Figure 7B A second configuration is optionally depicted in which a substrate for a well array includes a minor axis, a major axis, and a fluid channel network defining one or more flow paths that are preferentially aligned with the minor axis and coupled to a cavity in fluid communication with the microwell region. Due to the configuration in which the distance the fluid travels across the well array is shorter, Figure 7B The configuration shown can contribute to a lower risk of leakage of the well contents. Thus, with respect to blocks S230, S250, S260, S270, and S290 described above, due to the configuration of the fluid channels in communication with the microwell array, method 200 can include receiving one or more of a wash buffer, a lysis buffer, and a separation fluid into the fluid channel network along a shorter travel distance.

[0105] 2.5 Methods - Examples In one example, method 200 includes capturing cells in a set of microwells; capturing 20 micron particles (e.g., HDBB) in the set of microwells; washing away excess particles; soaking the microwells at 4 degrees Celsius for 11 minutes; flowing in cold (e.g., 4 degrees Celsius) lysis buffer for no more than 30 seconds; separating the wells using air and then oil; lysing the cells at room temperature for 25 minutes; ramping down the temperature to 2 degrees Celsius and soaking the contents of the wells for 11 minutes; flowing in a wash buffer (e.g., PBS containing an RNase inhibitor); removing the first wash buffer using an air wash; flowing in a second wash buffer; removing the wash buffer using an air wash; performing on-chip reverse transcription at 58 degrees Celsius for 16 minutes; removing the particles from the slide, and performing sequencing and data analysis. However, variations of the examples may be performed in any other suitable manner.

[0106] Additionally or alternatively, method 200 may include any other suitable processes performed in any suitable order.

[0107] The accompanying drawings illustrate the architecture, function and operation of the possible implementation of the system, method and computer program product according to the preferred embodiment, example configuration and its variation. In this regard, each frame in the flow chart or block diagram can represent a module, segment or part of the code, and the code includes one or more executable instructions for realizing one or more specified logical functions. It should also be noted that in some optional implementations, the function mentioned in the frame may not appear in the order mentioned in the accompanying drawings. For example, the two frames shown continuously can in fact be executed substantially at the same time, or the frame can sometimes be executed in the opposite order, depending on the function involved. It should also be noted that each frame of the block diagram and / or flow chart diagram and the combination of the frames in the block diagram and / or flow chart diagram can be realized by a special hardware-based system or a combination of special hardware and computer instructions that perform the specified function or action.

[0108] As those skilled in the art will recognize from the foregoing detailed description and from the accompanying drawings and claims, modifications and variations may be made to the preferred embodiments of the present invention without departing from the scope of the invention defined in the following claims.

Claims

1. A method, comprising: receiving a group of target cells in single cell form into an array of wells defined at a plane of a substrate surface; receiving a set of particles into the array of wells, thereby co-capturing the set of target cells and the set of particles across the array of wells; achieving a specific capture state of the well array, wherein a well of the well array in the specific capture state has a target cell from the set of target cells and a particle from the set of particles, and wherein the well has a width configured to prevent the target cell from being positioned laterally adjacent to the particle in the specific capture state after a wash fluid is received into a chamber in communication with the well array and excess particles from the set of particles are transferred out of the well array; providing the array of holes at a temperature below 15°C; receiving a lysis buffer into the well array in laminar flow; receiving a separation fluid into the chamber to displace the lysis buffer from the chamber in a laminar flow and separate each well in the array of wells from adjacent wells in the array of wells at the surface plane; transitioning the well array to a temperature above 25° C., thereby lysing the set of target cells; and The intercellular material of the group of target cells is retained within the well array separately with the group of particles, thereby mitigating leakage of intracellular material of a first well of the well array to a second well of the well array.

2. A method comprising: receiving a group of target cells in single cell form into an array of wells defined at a plane of a substrate surface; receiving a set of particles into the array of wells, thereby co-capturing the set of target cells and the set of particles across the array of wells; achieving a specific capture state of the well array, wherein a well of the well array in the specific capture state has a target cell from the set of target cells and a particle from the set of particles; receiving a lysis buffer into a cavity above the well array; receiving a separation fluid into the cavity, thereby displacing the lysis buffer within the cavity and separating each well in the array of wells from adjacent wells in the array of wells at the surface plane; and lysing the population of target cells; and The intercellular material of the group of target cells is retained within the well array separately with the group of particles, thereby mitigating leakage of intracellular material of a first well of the well array to a second well of the well array.

3. A method, comprising: transferring a biological sample comprising a target nucleic acid material and a treatment reagent into a fluid path defined above a well array at a plane of a substrate surface, wherein the treatment reagent comprises water, wherein the biological sample has a volume of 10 microliters to 50 microliters, wherein the well array comprises 10,000 to 250,000 wells arranged in a hexagonal tightly packed array, wherein each well in the well array extends vertically into the planar surface of the substrate and comprises a polygonal cross-section, and wherein the fluid path directs flow into the well array from a first subset of wells of the well array at a first end of the substrate to a second subset of wells of the well array at a second end of the substrate; and A water-immiscible liquid is dispensed into the fluid path to separate each well of the well array from adjacent wells of the well array at the surface plane and mitigate leakage of the target nucleic acid material from a first well of the well array to a second well.

4. A method comprising: transferring a biological sample comprising a target nucleic acid material and a treatment reagent into a fluid path defined above a well array at a plane of a substrate surface, wherein the treatment reagent comprises water, wherein the well array comprises between 10,000 wells and 250,000 wells, wherein each well in the well array extends vertically into the planar surface of the substrate, and wherein the fluid path directs flow into the well array from a first subset of wells of the well array at a first end of the substrate to a second subset of wells of the well array at a second end of the substrate; achieving a specific capture state of the well array, wherein a well of the well array in the specific capture state has a target from a group of targets of the target nucleic acid species; dispensing a water-immiscible liquid into the fluid path under positive pressure to separate each well of the well array from adjacent wells of the well array at the surface plane and mitigate leakage of the target nucleic acid material from a first well of the well array to a second well, regulating the temperature of the contents within the well array; and The contents of the well array are illuminated and optically imaged.

5. A method, comprising: transferring a biological sample comprising a target nucleic acid material and a treatment reagent into a fluid path coupled to an array of wells defined at a planar surface of a substrate, wherein the treatment reagent comprises water, wherein the array of wells comprises at least 10,000 wells, wherein each well in the array of wells extends perpendicularly into the planar surface of the substrate and comprises a polygonal cross-section, and wherein the fluid path conveys a flow into the array of wells from a first subset of wells of the array of wells at a first region of the substrate to a second subset of wells of the array of wells at a second region of the substrate; and A water-immiscible liquid is dispensed into the fluid path to separate each well of the well array from adjacent wells of the well array at the surface plane and mitigate undesired transfer of the target nucleic acid material from a first well to a second well of the well array.

6. A system for separating and analyzing a group of cells, the system comprising: A substrate having a wide surface; An array of holes is defined at a first side of the substrate, each hole in the array of holes includes an open surface defined at the first side, a bottom surface defined within the substrate adjacent a second side directly opposite the first side, and a set of walls extending between the bottom surface and the open surface to form a cavity of the hole, optionally the array of holes is arranged in an active area of ​​the substrate.

7. The system of claim 6, further comprising a fluid delivery module configured to couple with the substrate and transfer a sample containing the group of cells and / or another fluid to the well array.

8. The system of any one of claims 6-7, further comprising a flow control subsystem configured to control the flow of fluid through the system and any other suitable flow through the system that requires control and / or actuation.

9. The system according to any one of claims 6-8, further comprising a thermal control module for controlling the temperature of portions of the system, an imaging system configured to perform optical imaging, illumination or irradiation of the contents of the well array and to enable identification, localization and quantification of cells retained by the wells of the well array, and / or an extraction module capable of extracting one or more target cells, particles, cell-particle pairs, genetic complexes and / or genetic products from the well array.

10. A system for isolating and analyzing a biological sample containing a target nucleic acid species, wherein the system comprises: A substrate having a wide surface; an array of holes defined at a first side of the substrate, each hole in the array of holes comprising an open surface defined at the first side, a bottom surface defined within the substrate proximate a second side directly opposite the first side, and a set of walls extending between the bottom surface and the open surface to form a cavity of the hole, wherein the array of holes comprises between 10,000 holes and 250,000 holes arranged in a hexagonal tightly packed array; and A fluid path over the array of holes.

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