KR20220024734A - Systems and methods for recovering target material from microwells - Google Patents

Abstract

A system and method for target material recovery and processing, the system comprising: a first region configured to interface with a capture region of a capture substrate for capturing particles in a single particle format within a series of wells, the first region configured to interface with the capture region; an adapter comprising a second region and a cavity extending from the first region to the second region; and a support structure coupled to the adapter and providing a set of modes of operation for movement of the adapter relative to the capture substrate. The system enables magnetic and/or other force-based methods of recovery of a target material (eg, derived from a single cell).

Description

Systems and methods for recovering target material from microwells

[Cross-reference of related applications]

This application claims the benefit of U.S. Provisional Patent Application No. 62/866,726, filed on June 26, 2019, which is hereby incorporated herein by reference in its entirety.

In addition, this application is a US patent filed on May 5, 2020, each claiming the benefit of US Provisional Patent Application No. 62/844,470, filed on May 7, 2019, each of which is incorporated herein by reference in its entirety. Claims the benefit of application no. 16/867,235.

FIELD OF THE INVENTION The present invention relates generally to new and useful systems and methods for the recovery of target substances in the fields of cell capture and cell processing, and more particularly in the fields of cell capture and cell processing.

With the growing interest in cell-specific response drug testing, diagnostics and other assays, systems and methods that allow for isolation, identification and recovery of individual cells are becoming highly desirable. Single cell capture systems and methods have been shown to be particularly advantageous for these applications. However, the relevant processes and protocols for single cell capture and subsequent analysis often have to be performed in a specific order and with high precision to properly maintain the cells. In addition, there are many difficulties in efficiently recovering a target material from a high-density platform. As such, these processes are user-intensive, require extensive and iterative manual library preparation and selection processes, are impossible to automate, and can lead to cell damage or undesirable results if not performed properly. there is.

Therefore, in the field of cell capture and cell processing, there is a need to create new and useful systems and methods for sample processing and target material recovery, and to minimize the steps required for preparing a library of target biological materials.

1A shows a schematic diagram of an embodiment of a system for target material recovery.
1B shows a schematic diagram of an application to an embodiment of a system for target material recovery.
2A-2C show diagrams of modifications to a sample processing cartridge associated with a system for target material processing and retrieval.
3A-3C illustrate modes of operation of the lid opener associated with the sample processing cartridge shown in FIGS. 2A-2C ;
4A-4B illustrate modes of operation of the valve subsystem associated with the sample processing cartridge shown in FIGS. 2A-2C .
5 shows a modification of a process vessel for target material treatment, recovery and subsequent treatment.
6A-6B show schematic diagrams of an embodiment of a system for automated cell sample processing.
7A-7C show diagrams of variants of the system shown in FIGS. 6A-6B .
8A-8C show a first magnetostriction of a system for target material recovery.
9A-9C show a second magnetostriction of a system for target material recovery.
10A-10B show variations on a subset of the components used for material separation in a system for target material recovery.
11A-11J show modes of operation of a separation subsystem associated with a system for target material recovery and processing;
12A-12D show diagrams of components for variants of a separation subsystem associated with a system for target material recovery;
13A-13C show a second variant of the system for target material recovery.
14 shows a flow diagram for an embodiment of a method for target material recovery.
15A and 15B show schematic diagrams of variants of the method for target material recovery.
16 shows a flow diagram of a method for target material recovery.
17A-17D show schematic diagrams of a first example of a method for target material recovery.
18A-18E show schematic diagrams of a second example of a method for target material recovery.
19 shows a flow chart for a variant of the method for target material recovery.

The following description of preferred embodiments of the present invention is not intended to limit the present invention to these preferred embodiments, but is intended to enable those skilled in the art to make and use the present invention.

1. Advantage

The described system(s) and method(s) provide several advantages over conventional systems and methods.

The present invention(s) has the advantage of providing a mechanism for efficiently recovering a target material (eg, beads, cells, released nucleic acid material, etc.) from high-aspect ratio wells of a high-density capture platform. . Because the capture platform wells are dense, recovery in these scenarios is usually difficult and inefficient. In addition, the recovery mechanism described causes the target material to undergo an acceptable amount of shear, and other potential stresses that would otherwise interfere with subsequent processing steps.

In addition, the present invention(s) provides the advantage of reducing the burden on the system operator with respect to the process of recovering target material from the wells, where standard processes may require repeated aspiration and dispensing steps that require additional time. .

The invention(s) may also provide the advantage of increasing the efficiency with which target material is recovered (and non-target material is not). Thus, selective recovery efficiency can reduce processing burden and subsequent costs associated with processing reagents and other material costs (due to reduced requirements). For example, the invention(s) allow system operators to purchase smaller amounts of reagents, reduce the number of cleavages required for successful amplification of a target molecule, and contain no product but in one process step. It can eliminate the need to perform SPRI-based cleanup and size selection of the target oligonucleotide product on another oligonucleotide tag that is passed on to the next process step. Such improved target product recovery and reduced carryover of non-target products may also reduce the complexity of data analysis and may provide more useful data related to the desired biomarker analysis. This may serve to save cost, reduce reagent waste, or produce other suitable results.

The invention(s) may also provide the advantage of enabling, at least in part, automation of protocols related to single cell capture, target material recovery, and subsequent processing. For example, with respect to protocols that include repeated purification, washing and recovery steps, human operator users may not intervene in some or all of the methods. Further, the system(s) and/or method(s) may enable greater accuracy in protocol performance compared to conventional systems and methods. In addition, some of these inventions are amenable to full automation via liquid handling robots.

Additionally or alternatively, the systems and/or methods may provide other suitable advantages.

2. System

1A , an embodiment of a system 100 for target material retrieval is configured to interface/communicate with a capture region of a sample processing chip 132 for capturing particles in a single particle format within a series of wells. a cavity configured to include an adapter 110 , in response to an applied force, to facilitate target agent release from a series of wells into the adapter 110 for efficiently withdrawing the target agent from the chip 101 . (120) may be included. The applied force is preferentially applied perpendicular to the plane of the microwell (90°±30°). Embodiments of system 100 serve to provide a mechanism for efficiently recovering a target material from a high-density capture device (eg, a microwell chip), wherein the high-density capture device provides a measure of the efficiency of the captured single cell-bead pairing. Include a high-density array of high-aspect-ratio microwells to promote efficiency improvements. In addition, embodiments of system 100 may function to reduce the manual burden associated with retrieving target material from high-density capture devices. In addition, embodiments of system 100 may function to increase the efficiency with which target material is withdrawn from a high density capture device and the efficiency with which non-target material is retained in the capture device.

In an embodiment, as shown in FIG. 1B (top), a microwell of a high-density capture device can co-capture a single living cell carrying a target material and a functional particle. The system can then be capable of lysing the cells in the microwell and delivering the target substance to the functional particle, and the system can perform ligation or reverse transcription to link the target substance to an oligonucleotide tag on the functional particle. . The target material (and, in some embodiments, functional particles) may then be recovered from the microwell. FIG. 1B (bottom) depicting a top-bottom view of a high-density capture platform, some microwells containing functional particles, some microwells containing single cells, and multiple microwells of which some microwells were empty. Specific examples of embodiments are shown in

In connection with recovering the target material under one or more applied forces, the applied force may include magnetic force; gravity-related forces (eg, centrifugal forces, buoyancy forces, etc.); created by applying positive and/or negative pressure to the capture region (eg, through an input channel of the chip 101 , through an outlet channel of the chip 101 , or through an adapter manifold coupled to the chip 101 ). fluid pressure driving force; electric field-related forces (eg, due to applied voltage); ultrasonic power; acoustic power; pressure force generated by light; may include one or more of laser-generated impact forces and other suitable forces.

In an embodiment of a fluid pressure actuation mechanism, the system 100 utilizes an adapter 110 comprising a structure for interacting with a pipettor at a location where the aspirator is fluidly coupled to a fluid space that interfaces with the capture well. wherein the system 100 has a first mode of operation for dispensing a fluid into the fluid space and a second mode of operation for aspirating a fluid from the fluid space; include The first mode of operation generates a local convective force in the capture well to loosen and lift material within the capture well, the second mode of operation generates a convective current to deliver the material from the capture well to the aspirator, and the target material is It may then be transferred to an elution vessel. The system 100 iterates between the first mode of operation and the second mode of operation to increase the efficiency of recovering the target material from the capture well. A variation of this embodiment is capable of producing recovered target material within manual operation times of 10 to 15 minutes with recovery efficiencies of 85% to 90% (relative to the percentage of captured particles recovered). Embodiments, variations, and examples of fluid pressure actuation systems and methods are disclosed herein entitled "Systems and Methods for Recovering and Analyzing Particles", filed November 16, 2017, which is incorporated herein by reference in its entirety. It is described in more detail in US Patent Application Serial No. 15/815,532.

Other embodiments, variations and examples of systems involving different forces are described in more detail in Sections 2.2 and 2.3 below. Further, embodiments, variations, and examples of the system may be configured to implement embodiments, variations, and examples of the method(s) described in Section 3 below.

In the context of sample processing, embodiments of system 100 may be configured or include processing cells, cell-derived materials and/or cells, or other biological materials (eg, cell-free nucleic acids). Cells are mammalian cells (e.g., human cells, murine cells, etc.), embryos, stem cells, plant cells, microorganisms (e.g., bacteria, viruses, fungi, etc.) or some or all of a cell of any other suitable type. may include A cell may contain a target substance (eg, a target lysate, mRNA, RNA, DNA, protein, glycan, metabolite, etc.) that occurs within the cell and is selectively captured by the cell capture system for processing. . Additionally, a vessel containing cells may be prepared from a plurality of cell containing samples (eg, 12 samples, 24 samples, 48 samples, 96 samples, 384 samples, 1536 samples, different number of samples). Alternatively, the various samples may be hashed or barcoded prior to mixing together in a single container (or reduced number of containers). A plurality of samples may be distributed to the same microwell chip by dispensing to geographically distinct locations on the chip. This feature enables automated processing of multiple samples with the same automated execution for each single cell preparation task and library preparation task. Additionally or alternatively, system 100 interacts with particles (eg, beads, probes, nucleotides, oligonucleotides, polynucleotides, etc.), droplets, encapsulated cells, encapsulated biomarkers, reagents, or other suitable substances. can be configured to

System 100 may also additionally or alternatively be disclosed in U.S. Patent Application Serial Nos. 16/890,417, filed on June 2, 2020, each of which is incorporated herein by reference in its entirety; U.S. Patent Application Serial No. 16/867,235, filed May 5, 2020; U.S. Patent Application Serial No. 16/867,256, filed May 5, 2020; US Patent Application Serial No. 16/816,817, filed March 12, 2020; U.S. Patent Application Serial No. 16/564,375, filed September 9, 2019; U.S. Patent Application Serial No. 16/115,370, filed August 28, 2018; U.S. Patent Application Serial No. 16/048,104, filed July 27, 2018; U.S. Patent Application Serial No. 16/049,057, filed July 30, 2018; U.S. Patent Application Serial No. 15/720,194, filed September 29, 2017; U.S. Patent Application Serial No. 15/430,833, filed February 13, 2017; U.S. Patent Application Serial No. 15/821,329, filed November 22, 2017; U.S. Patent Application Serial No. 15/782,270, filed October 12, 2017; U.S. Patent Application Serial No. 16/049,240, filed July 30, 2018; and U.S. Patent Application Serial No. 15/815,532, filed on November 16, 2017.

2.1 System - Sample Cartridge and Sample Processing Chip

1A and 2A-2C , the sample processing chip 132 functions to provide one or more processing regions in which cells are captured and optionally processed for sorting, processing, or otherwise processed for subsequent applications, and subsequently The application may be performed on a sample processing cartridge 130 (eg, on-chip) that supports the sample processing chip 132 and/or remotely from the sample processing cartridge 130 (eg, off-chip). there is.

The sample processing cartridge 130 supports the sample processing chip 132 and functions to provide a fluid path for fluid transfer, capture, and sample processing in the sample processing chip 132 . In addition, the sample processing cartridge 130 may function to facilitate heat transfer to/from the sample processing chip 132 in connection with a sample processing procedure. Portions of the sample processing cartridge 130 may be configured within a single substrate, but may additionally or alternatively include multiple portions (eg, connected by a fluid pathway) across multiple substrates.

2A-2D, an example of a sample processing cartridge 130' may include a base substrate 131 to which other elements are coupled and/or to which other elements are defined. Further, with respect to processing a sample comprising a microfluidic element, the base substrate 131 for delivering a fluid to the microfluidic element, for accessing the sample processing space at various processing steps, and for carrying waste generated during sample processing. It can function as a manifold for In a variant, the base substrate 131 is a sample processing chip 132 , an input reservoir 133 for receiving sample material (eg, including cells, including particles) and delivering it to the sample processing chip 132 . ), an access region 134 for accessing one or more regions of the sample processing chip 132 , a lid 135 comprising a gasket 136 covering the access region and providing a sealing function, and a sample processing chip 132 . supports one or more of the waste containment areas 137 for receiving waste from The cartridge may also have an additional gasket port for connection with an off-cartridge pumping system. However, the deformation of the base substrate 131 may include other elements. For example, as described in more detail below, the base substrate is in addition to the sample processing chip 132 to collectively define a valve region for opening and closing flow through the sample processing chip 132 . It may include one or more openings, recesses, and/or projections that provide for engagement.

2A and 2C (bottom view), a sample processing chip 132 (also referred to herein as a microwell device or slide) is a series of wells 103 ( For example, microwells). Each well in a series may be configured to capture a single cell and/or one or more particles (eg, probes, beads, etc.), a suitable reagent, a plurality of cells, or other substances. In a variation, the microwells of the sample processing chip 132 may be configured to co-capture single cells with single functional particles to allow analysis of single cells and/or material from single cells without contamination throughout the well. . Embodiments, variations, and examples of the sample processing chip 132 are disclosed in U.S. Patent Application Serial Nos. 16/890,417, filed June 2, 2020, each of which is incorporated herein by reference in its entirety by reference above; U.S. Patent Application Serial No. 16/867,235, filed May 5, 2020; U.S. Patent Application Serial No. 16/867,256, filed May 5, 2020; US Patent Application Serial No. 16/816,817, filed March 12, 2020; U.S. Patent Application Serial No. 16/564,375, filed September 9, 2019; U.S. Patent Application Serial No. 16/115,370, filed August 28, 2018; U.S. Patent Application Serial No. 16/048,104, filed July 27, 2018; U.S. Patent Application Serial No. 16/049,057, filed July 30, 2018; U.S. Patent Application Serial No. 15/720,194, filed September 29, 2017; U.S. Patent Application Serial No. 15/430,833, filed February 13, 2017; U.S. Patent Application Serial No. 15/821,329, filed November 22, 2017; U.S. Patent Application Serial No. 15/782,270, filed October 12, 2017; U.S. Patent Application Serial No. 16/049,240, filed July 30, 2018; and U.S. Patent Application Serial No. 15/815,532, filed on November 16, 2017.

In material construction, the sample processing chip 132 may be constructed of microfabricated silicon or free fused silica material, which may, for example, have sharper edges (eg, thinner well walls, 90 degrees) in a series of wells. functions to enable higher resolution of a series of wells made possible by defining well walls arranged at an angle close to The material composition also facilitates optical examination of the contents of the sample processing chip 132 (eg, via the bottom surface, via the top surface) with respect to the imaging subsystem 190 , which is described in more detail below. can do. The materials and manufacturing processes described may further enable one or more smaller characteristic dimensions (eg, length, width, total footprint, etc.) of microwell cartridges as compared to conventional chip designs. In certain instances, the sample processing chip 132 may include a number of completed devices with an acceptable level of defects (eg, < 5%); Depth measured to within ±1 μm of nominal depth (eg, 25 μm); Fabricated using deep reactive ion etching (DRIE) techniques according to specifications relating to one or more of the ribs measured to within ±1 μm of nominal rib dimensions (eg, 5 μm). To alleviate problems during fabrication, specific examples of sample processing chip 132 include: a) the resist thickness and lithography required to etch a glass substrate having a nominal depth of 30 μm with a nominal width of 5 μm between microwells. decision of; b) determination of lateral resist erosion and mask bias; c) characterization of the vertical taper of the microwell sidewalls after etching; and d) optimization of the dicing process to achieve good yields of the final device.

Additionally or alternatively, the sample processing chip 132 may include, but is not limited to, other suitable materials such as, but not limited to, polymers, metals, biomaterials, or other materials or combinations of materials. The sample processing chip 132 may be fabricated by a variety of processes, such as precision injection molding, precision embossing, microlithographic etching, LIGA-based etching, or by other suitable techniques.

In some variations, one or more surfaces (eg, bottom, side, bottom and side, all sides, etc.) of a series of wells 103 may contain oligonucleotides for capturing biomarkers from individual cells into individual microwells. It can react with molecules. Oligonucleotide molecules present in each individual microwell can be barcoded so that biomarkers treated in each microwell can be linked back to specific wells and linked to specific single cells. In one variation, the series of wells comprises a series of microwells having a hexagonal cross-section taken transverse to the longitudinal axis of the wells as described in one or more of the patents incorporated by reference above.

In one variation, as shown in FIG. 2C , the sample processing chip 132 is configured to dispense fluid into an input opening 32 , a series of microwells 34 (eg, 1,000 to 10,000,000 wells). A first fluid distribution network 33 downstream of the input opening for 2 may include an outlet opening 36 coupled to the distal end of the fluid distribution network 35 . In this variation, the sample processing chip 132 is disposed on a first side (eg, lower side) of the base substrate 131 (eg, by laser welding, adhesive bonding, solvent bonding, ultrasonic welding, or other techniques). is coupled to Coupling the sample processing chip 132 to the side of the base substrate 131 may result in a series of microwells 34 and/or sample processing chip 132 in a heating and cooling subsystem 150 described in greater detail below. Heat can be transferred to other areas.

Also, as described above, the base substrate 131 is provided with an input reservoir 133 (eg, defined on a second side of the base substrate 131 opposite the first side to which the sample processing chip 132 is coupled). ) may be included. The input reservoir is a sample material (eg, a sample comprising cells, a sample comprising barcoded cells) from the aforementioned process vessel 20 ′ for delivery to the input opening 32 of the sample processing chip 132 . , a sample comprising encapsulated material, a sample comprising particles, etc.) and/or a sample processing material. In a variant, the input reservoir 133 may be defined as a recessed region in the surface of the base substrate 131 , the recessed region comprising an aperture that aligns and/or seals with the input opening 32 of the sample processing chip 132 . . The dosing reservoir 133 of the base substrate 131 may interface with an upstream fluid comprising components and/or bubble relief components as described in the patent, incorporated in its entirety by reference above.

In a variation, one or more of the inlet 32 of the sample processing chip 132 and the input reservoir 133 of the base substrate 131 are valve components that can be opened or closed by one or more components of the system 100 . may include In a first variant, the dosing reservoir 132 includes an aperture that can be accessed by a pipette tip or other suitable attachment of a fluid handling subsystem coupled to the gantry 170 (described in more detail below). In some embodiments, the aperture may be closed to prevent fluid from moving from the input reservoir 132 to the sample processing chip 132 . However, the input reservoir 133 may be configured in other suitable ways. The opening associated with the dosing reservoir 133 will have a conical surface open towards the top to interface and seal the pipette tip so that a fluid (aqueous solution or oil or air) can be pumped directly into the microchannel defined as 33 in FIG. 2C. can

2A and 2B , the base substrate 131 may also define an access region 134 for accessing one or more regions of the sample processing chip 132 , the access region comprising: Allows regions of 132 to be viewed and/or extracted from the sample processing chip 132 at various sample processing steps. 2A and 2B , the access region 134 may be defined as a recessed region within the base substrate 131 and an opening aligned with the region of the sample processing chip 132 comprising a series of microwells. (37) may be included. The sample processing chip 132 may have as few as 100 microwells to as many as 100 million microwells. As such, in variations in which the microwell region is open to the environment (eg, without a cover sealing the well), the opening 137 of the access region 134 (eg, as described in more detail below) As such, it may function as a microwell providing access to the contents of the microwell for observation and/or material extraction (by magnetic separation). The opening 37 may match the shape and space occupied by the microwell, and in the first variation may be a square opening as shown in FIG. 2B . However, in other variations, the opening 37 may have any other suitable shape.

2A-2C , the base substrate 131 may include or otherwise be coupled to a lid 135 covering the access area 134, the lid 135 providing a sealing function. A gasket 136 may be included, and the lid 135 prevents sample loss and/or contamination of the sample processing chip 132 contents during operation due to evaporation by switching the access area 134 between an open mode and a closed mode. function to prevent. The lid 135 additionally or alternatively protects the contents of a microwell or other processing region of the sample processing chip 132 from debris, and (eg, by isolating the region from the surrounding environment) the sample processing chip 132 . enables handling of the contents of the sample handling chip 132 (e.g., by opening it to receive reagents from a pipettor), initiating a protocol (e.g., by closing after adding all necessary reagents), and between a series of wells and a waste chamber (e.g., serving as a boundary for a fluid path adjacent a microwell region, serving as a top surface of the fluid path between a series of microwells and an inlet); It serves to define some or all of a fluid path, cavity, or reservoir (eg, as a sheath 135 ), which serves as the top surface of the fluid path, or to perform other suitable functions.

As shown in FIG. 2B , in at least one variation, the lid 135 is configured such that the lid 135 mates with the access region 134 while providing a gap with the sample processing chip 132 . may be complementary in features and form. Additionally, in a variant (shown in FIGS. 3B and 3C ), the lid 135 may be substantially flush with the base substrate 131 at its top surface when the lid is in the closed position. However, the lid 135 may be morphologically configured in other suitable ways.

In a variant, the protrusion 38 of the lid 135 may interface with the opening 37 of the access area 134 to substantially prevent access to the opening 37 when the lid is in the closed position. As shown in FIG. 2B , in some variations, the protrusion 38 may have a base (or other area) surrounded by a gasket 136 , which opens in the access area 134 in the closed position of the lid 135 . (37) It functions to seal. However, deformation of the lid 135 may omit the gasket and facilitate sealing of the access region 134 in any other suitable manner. In another embodiment, the entire bottom surface of the shroud closest to the microwells of the sample processing chip 132 may be covered with an elastomeric shroud covering the microwells to prevent evaporation or diffusion loss of molecules during thermocycling in each of the microwells. It may be an elastomeric substrate (eg, a flat elastomeric substrate) that allows

In some variations, the lid 135 may include a locking or latching mechanism that allows the lid 1235 to remain in a closed position with the base substrate until the locking/latching mechanism is released. 3A-3C , the periphery of the lid 135 may include one or more tabs 39 that interface with corresponding tab receptacles of the base substrate 131 , wherein the tabs 39 are It interfaces with the tab receiving portion of the base substrate 131 and is configured to bend when pushed into the base substrate 131 until it returns from the bent configuration to the latched state. Additionally or alternatively, in the variant shown in FIGS. 3A-3C , the locking/latching mechanism releases the tab(s) 39 from the tab receptacle and goes from the closed mode to the open mode with respect to the base substrate 131 . may include a release body 41 (eg, rod, recess, hook, etc.) that may be interfaced to convert the lid 135 . As such, the lid 135 provides an open mode in which the access area 134 is uncovered and a closed mode in which the access area 134 is covered. In the variant shown in FIGS. 3A-3C , the release element 41 comprises a concave bar remote from the access area 134 of the base substrate 131 , the bar being reversibly coupled to the lid opener 145 . can In a variation, the lid opener 145 is a first region (eg, a first area) that interfaces with an actuator (eg, an actuation tip, a pipettor of a fluid handling subsystem coupled to the gantry 170 described below, etc.) distal) and a second region (eg, a second end) comprising a connecting element 42 configured to interface with the release element 41 of the lid 135 . Thereafter, with movement of the pipette/pipette interface, the lid opener 145 pulls the release element 41 and/or depresses the lid 135 to switch the lid 135 between open and/or closed modes. It can be configured to As such, with respect to a fluid handling element coupled to the gantry 170 , described below, the system 100 is a lid opener that includes a connection element 42 to an actuator (eg, coupled to the gantry 170 ). (145); moving the lid opening tool to align with the release element 41 of the lid 135 reversibly coupling the release element 41 and the connection element 42; and applying a force to the release element to release the lid 135 from the latching state and to switch the lid 135 from the closed mode to the open mode. In order to effectively apply an unlatching force (eg, by an actuator coupled to the gantry 170 ), the base substrate 131 may be counteracted by an unlatching force applied via the lid opener 145 . Maintenance of the deck that passively or actively applies a counter force (e.g., by the retaining elements described in section 2.1.4, by the retaining elements of the heating and cooling subsystem, by the retaining elements of the fluid level sensing subsystem, element) can be held in the correct position.

However, in variations, the locking/latching mechanism may additionally or alternatively include or operate via a lock-key mechanism, magnetic element, or other suitable mechanism. Also, in alternative variations, the lid 135 may include other lid actuators including, for example, a motor that rotates the lid about access parallel to the large surface of the sample processing cartridge 130 . The actuator may additionally or alternatively move the lid 135 (eg, slide the lid 135 parallel to the large surface of the sample processing cartridge 130 , and move the lid 135 perpendicular to the large surface) ) or otherwise moving the lid 135 to selectively cover or not cover one or more predetermined regions (eg, a series of microwells). As such, the lid 135 operates in an automated or semi-automated manner such that one or more triggers (eg, a cell capture protocol is initiated by a user, a cell handling protocol is initiated by a user, and all reagents for a selected protocol are processed. Lid 135 automatically closes upon being added from container 20, etc.) and one or more triggers (eg, cell capture protocol completed, cells confirmed as viable per user request, single cells captured confirmed to have been opened, etc.). Additionally or alternatively, actuation of lid 135 may be initiated and/or completed by a user, actuated according to a schedule or other temporal pattern, or otherwise actuated.

2A-2C , the base substrate 131 may also include a waste containment area 137 for receiving waste from the sample processing chip 132 . The waste containment area 137 also maintains a desired pressure (eg, vacuum pressure, etc.) within the sample treatment chip 132 so that liquid is transferred from the input reservoir 133 through the sample treatment chip 132 to the waste containment area. It can function to allow flow to (137). A waste containment area 137 extends from the base substrate 132 (eg, coupled to an outlet of the base substrate 131 ) for receiving waste or other material from the sample processing chip 132 . 131) concave into, etc.) space. In the variant shown in FIGS. 2A-2C , the waste containment area 137 is a waste containment area 137 where waste is removed from the sample processing chip 132 by the force of a pumping subsystem 157 , which is described in more detail below. It is defined on the side of the base substrate 131 opposite to the side to which the sample processing chip 132 is coupled so as to be pushed up or pulled by the . However, waste containment vessel 137 may additionally or alternatively be configured at other suitable locations relative to sample processing chip 132 and base substrate 131 to receive waste. The waste containment area 137 may have a volumetric capacity between 10 ml and 100 ml or other suitable volumetric capacity.

As shown in FIGS. 2A-2C , waste containment area 137 is substantially flush with cover 48 (eg, cover 135 ) that facilitates containment of waste within waste containment area 137 . cover) may be included. Alternatively, the waste containment area 137 may not include a cover. Also, as shown in FIG. 2C , an example of a waste containment area 137 may include a pump outlet 51 that is distinct from the cover, wherein the pump outlet 51 allows residual air in the waste chamber to pass to the off-cartridge pump. A modification of waste containment area 137 may alternatively omit a waste outlet, although it may be pressurized by (eg, by a pumping mechanism).

With respect to the waste containment area 137 , the system 100 may also include a valve 43 configured to permit and/or prevent flow from the sample processing chip 132 to the waste containment area 137 . there is. The valve 43 may interface with the vent opening 36 of the sample processing chip 132 described above to allow and/or block flow to and from the waste containment area 137 and out of the vent opening 36 . . The valve 43 may be normally in an open state and transition to a closed state when interacting with the valve actuating mechanism. Alternatively, the valve 43 may be normally in a closed state and transition to an open state when interacting with the valve actuating mechanism.

In the variant shown in FIGS. 2A and 4A-4B , the valve 43 may comprise an elastic body and an opening 44 of the sample processing chip 132 that aligns with a corresponding valve receptacle of the base substrate 131 . ) through which the sample processing chip 132 is coupled to the base substrate 131 . In this variant, the switchable portion of the valve 43 follows the flow path from the outlet opening 36 of the sample processing chip 132 to the inlet of the waste containment area 137 of the base substrate 132 (eg, , along a flow path from the microwell area to the outlet of the sample processing chip) into a waste containment area of the sample processing cartridge. In one example, the opening 44 of the sample processing chip 132 is adjacent to the exhaust opening 37 of the sample processing chip 132 , but in other variations, the exhaust opening 37 and the opening 44 are displaced from each other. and can be connected by other microfluidic channels. As such, closing of valve 43 may block flow from discharge opening 37 to waste containment area 137 , and valve 43 may cause flow from discharge opening 37 to waste containment area 137 . may be open to allow

In the variant shown in the cross-sectional images of the base substrate 131 shown in FIGS. 4A-4B , the valve actuator 45 can access the base substrate 131 from below (eg from under the deck), It may pass through a channel or other recess/opening in the base substrate 132 to interact with the valve 43 . In particular, when the tip 46 (aligned with the opening into the base substrate) of the valve actuator 45 pushes the valve (eg, the elastic membrane of the valve 43), as shown in FIG. 4B (top), , valve 43 can be switched to a closed state to fluidly isolate the outlet opening 37 of the sample processing chip 132 from the waste containment area 137 . Additionally or alternatively, as shown in FIG. 4B (bottom), removal of force by valve actuator 45 relieves pressure at valve 43 and transitions to an open state for sample from waste containment area 137 . The discharge opening 36 of the treatment chip 132 may be fluidly coupled. As such, the valve actuation subsystem includes an engaged mode in which the tip extends into the valve opening to deform the resilient valve to close the flow path, and a disengage mode, in which the tip retracts to open the flow path. However, the valve 43 may additionally or alternatively be configured in any other suitable manner.

In other variations, the system may include a similar mechanism for coupling the valve to other flow paths of the base substrate 131 and/or the sample processing chip 132 .

However, the deformation of the base substrate 131 may include other elements. For example, as described in more detail below, the base substrate 131 is one that provides additional coupling with the sample processing chip 132 to facilitate or inhibit flow through the sample processing chip 132 . It may include the above openings, recesses and/or protrusions. For example, as shown in FIG. 4A , the base substrate 131 is a pumping sub (eg, via the deck 110 ) to drive and/or stop fluid flow through the sample processing chip 132 . A pump opening 46 coupling the base substrate 131 to the pumping element of the system 157 may be included. However, the base substrate 131 of the sample processing cartridge 130 may include other suitable elements.

Embodiments, variations, and examples of chip 101 may include embodiments, variations, and examples of capture devices described in one or more patents incorporated by reference above.

2.2 System - vessel for disposing of recovered material

With respect to processing the recovered material using the described separation system (eg, purification, washing, extraction, amplification, etc.), system 100 may also include a process vessel 20 . The process vessel 20 functions to process the recovered target component of the sample according to one or more workflows for various applications, as described in more detail below. As such, material may be withdrawn from the aforementioned sample processing chip 132 and delivered to the process vessel 20 for further processing, as will be described in greater detail below. Additionally or alternatively, in some variations, the process vessel 20 may include in one or more compartments materials for cell capture and sample handling in the context of a fully automated system. As such, process vessel 20 may define a series of storage spaces distributed throughout a series of domains, which series of domains may be configured to provide an environment suitable for the material content of each domain. The series of storage spaces may directly contain the sample processing material, and/or may alternatively be configured to receive and maintain the location of individual containers (eg, tubes, etc.) containing the sample processing material. The storage space of each domain may be distributed in an array or otherwise arranged.

The individual storage spaces of the series of storage spaces of the process vessel 20 separate the materials within the process vessel 20, prevent cross-contamination between materials within the individual storage spaces, prevent contaminants from entering the individual storage spaces, and It may further include one or more sealing parts that function to prevent volatilization and loss during storage and/or shipping. The sealing portion(s) may be pierceable sealing portion(s) (eg, composed of paper, composed of metal foil, and/or composed of other suitable materials). However, the sealing portion(s) may alternatively be configured to be impenetrable (eg, the sealing portion(s) may be configured to be releasable from the process vessel 20 ). In embodiments, certain reagent vessels may be sealed with a hinged lid that can be tooled open or closed as needed (as described in more detail below) for processing at the appropriate stage of the protocol.

In a variation, the series of domains comprises a first domain for storing reagents requiring a refrigerated environment (eg, at a temperature between 1 °C and 15 °C), a second domain for storing substances that can be stored at room temperature conditions, as described below. A third domain, functionalized particles (eg, as described in U.S. Patent Application Serial No. 16/115,370, , a fourth domain to store a bead with a probe having a barcoding region and other functional regions, and a fifth domain to perform a separation operation (e.g., separation of a target material from a non-target material by a magnetic field). may include In a variant, domains providing different environments for storage may be configured differently. For example, the first domain (ie, for cold storage) may be comprised of an insulating material and/or may include an insulating material for the storage space of the domain (eg, individually for the entire domain). . Additionally or alternatively, the domain for isolation may include a magnetically conductive material configured to provide suitable magnetic field properties for isolation. Additionally or alternatively, domains for thermal cycling or other heat transfer applications may be constructed of a thermally conductive material to facilitate efficient heat transfer to/from the process vessel 20 . In embodiments, the various domains may be optimally placed such that crosstalk between specific tasks is minimized. For example, domain(s) for refrigerated reagent storage can be maintained at run-time temperature (e.g., 4 °C) and domain(s) for PCR reactions can be heated (e.g., maximal during denaturation) 95 °C) may be required. As such, to minimize the effect of PCR thermocycling on refrigerated reagents, the domain(s) comprising the reagents stored at room temperature may be configured to be between the PCR thermocycling domain(s) and the refrigerated storage domain(s). there is. To further prevent thermal crosstalk, additional buffer tubes with only air between specific domains requiring independent temperature control can be used.

In variations, the process material supported by the domains of the process vessel 20 is a buffer (eg, ethanol, priming buffer, lysis buffer, custom lysis buffer, sample wash buffer, saline with RNAase inhibitor, bead wash buffer, RT buffer, buffer, etc.), oil (e.g., perfluorinert oil), PCR master mixture, cells, beads (e.g., functionalized beads), or any other suitable material used for cell capture and/or sample processing. may include more than one. Additionally or alternatively, one or more of the series of storage spaces may be empty (eg, initially empty, empty throughout one or more processes, empty before an operator fills it, etc.). The different storage areas of the various domains of the process vessel 20 may have initial reagent volumes of several milliliters (eg, 5 milliliters) to 50 milliliters.

In a particular example, as shown in FIG. 5 , the process vessel 20 ′ has a first domain 21 ′ in a first peripheral region of the process vessel 20 ′ for storing reagents requiring a refrigerated environment. , a second domain 22' in the central region of the process vessel 20' for storing material that can be stored at room temperature, a tube containing the material for performing a polymerase chain reaction (PCR) operation. A third domain 23' in the peripheral region of the process vessel 20', near the second domain 122 for a fourth domain 24' in the peripheral region of the process vessel 20' for storing probes with probes having such barcoded regions and other functional regions, and a separation operation (eg, by magnetic force). and a fifth domain 25' in the peripheral region of the process vessel 20' for performing target material separation). In certain instances, the fourth domain 24' may be a modular element, such that the fourth domain 24' engages the process vessel 24' with the fourth domain 24' set in place. At that point, the functionalized particles may be stored separately from the rest of the process vessel 20' until ready for use.

In a specific example, the first domain 21' and the second domain 22' are covered with a first sealing portion comprised of a metal foil, and the third domain 23' and the fifth domain 25' are made of paper. is covered with a second sealing portion constituted, and the fourth domain 24' is covered with a third sealing portion constituted of a metal foil. However, variations on the example of process vessel 20' may be constructed in other suitable manners.

Also, variations 20' of process vessel 20 may omit various domains and may be configured for processing and separation of recovered target material, as described in more detail below.

The process vessel 20 may additionally or alternatively further comprise aspects described in the patents incorporated by reference above.

2.3 System - Deck, Separation Subsystem, and Gantry Sides

In variations, the side of the sample processing cartridge 130 and process vessel 20 may be supported by, or otherwise interact with, other system elements (eg, of a system for automating sample processing). 6A-6B and 7A-7C , in an embodiment, system 100 is configured for automated sample processing (eg, at the upper broad surface, at the upper and lower broad surfaces, at the side in, etc.) may include a deck 10 that serves as a platform for supporting and placing one or more components of the system 100 . Deck 10 also includes a fluid processing subsystem, an imaging subsystem, a heating subsystem, a separation subsystem (eg, a magnetic separation subsystem), and/or a gantry 170 and/or base ( 180 may function to place one or more components of system 100 that are aligned with or interact with other subsystems coupled thereto. In this regard, the deck 10 may be secured as a reference platform, while other components are actuated into position to interact with the elements of the deck 10 . Alternatively, deck 10 may be coupled with one or more actuators for positioning elements of deck 10 for interaction with other subsystems.

6A-6B, the deck 110 includes a sample processing cartridge 130, a process vessel 20, a tool vessel 40 (described in a patent incorporated by reference), and a heating and cooling sub. Provides a platform to support one or more units of system 50 , pumping subsystem 57 , fluid level sensing subsystem 59 , isolation subsystem 160 , and imaging subsystem 90 . The sample processing elements may be supported by the deck 110 in a coplanar manner or alternatively in different planes. Preferably, the individual elements supported by the deck do not overlap but alternative embodiments of the deck 10 can support the sample processing elements in an overlapping manner (eg, to save space, for operational efficiency). .

As such and as shown in FIGS. 7A-7C , the deck 10 also includes at least one area for supporting a unit of the sample processing cartridge 130 , the area including the heating and cooling subsystem 50 . ), the pumping subsystem 57 , the fluid level sensing subsystem 59 , and/or the imaging subsystem 90 ). In this regard, the region includes the associated portions of the heating and cooling subsystem 50 , the pumping subsystem 57 , the fluid level sensing subsystem 59 , and the imaging subsystem 90 and of the sample processing cartridge 130 . One or more openings, recesses, and/or protrusions may be included to provide an interface between complementary portions, and to promote and maintain alignment between such portions.

Likewise, as shown in FIGS. 7A-7C , the deck 10 may include at least one area for supporting the units of the process vessel 20 , the area being heated, described in more detail below. and positioning the process vessel 20 relative to portions of the cooling subsystem 50 and the separation subsystem 160 . In this regard, the region provides an interface between relevant parts of the heating and cooling subsystem 50 and separation subsystem 60 and complementary parts of the process vessel, and additionally facilitates alignment between such parts and It may include one or more openings, recesses, and/or projections for retention.

7A and 7B , the deck 10 may also include at least one area for supporting a unit of the tool container 40, the area including a gantry (described in more detail below) Functions to position the tool container 40 relative to the fluid handling device of 170 . Embodiments, variations and examples of the tool container 40 are described in the patent incorporated by reference above.

Embodiments, variations and examples of heating and cooling subsystem 50 , pumping subsystem 57 , fluid level sensing subsystem 59 , and imaging subsystem 90 , and base 180 and gantry 170 . Couplings related to (including pipettor 174) are also described in greater detail in the patents incorporated by reference. Aspects of isolation subsystem 160 are further described below.

2.4 System - first variant for recovery by magnetic force

As shown in FIGS. 8A-8C , a variation of system 200 is a capture region of a sample processing cartridge (eg, the embodiment of sample processing cartridge 130 described above) to capture particles in a single particle format. a first region 311 configured to couple to, a second region 212 , and an interior cavity 220 passing from the first region to the second region (eg, the separation subsystem 160 described above). ) adapter 21; a magnet (230) configured to pass into the interior cavity (220) of the adapter (210) during operation and to apply an attractive force to the capture region of the sample processing cartridge (130); and a support structure 240 reversibly coupled to the magnet 230 and the second region 212 of the adapter 210 .

In addition, the support structure 240 of the system 200 may include a plunger subsystem 250 coupled to an ejector proximal to the magnet 230 , wherein in a baseline mode of operation, the adapter 210 supports the support structure 240 . and the plunger subsystem 250 is not activated, and in the ejection mode the adapter 210 disengages from the support structure in response to activation of the plunger subsystem 250 . In a variation, the plunger subsystem 250 may also include a structure that functions to facilitate fluid dispensing and aspiration functions for dispensing and/or retrieving a substance from the capture region of the sample processing cartridge 130 . In addition, the system 200 prevents physical contact between the capture area of the sample processing cartridge 130 and the magnet 230 during operation and the support structure 240 in position relative to the capture area of the sample processing cartridge 130 . and a guide 260 configured to hold the

The system 200 is capable of controlling a magnetic force in the capture region of the chip 210 to provide an attractive force to attract a target material bound (directly or indirectly) to a magnetic component in the capture region, into the adapter 210 . It functions to authorize. An embodiment of the method implemented by the system 200 is a manual operation time of 5 to 8 minutes (and a total time of 10 to 45 minutes) with a recovery efficiency of >90% in which only magnetic particles bound to the target material in the sample are recovered. It is possible to recover the target material within. Thus, system 200 can serve to increase selective recovery efficiency, thereby reducing processing burden and subsequent costs associated with processing reagents and other material costs (due to reduced requirements, reduced fractionation of biochemical reactions). System 200 may implement one or more embodiments, variations, or examples of the method(s) described below, and/or may be used to implement other methods.

2.4.1 First Magnetostriction - Adapter

8A-8C , the adapter 210 includes a first region 211 , a second region 212 configured to couple to a capture region of a sample processing chip 132 for capturing particles in a single particle format. ), and an interior cavity 220 passing from the first region to the second region. The adapter 210 provides a structure for separating the magnet 230 from a well or other sensitive material in physical contact with the capture area of the sample processing cartridge 130 , and the adapter interfaces with the capture area of the sample processing cartridge 130 . By transferring a magnetic force to the region of 210 serves to support application of a magnetic field to the capture region for target material recovery of the sample processing cartridge 130 by the system 200 . The adapter 210 may also function to prevent sample cross-contamination by serving as a disposable component that may be discarded between uses of the system 200 . The unit of adapters may be stored in the embodiment of the tool container 40 supported by the deck 10 described above, or may be stored or prepared by the system in any other suitable manner. As shown in FIG. 8A , units of adapters 210 can be held in place within a portion of a rack or tool container 40 until they need to be used.

The adapter 210 may be morphologically prismatic with an interior cavity 220, and the cross-section of the adapter 210 along its longitudinal axis may have a polygonal perimeter, an ellipsoidal perimeter, an amorphous perimeter, or other suitable shape (eg, closed shape, open shape). The cross-section of the adapter 210 may complement the shape of the space occupied by the capture area of the sample processing cartridge 130 , but may alternatively not complement the shape corresponding to the capture area of the sample processing cartridge 130 . The adapter 210 may have a width of 0.2 cm to 4 cm and a length of 0.5 cm to 8 cm (eg, corresponding to the shape of the capture area of the chip 201 ). Preferably, the adapter 210 has a wall thickness that supports the application of a magnetic force from the magnet 230 to a capture region that interfaces with the first region 211 of the adapter 210 . The wall thickness may or may not be constant along the length of the adapter 210 . In an example, the wall thickness may be between 0.2 mm and 3 mm thick, although in other examples, the wall thickness may have any other suitable thickness. The surface of the adapter receiving the magnetic particles is smooth (e.g., with a better surface condition than SPIB1) so that small magnetic particles (1 μm to 3 μm) are not trapped on the surface while the beads are trapped on the surface and then released to another container. is made

Adapter 210 may additionally or alternatively include structural features that enable modes of operation of system 200 . For example, in connection with detaching the adapter 210 from the support structure 240 (described in more detail below), the adapter 210 includes a protrusion 214 configured to interface with the plunger subsystem 250 . and a trigger of the plunger subsystem 250 may push the protrusion 214 to disengage the adapter 210 from the support system 240 when the plunger subsystem 250 is activated. The protrusion 214 may be a border for the second region 212 of the adapter 210 , or alternatively may be defined by any other suitable shape.

As noted above, the adapter 210 facilitates application of a magnetic field to the capture region and is capable of attracting a target material (eg, a target material bound to magnetic particles) into the adapter 210 for further subsequent processing. the exposed capture region and first region 211 of the sample processing cartridge 130 (eg, with the lid 134 open to provide access to the access region 134 ) interface in Accordingly, the adapter 210 may include a sealing portion in the first region 211 to facilitate a mechanism for attracting a target material from the sample processing chip 132 to the adapter 210 . The sealing portion may be an integral element with the adapter 210 or a separate element. However, the adapter 210 may omit the sealing portion in the first region 211 . The adapter 210 also couples to the support structure 240 in the second region 212 to hold it in position relative to the magnet 230 and to reversibly couple and remove the support structure 240 from it. . The coupling of the adapter 210 to other components may occur with one or more of a press fit, a snap fit, a female-male mating interface, screws, other fasteners, magnetic mechanisms, and other suitable mechanisms.

The adapter 210 may be made of a polymer material (eg, plastic) that does not adversely affect the magnetic field applied by the magnet 230 during operation. The adapter 210 may additionally or alternatively be comprised of or include (eg, a particle thereof) a material (eg, a metallic material) that is magnetic or capable of generating an induced magnetic field to support a usage application of the system 200 . can be included). Adapter 210 may additionally or alternatively be constructed from other suitable materials. The distribution of the material(s) of the adapter 210 may be homogeneous or non-homogeneous through the body of the adapter with respect to the desired magnetic effect in the capture region of the chip 201 . The inner cavity 220 of the adapter 210 may include a medium (eg, a magnetic medium, etc.), or alternatively may not include a medium.

2.4.2 First Magnetostriction - Magnet

As shown in FIGS. 8A-8C , the magnet 230 passes into the inner cavity 220 of the adapter 210 , and during operation exerts an attractive force on the capture area and the target material captured by the sample processing cartridge 130 . is composed The magnet 230 generates a magnetic field capable of attracting a target material captured in the capture region of the sample processing chip 132 (eg, associated with magnetic particles in the capture region) towards the adapter 210 for further processing. function to The magnet shape and pole configuration is such that a magnetic force is applied that is almost perpendicular to most target microwells from which trapped particles are being removed.

The magnet 230 may be morphologically prismatic, and the cross-section of the magnet 230 along its longitudinal axis may be of a polygonal perimeter, an ellipsoidal perimeter, an amorphous perimeter, or other suitable shape (eg, a closed shape, an open shape). defined by boundaries. In a variation, the magnet may have a length of 0.25" to 5" and a width of 0.1" to 1". In a particular example, the magnet has a square cross-section along its length and has sides of 2" long and 0.25" wide. The particular example magnet 230 weighs 15.4 grams.

The magnet 230 couples to the support structure 240 at the first end and passes into the interior cavity 210 of the adapter 210 . In a variant, as shown in FIG. 2B , the units of magnet 230 (and corresponding adapter 210 ) may have different dimensions to support different variants of chip 201 . For example, a unit of magnet 230 may have a small cross-section (eg, 0.25″×0.25″) to support chip deformation with a small number of microwells, and a unit of magnet 230 may have a larger unit of magnet 230 . It can have a cross-section (eg, 0.375" x 0.375" to support chip variants with more microwells).

The magnet 230 is constructed of a permanent magnetic material, but may alternatively be an electromagnet. In variations, the magnet 230 may be constructed of one or more of alnico, neodymium, neodymium iron boron, samarium cobalt, ferrite, and other suitable magnetic materials. Magnet 230 may additionally or alternatively include a plating material to facilitate operations involving processing of a biological or other sample. In a specific example, the magnet 230 is constructed of neodymium iron boron (NdFeB, grade 42) with a nickel-based coating (eg, a nickel-copper-nickel coating).

Magnet 230 may have more than one magnetization direction, with a surface field of up to 12,000 Gauss in strain, an internal field of up to 30,000 Gauss (eg, BRmax of 30,000 Gauss), and an energy of up to 90 MGOe. It can generate pulling and/or pushing forces of up to 10 lbs with density (BHmax). In the specific example, the magnet 230 has a magnetization direction through its thickness, a pulling force of 5.58 lbs, a surface field of 6584 Gauss, a BRmax of 13,200 Gauss, and a BHmax of 42 MGOe. In terms of magnetic field, the magnet 230 of the particular example is magnetized through its length, so the pole is one of the 0.25" x 0.25" ends of the magnet 230 . However, magnet 230 may alternatively be configured to create other suitable fields.

2.4.3 First magnetostriction - support structure

8A and 8C , the support structure 240 is reversibly coupled to the magnet 230 and the second region of the adapter 210 . The support structure holds the magnet 230 in position and functions to switch between the working modes for engaging and disengaging the adapter 210 and the magnet 230 . In addition, the support structure 240 may function to switch between operating modes for taking material out of the capture area of the sample processing cartridge 130 for subsequent processing.

The support structure 240 may have a housing having a form factor similar to that of a manual pipettor, the housing having a gripping area (eg, a series of protrusions and recesses) configured to complement a user's hand. has a surface with Alternatively, the support structure 240 may be configured not to be handled by a human operator, and additionally or alternatively a robotic device (eg, a robotic device (eg, for automated target material retrieval from the capture area of the sample processing cartridge 130 ) It may include a function for interfacing with the interface of the pipettor 174 coupled to the gantry 170 described above). Variations of this embodiment are described in more detail below along with various modes of operation for material recovery and disposal.

As noted above, the support structure 240 of the system 200 may also include a plunger subsystem 250 coupled to an ejector proximal to the magnet 230 , in which the adapter 210 is supported in a baseline mode of operation. It is coupled to the structure and the plunger subsystem 250 is not activated, and in the ejecting mode the adapter disengages from the support structure 240 in response to activation of the plunger subsystem 250 . As noted above, the ejector may interface with the protrusion 214 of the adapter 210 to disengage the adapter 210 from the support structure 240 in an ejecting mode. Also in variations, the plunger subsystem 250 may also include a structure that functions to facilitate fluid dispensing and suction functions to dispense and/or retrieve material from the capture region of the chip 201 . As such, a variant of the plunger subsystem 250 may perform a function similar to that of a pipettor, along with assisting in applying a magnetic field and disconnecting the adapter.

Support structure 240 may be comprised of one or more polymeric materials (eg, plastics) that are sterilizable (eg, autoclavable, resistant to damage by ethanol, etc.) between uses of system 200 . can However, support structure 240 may alternatively be constructed of other suitable materials.

2.4.4 First Magnetostriction - Guide

8A and 8C , the system 200 maintains the support structure 240 in the correct position relative to the capture area of the sample processing chip 132 and captures the sample processing chip 132 during operation. and a guide 260 configured to prevent physical contact between the region and the magnet 230 . Accordingly, the guide 260 serves to provide support for the support structure 240 and/or the chip 201 during the retrieval process. As such, in one variation, the guide 260 is coupled to the chip 201 as well as the coupled magnet 230 and adapter to secure the relative alignment between the adapter 210 and the capture region of the sample processing chip 132 . a recessed region configured to receive a support structure 240 having a 210 . The guide guides the adapter surface at a fixed distance (e.g., 25 μm, 100 μm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, etc.) into the micro- of the sample processing cartridge 130 . to be placed over the well. In another variation, as shown in FIG. 8C , the guide 260 is coupled to the sample processing cartridge 130 and is configured to position the adapter 210 relative to the capture area of the chip 201 simply by connecting to the adapter. can be configured. However, the guide 260 may be coupled to other suitable portions of the system 200 to provide support.

Guide 260 may be comprised of one or more polymeric materials (eg, plastic) that are sterilizable (eg, autoclavable, resistant to damage by ethanol, etc.) between uses of system 200 . there is. However, the guide 260 may alternatively be constructed of other suitable materials. Also, the guide 260 may be a disposable part of the system 200 .

2.5 System - Second variant of recovery and disposal by magnetic force

6A, 7B, and 9A-9C, variations of system 200' use magnetic forces to facilitate target material separation and recovery from non-target material adapter 210. '), magnets 230' and support structure 240' (eg, of separation subsystem 160). In variations, separation subsystem 160 may include embodiments, variations, and examples of components described in greater detail below and in patents incorporated by reference above. However, other variations of separation subsystem 160 may additionally or alternatively include other components.

2.5.1 Second Magnetostriction - Adapter

As shown in FIGS. 9A-9C , the adapter 210 ′ may be configured to enable target material retrieval from the sample processing chip 132 , for example through an access region 134 , the sample processing chip 132 . and a first region 211 configured to interface with the . The adapter 210' also provides an interior cavity 220' passing from the first region to the second region, and for engaging the magnet 230' (eg, magnetic distal end) of the support structure 240'. It may include a second region 212 ′. Adapter 210 ′ supports application of a magnetic field to a desired area for retrieval of target material (or non-target material) and magnet 230 from physically contacting wells or other sensitive materials of sample processing chip 132 . ') and/or serve to provide a structure separating the support structure 240'. The adapter 210' may also function to prevent sample cross-contamination by serving as a disposable component that may be discarded between uses of the system 200'.

The adapter 210' may be morphologically prismatic with an interior cavity 220', and the cross-section of the adapter 210' along its longitudinal axis may have a polygonal perimeter, an ellipsoidal perimeter, an amorphous perimeter, or other suitable shape (e.g., For example, a closed shape, an open shape) is defined by the boundary. The cross-section of the adapter 210 ′ may complement the shape of the space occupied by the microwell region of the sample processing chip 132 , but alternatively may not complement the shape corresponding to the sample processing chip 132 . Preferably, the adapter 210' has a wall thickness that supports the application of a magnetic force from the magnet 230' to the sample processing chip 132 that interfaces with the first region 211' of the adapter 210'. The wall thickness may or may not be constant along the length of the adapter 210 . In an example, the wall thickness may be between 0.2 mm and 3 mm, although in other examples, the wall thickness may have any other suitable thickness. The surface of the adapter 210 ′ that receives the magnetic particles allows small functionalized particles (eg, with characteristic dimensions of 1 μm to 3 μm) to be captured and then released to another vessel (eg, process vessel 20 ). It is made smooth (e.g., a better surface finish than SPIB1) so that it does not get trapped on the surface while it is being molded.

Additionally or alternatively, adapter 210 ′ may include architectural features that enable a detach mode of operation of detach subsystem 160 , which is described in greater detail below. For example, with respect to adapter 210 ′ separation from support structure 240 ′, adapter 210 ′ can be used to separate adapter 210 ′ from support structure 240 ′ with another object (eg, , a sleeve stripping tool 165 , described in greater detail below, may include a protrusion 214 ′ configured to provide a force to the protrusion 214 ′.

As described above, the adapter 210 ′ interfaces with the capture region of the sample processing chip 132 exposed through the access region 134 at the first region 211 ′ to facilitate application of a magnetic force to the region, and , to enable attraction of a substance (eg, a target or non-target substance bound to a magnetic particle) to the adapter 210' for further subsequent processing. Accordingly, the magnetic sleeve 1410 may include a sealing portion in the first region 211 ′ to facilitate a mechanism for drawing a target material from the sample processing chip 132 to the adapter 210 ′. The sealing portion may be an integral element with the adapter 210' or a separate element. However, the adapter 210 ′ may omit the sealing portion in the first region 211 ′.

The adapter 210' may be constructed of a polymeric material (eg, plastic) that does not adversely affect the magnetic field applied by the magnet 230' during operation. Additionally or alternatively, adapter 210 ′ may be constructed of or contain (eg, contain particles thereof) a material that is magnetic or capable of generating an induced magnetic field to support a usage application of system 200 ′. can Additionally or alternatively, adapter 210' may be constructed from other suitable materials. The distribution of the material(s) of the adapter 210 ′ may be homogeneous or non-homogeneous through the body of the adapter with respect to a desired magnetic effect in the capture region of the sample processing chip 132 . The inner cavity 220' of the adapter 210' may include a medium (eg, a magnetic medium, etc.), or alternatively may not include a medium.

2.5.2 Second magnetostriction - support structure and magnet

In the variation shown in FIGS. 9A-9C , the system 200 ′ includes a support structure 240 ′ that includes an interface 162 (eg, a pipette interface) to the fluid handling subsystem of the gantry 170 described above. ), and a magnet 230 ′ configured to provide a magnetic force for target material separation. In this variation, the magnet 230' may be configured to engage one or more units of the adapter 210', in a variation where the adapter 210' is a disposable element. Interface 162 also couples to a pipetting head coupled to gantry 170, described in greater detail below, to target or rain through magnetic forces, fluid suction, and/or a fluid transfer operation provided by the pipetting head. It may be configured to facilitate automation of target material recovery. As such, the system 100 includes a gantry 170, coupled to an adapter 210', between the units of the process vessel 20 and the sample processing cartridge 130 for magnetic separation and processing of a target material from a sample. It may include a detached mode carrying support structure 240'. In addition, an embodiment of the method implemented using the separation subsystem 160 can rapidly recover the target material with a recovery efficiency of >90% in which only magnetic particles bound to the target material (or non-target material) of the sample are recovered. there is. Thus, separation subsystem 160 can serve to increase selective recovery efficiency, thereby reducing processing burden and subsequent costs associated with processing reagents and other material costs (due to reduced requirements, reduced fractionation of biochemical reactions). .

As shown in FIG. 9A , the support structure may include an interface 162 to a fluid handling subsystem of the gantry 170, described below, wherein the interface is an engagement region that complements a corresponding engagement region of the fluid handling subsystem. includes The coupling region of interface 162 may include a magnetic coupling mechanism; press fit; snap fit; mechanism for tightening screws; male-female connection; or any other suitable mechanism to provide a reversible coupling with the fluid handling subsystem.

The magnet 230' of the support structure 240' may comprise or consist of a material to provide permanent magnetism, or alternatively be an electromagnet (coupled to suitable electronics of the system 100). can be configured. In a variation, the magnet distal region 163 may be comprised of one or more of alnico, neodymium, neodymium iron boron, samarium cobalt, ferrite, and other suitable magnetic materials. In shape, magnet 230 ′ may complement the shape of adapter 210 ′ such that a unit of adapter 210 ′ may couple (eg, reversibly engage) with magnet 230 ′. Also, the shape and pole configuration of the magnet 230' is such that a magnetic force that is nearly perpendicular to most of the target microwells from which the trapped particles are being removed is applied.

2.5.3 Second Magnetostriction—Optional Separating Elements Including Deck, Gantry, and/or Base

6A , 7B and 10A-10B , in a variant, the separation subsystem 160 may include a magnet subsystem 166 comprising a series of magnets 167 within a housing 168 . In addition, magnet subsystem 166 moves a series of magnets 167 relative to deck 10 (eg, through an opening in deck 10 ), one of process vessel 20 described above. It further includes a magnetic actuator 169 configured to be aligned/unaligned with the above separation reservoirs 129a and 129 . Also, the magnetic actuator 169 may be coupled to the control circuit (eg, at the base 180 ). Further, the magnetic actuator 169 may be configured to toggle the series of magnets between a retracted state and an expanded state, wherein in the expanded state, the series of magnets (eg, as shown in FIGS. 10A and 10B ) It becomes part of the first area of the deck. As such, separation subsystem 160 may also include elements supported by deck 10 and/or base 180 to facilitate operations to separate target material from non-target material.

In variations, the series of magnets 167 may include one or more permanent magnets and/or electromagnets (eg, coupled with suitable electronics of the system 100 ). The permanent magnet may be composed of one or more of alnico, neodymium, neodymium iron boron, samarium cobalt, ferrite, and other suitable magnetic materials.

In the example shown in FIGS. 10A-10B , the series of magnets 167 is a first subset of magnets 167a arranged in a linear array (eg, to perform a refining operation in the process vessel 20 ′). ), the location of the first subset of magnets 167a in a fifth domain for particle separation/purification described in relation to the process vessel 20' and the workflow described in Section 3 below. It corresponds to the spatial position of (25'). In the example shown in FIGS. 10A-10B , the series of magnets 167 are configured to interact with the separation reservoirs 129 , 129a of the process vessel 20 (eg, for initial bead recovery) of the magnets. It may also include a second subset of magnets 167b (eg, one or more magnets) displaced or offset from an axis associated with the first subset 167a. However, the set of magnets 167 may be (eg, in terms of number, in terms of a distributed array) in connection with providing appropriate interaction with the separate reservoir 129 of the process vessel 20 or other vessel. It may be arranged in any other suitable manner.

A housing 168 encloses the set of magnets 167 and provides a smooth operation with respect to converting the set of magnets 167 into alignment/non-alignment with corresponding portions of the process vessels 20, 20'. function. Thus, with respect to a configuration in which a first subset of magnets 167a and a second subset of magnets 167b are present, as shown in FIG. 10B , the housing 168 holds the first subset 167a. may include a first surface (eg, a first plane) to track and a second surface (eg, a second plane) to track to a second subset 167b, the first surface 168a and The second surfaces 168b are spaced apart from each other. In this variation, a pair of opposing walls are provided to facilitate smooth movement (eg, sliding operation) of the magnet and housing 168 through the deck 10 to interface with the process vessel 20, 20'; It may extend from the first surface and the second surface.

With respect to the process vessel 20', as shown in FIG. 10B, the space of the process vessel 20' configured for magnetic separation is, respectively, a plane 128a, or (eg, in an expanded magnet state). Other surfaces complementary to housing 168 may be included on the side configured to be closest to housing 168 during operation. In addition, the space of the process vessel 20 ′ configured for magnetic separation may each have a second surface 128b displaced away from the housing 168 for suction and/or delivery of fluid by a pipettor coupled to the gantry 170 . ) (eg, a curved surface). The cross-section taken longitudinally through the separation spaces/reservoirs 129, 129a of the reagent cartridge 120 allows the separation operation to require less fluid and/or more efficient aspiration and separation of the target material from the non-target material. may taper towards the base of the process vessels 20, 20' to provide

2.5.4 Second Magnetostriction - Working Mode

11A-11J, the separation subsystem 160 may provide a series of operating modes for material separation, and as shown in FIG. 12A, the operating modes may include a pipetting head or other operable mode of operation. a specific system configuration configuration of a support structure 240 ′ coupled with a component (eg, an interface 174 of a pipettor coupled to the gantry 170 ), the support structure 240 ′ comprising a magnet 230 . '), the unit of adapter 210', sleeve stripping tool 165, separation reservoir 129, and magnet 167b of the series of magnets 167 described above.

More specifically, as shown in FIG. 11B , the separation subsystem 160 may provide a first mode of operation 164a, wherein the first mode of operation 164a may be configured such that the support structure 240' is a pipette interface or Baseline operation in which magnet 230' of support structure 240' is disconnected from adapter 210', and decoupled from other operable components (eg, described with respect to gantry 170 below). is the mode The magnetic sleeve 1410 is further held by a sleeve stripping tool 165 above the separation reservoir 129 (or at another location in a variant), and the magnet 167b is attached (eg, to the magnetic actuator 169 described above). by) away from the separation reservoir 129 . As such, in the first mode of operation 164a, the system may install the adapter 210' at a location near the separation reservoir 129, and a magnet 230' for separation and recovery of the target material from the sample. A support structure 240 ′ coupled to may be installed.

As shown in FIG. 11C , the separation subsystem 160 may provide a second mode of operation 164b , wherein the support structure 240 ′ may be connected to a pipette interface or (eg, , gantry 170 and another actuator interface 6 (described in relation to pipettor 174 ), in which the magnet 230 ′ of the first body 161 is detached from the adapter 210 ′. is the mode The adapter 210 ′ is further retained by a sleeve stripping tool 165 above the separation reservoir 129 , and the magnet 167b (eg, by the magnetic actuator 169 described above) in the separation reservoir 129 . away from As such, in the second mode of operation 164b, the system installs the adapter 210' at a location near the separation reservoir 129, and prepares for separation and recovery of the target material from the sample (eg, an actuator) The support structure 240 ′ is coupled to the pipettor 174 using a magnet 230 ′ (via interface 6 ).

As shown in FIG. 11D , separation subsystem 160 may provide a third mode of operation 164c, in which support structure 240' may be configured to be connected to a pipette interface or ( For example, it engages with another actuator interface 6 (described in relation to the gantry 170 and pipettor 174 ) and moves into alignment with the separation reservoir 129 . In the third mode of operation 164c , the magnet 230 ′ of the support structure 240 ′ engages the adapter 210 ′ above the separation reservoir 129 in the retaining position of the adapter 210 ′. In the third mode of operation 164c , the magnet 167b is moved away from the separation reservoir 129 (eg, by the magnetic actuator 169 described above). As such, in the third mode of operation 164c, the system is configured to engage an adapter 210' held near the separation reservoir 129 (eg, in preparation for the separation and recovery of the target material from the sample). , via pipettor 174) to switch support structure 240' and magnet 230'.

As shown in FIG. 11E , separation subsystem 160 may provide a fourth mode of operation 164d, in which support structure 240' may be configured to operate at a pipette interface or ( In conjunction with another actuator interface 6 (eg, described with respect to gantry 170 and pipettor 174 ), magnet 230 ′ of support structure 240 ′ is positioned above separation reservoir 129 . It is coupled with the adapter 210'. In a fourth mode of operation 164d , the pipetting head (or other operable component) is configured with a sleeve stripping tool to prepare for drawing in sample-derived material (eg, functionalized particles from a sample processing cartridge). Move the magnet 230' coupled to the adapter 210' and the support structure 240' out of the retaining position provided by 165. In the fourth mode of operation 164d, the magnet 167b is moved away from the separation reservoir 129 (eg, by the magnetic actuator 169 described above). As such, in the fourth mode of operation 164d, the system provides a magnet 230' coupled to the adapter 210' to the separation reservoir 129 and out of the holding position, in preparation for the separation and recovery of the target material from the sample. ) and support structure 240 ′ (eg, via pipettor 174 ).

11F , separation subsystem 160 may provide a fifth mode of operation 164e, in which support structure 240' may be configured as a pipette interface or ( coupled with another actuator interface 6 (eg, described with respect to gantry 170 and pipettor 174 ), and magnet 230 ′ coupled with adapter 210 ′ in separation reservoir 129 . . In a fifth mode of operation 164e , the pipette interface (or other operable component) delivers a sample-derived fluid (eg, a dissolved target substance bound to a target particle) to the separation reservoir 129 and , the adapter 210 ′, still coupled with the support structure 240 ′, is submerged in the fluid of the separation reservoir 129 to attract the functionalized particles bound to the target contents. In the fifth mode of operation 164e, the magnet 167b is moved away from the separation reservoir 129 (eg, by the magnetic actuator 169 described above). As such, in the fifth mode of operation 164e , the system configures the support structure 240 ′, the magnet 230 ′ and the adapter 210 ′ to attract the target material delivered to the separation reservoir 129 . .

As shown in FIG. 11G , separation subsystem 160 may provide a sixth mode of operation 164f, in which support structure 240 ′ may be configured to be connected to a pipette interface or ( Magnets 230 of first body 161 that are still coupled with adapter 210' and coupled with other actuator interfaces 6 (eg, described in relation to gantry 170 and pipettor 174). ') moves back to the holding position in the sleeve stripping tool 165 . In the sixth mode of operation 164f, the adapter 210' (still associated with the target substance/functionalized particle) is immersed in the fluid in the separation reservoir 129 . In the sixth mode of operation 164f, the magnet 167b is moved away from the separation reservoir 129 (eg, by the magnetic actuator 169 described above). As such, in the sixth mode of operation 164f, the system configures the support structure 240', the magnet 230', and the adapter 210' for processing of a target material coupled to the adapter 210'. . For example, while the material is being bound to the adapter, the system may perform a washing step or other process to remove non-target material. Additionally, or alternatively, a sixth mode of operation 164f may include magnets 167b proximal to separation reservoir 129 in adapter 210 ′ for further processing (eg, suction, delivery for amplification, etc.). It is possible to prepare a target material for delivery to the region of

As shown in FIG. 11H , separation subsystem 160 may provide a seventh mode of operation 164g, in which support structure 240 ′ may be configured to be connected to a pipette interface or ( In conjunction with another actuator interface 6 (eg, described with respect to gantry 170 and pipettor 174 ), magnet 230 ′ engages adapter 210 ′ in the holding position of detachment reservoir 129 . is combined with In the seventh mode of operation 164g, the adapter 210' still engaged with the support structure is held in position in the sleeve stripping tool 165, and the adapter 210 (along with the magnetically coupled functionalized particles) is ') is immersed in the fluid of the separation reservoir 129 . In the seventh mode of operation 164g, the magnet 167b is attached to the wall 128a of the separation reservoir 129 to prepare for attraction and retention of target or non-target substances bound to the functionalized particles of the fluid (e.g., (eg, by the magnetic actuator 169 described above) towards the separation reservoir 129 . As such, the seventh mode of operation 164g transfers from the adapter 210' to the region of the magnet 167b proximal to the separation reservoir 129 for further processing (eg, suction, delivery for amplification, etc.). Prepare the target material for

11I , the separation subsystem 160 may provide an eighth mode of operation 164h, in which the support structure 240' may be configured to provide a pipette interface or ( It engages with another actuator interface 6 (eg, described with respect to gantry 170 and pipettor 174 ) and is moved away from separation reservoir 129 for replacement with a suitable tip of the tool container described above. In the eighth mode of operation 164h, magnet 230' holds adapter 210' through pipetting head support structure 240' while adapter 210' is held in position in sleeve stripping tool 165. It is disengaged from the adapter 210 ′ above the separation reservoir 129 by moving it away from the In the eighth mode of operation 164h , the adapter 210 ′ is immersed in the fluid of the separation reservoir 129 . In the eighth mode of operation 164h, the magnet 167b is attached to the wall 128a of the separation reservoir 129 to retain the target or non-target material bound to the functionalized particles of the fluid (eg, It is still placed near the separation reservoir 129 (by the magnetic actuator 169 described above). In the eighth mode of operation 164h, the magnet 167b may be engaged (eg, for later extraction from the bottom by the pipettor, to retain material while the pipettor attracts material not bound to the magnet 167b). ) draws the target material towards the bottom of the separation reservoir 129 . As such, the eighth mode of operation 164h may allow material captured in adapter 210' to be transferred and temporarily held in the region of separation vessel 129 for further processing.

11J , the separation subsystem 160 may provide a ninth mode of operation 164i, in which the pipetting head/actuator interface 6 is appropriately It engages the hap tip and moves to the separation reservoir 129 to suck material from the separation reservoir 129 . In the ninth mode of operation 164i , the adapter 210 ′ is still held in position above the separation reservoir 129 in the sleeve stripping tool 165 and is immersed in the fluid in the separation reservoir 120 . In the ninth mode of operation 164i, the magnet 167b moves away from the separation reservoir 129 (eg, by the magnetic actuator 169 described above) with suction of material by the pipetting head. As such, the ninth mode of operation 164i may allow material captured in adapter 210' to be transferred and temporarily held in the region of separation vessel 129 for further processing.

However, variations of the steps shown in FIGS. 11A-11J related to target material recovery and subsequent processing can be achieved using variations of the system 200 described above without intervention of the interface of the pipettor 174 and/or the gantry 170 . can be implemented.

12A-12D show additional views of the configuration of the magnetic sleeve 1410 relative to the sleeve stripping tool 165 of the separate reservoir 129 of the process vessel 20 in connection with the modes of operation described above.

However, variations of the separation subsystem 160 may include elements and provide modes of operation for recovering a target material based on one or more of gravity, buoyancy, centrifugal force, chemical separation, and/or other suitable separation methods. . In another embodiment, the target material retrieval operation by the separation subsystem 160 transfers the target particles from the microwell chip to another substrate or another empty new microwell chip while maintaining the relative spatial position of the different particles being delivered. can be used for

2.6 Systems - Examples for Recovery by Gravity-Related Forces

13A-13C , variations of the system 300 include a first region 311 , a second region configured to couple to a capture region of the sample processing cartridge 130 to capture particles in a single particle format. an adapter 310 comprising ( 312 ) and an interior cavity ( 320 ) passing from the first region ( 311 ) to the second region ( 312 ); and a support structure 340 reversibly coupled to the second region of the adapter 310 . The adapter 310 may also include an operable vent 318 to prevent air bubbles from being retained between the capture region of the sample processing chip 132 and the interior cavity 320 of the adapter 310 .

The system 300 also includes a series of plugs 350 configured to couple an inlet and/or outlet fluidly coupled to the capture region of the sample processing chip 132 (eg, directly or via a manifold device). ); and a guide 360 comprising a recess complementary to the adapter 310 and the sample processing chip 132 , wherein the guide 360 is configured to capture the adapter 310 and the sample processing chip 132 . and retain the sample processing chip 132 and the coupled adapter 310 within the centrifugation device for applying a gravity-related force through centrifugation to the contents of the region. In addition, the guide 360 may function to prevent physical contact between the sample processing chip 132 and the centrifugation device during operation.

The system 300 applies a gravity-related force applied to the capture region of the sample processing chip 132 , as shown in FIG. 13B , to the adapter 310 to provide a directional force to deliver the target material within the capture region. function that allows. An embodiment of the method implemented with system 300 is capable of recovering target material in a manual operation time of 2 minutes to 3 minutes (and a total time of ~15 minutes) with recovery efficiencies of ˜85% to 95%. Accordingly, the system 300 may function to provide a fast method (with respect to manual work time and total work time) with high recovery efficiency. System 300 may implement one or more embodiments, variations, or examples of the method(s) described below, and/or may be used to implement other methods.

2.6.1 adapter

13A-13C , the adapter 310 has a first region 311 , a second region 312 configured to couple to a capture region of the sample processing cartridge 130 to capture particles in a single particle format. ), and an interior cavity 320 passing from the first region to the second region. The adapter 310 provides a structure that allows the target material to be delivered from the capture region of the sample processing cartridge 130 in a manner that facilitates easy recovery of the target material from the adapter 310 , and to the adapter 310 . It serves to support application of a gravity-related force to the capture region to deliver the target material of the sample processing chip 132 . The adapter 310 may also function to prevent air bubbles and/or other obstructions from forming an obstruction between the interior cavity 320 of the adapter 310 and the capture area of the sample processing cartridge 130 . Adapter 310 may also function to prevent sample cross-contamination by serving as a disposable component that may be discarded between uses of system 300 .

The adapter 310 has an interior cavity 320 having a concave surface facing the capture area of the sample processing chip 132 (when the adapter 310 is coupled to the sample processing chip 132 ). The concave surface serves to define a space that allows for force-based separation and receipt of target material from other components captured within the capture area of the chip. In variations, the volume of the interior cavity 320 may be between 0.1 mL and 5 mL, although in alternative variations the volume of the interior cavity may define other volumes.

In variations, the surface of the interior cavity 320 may be textured (eg, dents or other indentations, chambers, etc.), a binder (eg, a chemical agent, an antistatic agent, etc.), and/or applied with an applied force. It may include other features to facilitate preferential retention of the target material in the interior cavity 310 of the post adapter 320 .

As shown in FIG. 13A , the adapter 310, upon application of an applied force, causes air (or other gas) to create an obstacle that prevents target material separation from the wells of the capture region of the sample processing chip 132 . To prevent this, it also includes a vent 318 configured to allow air (or other gas) to escape within the interior cavity 320 after the adapter 310 is coupled to the chip 132 . The vent 318 may be disposed in a peripheral area of the adapter 310 , or alternatively may be disposed in another suitable area of the adapter. With respect to the applied force, the vent may be positioned in a direction that prevents the target material from exiting the interior cavity 320 through the vent 318 , although the vent 318 may alternatively be positioned in another orientation. there is. In addition, adapter 310 may include a plurality of vents or other bubble release functions (eg, valves) and/or self-sealing material sections that may be needle-pierced and deflated.

Preferably, the adapter 310 has a wall thickness suitable for the magnitude of the force used to separate the target material. In an example, the wall thickness may be between 0.2 mm and 3 mm, although in other examples the wall thickness may have other suitable thicknesses.

Additionally or alternatively, adapter 310 may include structural features that enable a mode of operation of system 300 . For example, with respect to coupling and disengaging the adapter 310 to the capture region of the sample processing cartridge 130 , the adapter 310 facilitates coupling and disengagement of the adapter 310 from the sample processing chip 132 . may include a protrusion 314 (eg, a tab) that may be used to

As described above, the adapter 310 forms a space for separation and applies a gravity-related force to the exposed portion of the sample processing cartridge 130 in the first region 311 to retrieve the target material from the capture region. binds to the capture region. The adapter 310 may include a sealing portion in the first region 311 to prevent material leakage at the interface between the sample processing chip 132 and the adapter 310 . The sealing portion may be an integral element with the adapter 310 or a separate element. However, the adapter 310 may omit the sealing portion in the first region 311 . The adapter 310 also holds the adapter 310 in position in the sample processing chip 132 and provides a second region 312 for reversible coupling and separation from the sample processing chip 132 and the support structure 340 . ) is coupled to the support structure 340 . The coupling of the adapter 310 to other system components may occur by one or more of a press fit, a snap fit, a compression fit, a friction fit, a female-male mating interface, a screw, another fastener, a magnetic mechanism, and other suitable mechanism. there is.

The adapter 310 may be constructed of a polymeric material (eg, plastic, elastomer) that can undergo elastic deformation to facilitate removal of air or gas trapped within the adapter 310 . Additionally or alternatively, the adapter 210 may include another material (eg, a non-polymer material, a metal, , ceramic, etc.) or may contain (eg, include particles thereof). Additionally or alternatively, adapter 310 may be constructed from other suitable materials.

2.6.2 Support structure

13A and 13C , the support structure 340 is reversibly coupled to the second region 312 of the adapter 310 . The support structure 340 holds the assembly of the adapter 310 and sample processing chip 132 in position, and the sample processing chip 132 , adapter 310 and/or guide 360 (more detail below) It functions to switch between working modes for combining and disengaging technologies).

The support structure 340 is a form factor for clamping the assembly of the adapter 310 and the sample processing cartridge 130 together in a manner that prevents material from leaking at the interface between the sample processing cartridge 130 and the adapter 310 . can have In one variation, the support structure 340 takes the form of a clamshell such that the distally opposed region includes a clamping structure for clamping the assembly of the sample processing chip 132 and the adapter 310 together. can have In addition, the support structure 340 may have an opening that allows viewing of the contents of the capture region of the sample processing chip 132 and/or the adapter 310 during processing.

Support structure 340 may be comprised of one or more polymeric materials (eg, plastics) that are sterilizable (eg, autoclavable, resistant to damage by ethanol, etc.) between uses of system 300 . can However, support structure 240 may alternatively be constructed of other suitable materials. Additionally, support structure 340 may be a disposable or non-disposable component of system 300 .

2.6.3 Guide and plug set

As shown in FIG. 3A , system 300 is configured to couple an inlet and/or outlet fluidly coupled to a capture region of a sample processing chip 132 (eg, directly or via a manifold device). It may also include a configured series of plugs 350 . In embodiments where the sample processing chip 132 (described above) comprises a manifold or other substrate for fluid transfer to/from the sample processing chip 132 , the series of plugs 350 may be connected to the inlet(s) of the manifold. ) and outlet(s). Additionally or alternatively, the series of plugs 350 may couple directly to the inlet and/or outlet of the sample processing chip 132 . The engagement of the series of plugs 350 to other system components may occur by one or more of a press fit, a snap fit, a friction fit, a female-male mating interface, a screw, another fastener, a magnetic mechanism, and other suitable mechanism. .

The series of plugs 350 are sterilizable (eg, autoclavable, resistant to damage by ethanol, etc.) between uses of the system 300 and one or more polymeric materials that are resilient (eg, plastic) may be used. However, the series of plugs 350 may alternatively be constructed of other suitable materials. Additionally, the set of plugs 350 may be a single-use or non-disposable component of the system 300 .

13A and 13C , system 300 may also include a guide 360 comprising recesses complementary to adapter 310 and sample processing chip 132 , guide 360 . is configured to retain the sample processing chip 132 and the coupled adapter 310 within the centrifugation device for applying a gravity-related force via centrifugation to the adapter 310 and the contents of the capture region of the sample processing chip 132 . . In addition, the guide 360 may function to prevent physical contact between the sample processing chip 132 and the centrifugation device during operation. Preferably, the recesses in the guide 360 allow the entire sample processing chip 132 and adapter 310 assembly to be seated within the recesses while the recesses in the guide 360 alternatively include the sample processing chip 132 and/or the recesses in the guide 360 . It may be configured to receive only a portion of the adapter 310 .

As shown in FIG. 13C , the recess may include an extended area that does not contact the sample processing chip 132 and/or the adapter 310 , wherein the expanded area allows an operator to place and remove the chip assembly within the recess. make it easy The engagement of the sample processing chip 132 and/or adapter 310 to the recess of the guide 360 may be a press fit, snap fit, compression fit, friction fit, male-female mating interface, screw, other fastener, magnetic mechanism. , and other suitable mechanisms.

Guide 360 is one or more polymeric materials (eg, plastic) that are rigid and sterilizable (eg, autoclavable, resistant to damage by ethanol, etc.) between uses of system 300 . can be composed of However, guide 360 may alternatively be constructed of other suitable materials. In addition, guide 360 may be a disposable or non-disposable component of system 300 .

2.7 System - Conclusion

The described system(s) may additionally or alternatively include other components that facilitate target material retrieval from the capture region of the chip. The described system(s) may implement one or more embodiments, variations and examples of the method(s) described below, or other suitable methods.

3. Method

As shown in FIG. 14 , an embodiment of a method 400 for target material recovery includes capturing 410 a series of particles in a single particle format in a series of wells dispersed throughout the substrate in a capture region; supporting (420) an environment for processing a target material of a set of particles within the capture region according to the set of operations; forming 430 an assembly with an adapter configured to interact (eg, couple) with the substrate; ejecting (440) a series of particles of target material into the adapter by transmitting a force to the adapter and the capture region; and releasing (450) a series of particles of target material for capture by the adapter.

Embodiments, variations, and examples of method 400 serve to provide a mechanism for efficiently recovering a target material from a high-density capture device (eg, a microwell chip), wherein the high-density capture device comprises captured single cells. To facilitate an increase in the efficiency of bead pairing efficiency, a high-density array of high aspect ratio microwells is included. Additionally, embodiments of method 400 may serve to reduce the manual burden associated with target material recovery in high-density capture devices. Additionally, embodiments of method 400 may function to increase the efficiency with which target material is withdrawn from the high density capture device and the efficiency with which non-target material is retained in the capture device.

Method 400 may process target material from captured cells in a single cell format in the capture region of the chip, as described above. Cells may include some or all of a mammalian cell (eg, a human cell, a murine cell, etc.), an embryo, a stem cell, a plant cell, a microorganism or other suitable type of cell. A target substance is a cell, tissue, nuclear or cell-free nucleic acid (eg, target lysate, mRNA, RNA, DNA, protein, glycan, metabolite, etc.) or a substance associated with a particle associated with a cellular or cell-free biomarker. may include Additionally or alternatively, method 400 can be configured to treat particles (eg, beads, probes, nucleotides, oligonucleotides, polynucleotides, etc.), reagents, or other suitable substances as target substances for further processing. there is. Further, the method selectively combines a target particle with another carrier particle that can be transported to a different location by moving the carrier particle with a mechanism for moving the carrier particle to simultaneously selectively move a plurality of target particles on a surface seeded with a plurality of particles. can be configured to remove.

Method 400 may be implemented by embodiments of the system described above and/or other suitable system components.

4.1 Method - Capture and processing of target material

Block 410 refers to capturing a series of particles in a single particle format in a series of wells that are dispersed throughout the substrate in the capture region. Block 410 provides for co-capture of particles (e.g., single cells, functional particles and co-captures) in single particle format within individual capture chambers of the chip, to separate the target material from the individual target particles in a manner that facilitates further subsequent processing. It functions to process the contents of the sample to separate old cells, etc.). While block 410 may be implemented by embodiments, variations, or examples of sample processing cartridge 130/sample processing chip 132 described above, block 410 may additionally or alternatively include single cell types and single cell types. receiving the biological sample in another suitable system configured to capture the cells in at least one of the cluster formats (eg, co-capture the cells in a single cell format with one or more functional particles corresponding to each single cell). .

At block 410, the biological sample comprising the target particle is dispensed (eg, by pipetting, by fluid transfer through a fluidic channel coupled to the array) for distribution throughout the series of wells of the capture region of the chip. may be delivered and/or received directly and/or in any other suitable manner into the inlet of the chip. Embodiments, variations, and examples of block 410 may be implemented as described in one or more patents incorporated by reference above.

Block 420 refers to supporting an environment for processing a target material of a set of particles within the capture region according to a set of operations. Block 420 functions to create an environment in which a target material of a sample may be prepared for retrieval with application of a force applied according to subsequent blocks of method 400 . As such, block 420 may include lysing the captured cells to disrupt the membranes of the captured cells; releasing a target agent (eg, nucleic acid content) from the captured cell; isolating unwanted elements (eg, RNA, protein) from the captured sample material; performing a washing step, and co-capturing individually captured cells and/or their target material and functional particles (eg, non-magnetic beads, magnetic beads); performing a barcoding step; attaching the relevant adapter molecule to the released nucleic acid content; hybridizing a target substance (eg, mRNA) to the functional particle; performing reverse transcription; delivering a retrieval buffer to the chip to prepare the target material for release from the capture region; sonicating or physically perturbing the contents of the capture area relative to the chip to prepare the target material for release from the capture area; and/or creating a physical environment (eg, within a chamber, using appropriate process reagents) for performing other suitable steps to enable efficient recovery of the target material from the capture region of the chip. may include the step of As will be described in more detail below, certain steps may be taken to implement a magnetic force recovery mode and/or a gravity related recovery mode.

Additionally or alternatively, embodiments, variations, and examples of block 420 may be implemented as described in one or more patents incorporated by reference above.

4.2. Method - Magnetic force recovery mode

With respect to embodiments, variations, and examples of systems 200 , 200 ′ described above, method 400 may include using a magnetic force recovery mode to recover a target material from a capture region of a sample processing chip. there is.

In particular, block 430 refers to forming an assembly with an adapter configured to couple to a substrate. Preferably, block 430 is implemented via embodiments, variations, or examples of adapters 210, 210' described above, whereby the adapters are magnetized from wells or other sensitive materials in physical contact in the capture region of the chip. and the ability to transmit a force associated with a magnetic field to the capture region to separate the target material from the chip. Forming the assembly may include a guide or other support structure for maintaining the relative orientation between the chip and the adapter during the process of transferring the captured target material through the structural features of the chip and/or adapter and from the capture area of the chip for retrieval. This can be facilitated through the use of

Block 440 refers to ejecting a series of particles of target material towards the adapter by transmitting a force to the adapter and capture region. Block 440 serves to transmit a force in a controlled manner to the capture region of the chip using the adapter to facilitate target material release from the chip for retrieval. With respect to the magnetic recovery mode, the force is a magnetic force generated through the use of a magnet (eg, such as the magnet described above), but additionally or alternatively the force may include other suitable forces. Further, the force is preferably applied as a pulling force in a direction perpendicular to the plane in which the series of wells are defined, although the force may alternatively be oriented in any other suitable direction.

Block 450 refers to releasing a series of particles of target material for capture by the adapter. Block 450 functions to facilitate target agent delivery towards the adapter (eg, using magnetic beads coupled to functional particles to which the target agent is bound) to facilitate target agent recovery from the chip in an efficient manner. do The target material can then be extracted for further subsequent processing.

Variations and examples of magnetic recovery modes associated with blocks 420 through 450 are further described in Section 4.2.1 below.

4.2.1 Magnetic recovery method variants and examples

In particular, as shown in FIGS. 15A and 15B , a variant of the magnetic recovery method 500 includes the steps of co-capturing 502 single cells and barcoded beads within individual wells of the chip; Lysing the cells and delivering 504 mRNA from the cells to the co-captured beads; ligating the captured mRNA/protein to the barcoded oligonucleotide sequence of the beads via reverse transcriptase or ligation (during this process, only biotinylated TSO primers are attached to the beads in microwells bearing the target cells). (506); In this way, binding (510) the biomarker from a single cell to the functionalized nonmagnetic beads in a single bead format within a series of wells of the chip; binding a series of magnetic particles (e.g., 0.5 μm to 3 μm particles) via specific interactions to functionalized non-magnetic beads within a series of wells (e.g., biotinylation of 0.5 μm to 3 μm) the magnetic beads that have been made bind to the target bead through streptavidin interaction, and the remaining excess beads float freely and either lay on the bottom or bind non-specifically to other beads); and an adapter bound to the captured target material (e.g., directly bound to functionalized non-magnetic microspheres in the well) to recover the target material from within a series of wells, using a molecular scissors process. applying a magnetic force to the series of magnetic particles (directly bonded to the material) 530 . The described magnetic recovery method recovers the target material within a manual operation time of 5 minutes to 8 minutes (and a total time of 15 minutes to 45 minutes) with a recovery efficiency of >90% in which only magnetic particles bound to the target material in the sample are recovered. can do. In addition, the self-recovery method reduces reverse transcription-derived concatemers, provides an automation friendly protocol, reduces the number of divisions required for subsequent cDNA amplification, reduces the need for SPRI-based cleanup and size selection (e.g. , which can reduce the effort and use of processing reagents in subsequent steps (related to exonuclease treatment and cDNA amplification). Contamination of the exonuclease enzyme to instruments, laboratory benches, and equipment used during one single cell preparation can inhibit reactions used in subsequent single cell preparation for the next sample using exonuclease contaminating elements. It is very advantageous to eliminate the exonuclease treatment step in the library preparation from single cells because there is.

First Example: In a first example of method 400 and method 500, binding of barcoded nonmagnetic microspheres comprising mRNA product or binding to an mRNA product as a target substance from a single cell originally captured in a chip and A magnetic force may be transferred to the chip to enable a mechanism for selective removal. More specifically, as shown in FIG. 16 , method 600 includes co-capturing 610 single cells and single barcoded beads using different partitions (microwells, droplets); Lysing the cells in the partition and delivering (620) the biomarker to the barcoded beads via binding interaction; Linking biomarkers to barcoded oligo tags on beads through molecular reactions (reverse transcription, ligation, etc.), during molecular ligation reaction, biotinylated primers are added to beads containing only the target biomarker and these ligation reactions are partitioned step 630 , which may be performed at or after the partition is removed; washing 640 unbound biotinylated primers; adding streptavidin-encoded magnetic beads (650); The oligonucleotide sequence is selectively cleaved from the bead using a cleavage mechanism (e.g., molecular scissors, photocleavage, thermal shear, etc.), and the cleaved product is subjected to biotin-streptavidin interaction to target biomolecular beads. having marker binding (660); and separating ( 670 ) the complexed target oligonucleotide from the remaining oligonucleotide tag using a magnetic force. Embodiments, variations, and examples of workflow aspects may be practiced as described in connection with the patents incorporated by reference above.

17A-17D , an example method 700 includes capturing 710 functionalized non-magnetic beads in a single bead format and functionalization to mRNA material from a single cell captured simultaneously in a corresponding well. It may include a step 720 of crossing the non-magnetic beads. As shown in Figure 17a (top), the hybridization is a set of thymine (T) bases bound to functionalized non-magnetic beads, a template switching oligonucleotide (TSO) sequence at the 5' end, a barcode sequence, a unique molecular identifier (UMI) , and the use of molecules with tails at the 3' end.

Then, as shown in FIG. 17 (bottom), a first example of method 600 uses a first molecule having a 3' terminal non-template cytosine (C) base and corresponding to the target mRNA molecule, the target mRNA substance It may include performing a reverse transcription reaction (RT reaction) with the step 730 . Thus, the RT reaction incorporates a TSO with a biotin tag at the 5' end. Following block 730, method 700 may include a washing step to remove unbound biotinylated TSO primer as described in the patents incorporated by reference above.

Then, as shown in FIG. 17B , a first example of method 600 uses the template conversion mechanism of the Moloney Murine Leukemia Virus (MMLV) RT enzyme to completely capture the 5' end of the target substance, the 3' and implementing ( 740 ) the cDNA with template switching oligonucleotide (TSO) sequence extensions in the first strand with terminal non-template cytosine bases. The cDNA TSO sequence extension corresponds to the biotin TSO at the 5' end of the target mRNA.

Then, as shown in Figures 17B and 17C, a first example of method 700 involves magnetic streptavidin beads at the 5' biotin terminus of the molecule generated in block 740, via a biotinylated TSO moiety ( For example, binding 750 of functionalized non-magnetic beads to dynabeads. More specifically, the magnetic streptavidin beads can be added to the capture chamber and mixed with an internal material with an incubation period (eg, 20 minutes) prior to recovery. As shown in FIG. 17C , method 700 includes applying 660 an attractive magnetic force to attract a bead complex to an adapter using an embodiment, variation, or example of system 200 described above. may include more. 17D , the captured bead complexes can then be transferred to a vessel (eg, a tube) for further processing.

Second Example: In a second example of methods 400 and 500, a barcoded nucleic acid material product that is a target material in a single cell originally captured in the chip, leaving functionalized non-magnetic particles on the chip during the recovery process. Magnetic forces can be transferred to the chip to enable a mechanism for the coupling and selective removal of . More specifically, as shown in FIGS. 18A-18E , an example of method 800 includes capturing functionalized non-magnetic beads in a single bead format and simultaneously capturing mRNA material from a single cell captured in a corresponding well. crossing the functionalized non-magnetic beads. Then, instead of recovering the functionalized non-magnetic beads from the chamber of the chip, the method 800 may be photocleaved or cleaved to release the target cDNA-RNA hybrid for capture in a secondary magnetic particle, for recovery by an applied magnetic force. Molecular scissors (eg, double-stranded molecular scissors molecules, single-stranded molecular scissors molecules) may be implemented. Examples of molecular scissors include Btu endonucleases that are capable of specifically cleaving a single starting oligonucleotide sequence that contains a modified base (eg, deoxyuridine, dSpacer, or dexylnosine). Other examples of molecular scissors include uracil-specific excision reagent (USER) enzymes capable of specifically cleaving single-stranded oligonucleotide sequences comprising the modified base uracil. In an example, the enzyme is activated at a temperature of ~37 °C. An example of a photocleavable linker (PC linker) includes a non-nucleoside moiety that can be used to link two nucleotide sequences via a short UV photocleavable C3 spacer arm. Photocleavage of the PC linker by UV light yields one 5' phosphorylated oligo and one 3' phosphorylated oligo. This process reduces damage to the target cDNA-RNA hybrid due to the reduced stress that occurs when selecting a molecule containing product and pulling a single bead type from the capture well, as opposed to pulling both bead types from the well. can do it

More specifically, as shown in Figure 18a (top), hybridization is a molecule with a set of thymine (T) bases (e.g., 5 or 10 T bases) bound to functionalized non-magnetic beads, (e.g., A set of modified non-natural bases configured to be targeted by molecular scissors (e.g., dU as shown at the top of Figure 18A; dSpacer as shown), the use of a modified base region, a template switching oligonucleotide (TSO) sequence, a barcode sequence, a unique molecular identifier (UMI) and a tail at the 3' end.

A second example of method 700 is similar to blocks 730 and 740 described above (Fig. 18B and 18C ) and blocks 830 and 840 .

Then, as shown in FIG. 18C , a second example of method 800 uses single- or double-stranded molecular scissors to limit the bead-oligonucleotides in the modified base region to the wells of the capture region of the chip. releasing the cDNA hybrid (850).

Thereafter, as shown in FIGS. 18C and 18D , a second example of method 800 generates a target RNA-cDNA hybrid released to magnetic streptavidin beads at the 5′ biotin terminus of the molecule generated in block 840 . It may include a step of combining. As shown in FIG. 18D , the method 800 further includes applying 860 an attractive magnetic force to attract the bead composite to the adapter using an embodiment, variation, or example of the system 200 described above. may include 18E , the captured bead complexes can then be transferred to a vessel (eg, a tube) for further processing.

Although examples described above, other enzymes (eg, non-MMLV enzymes), other transcriptional processes, and/or other suitable processes may be used to process other suitable target substances (eg, non-mRNA substances).

4.3 Method - Gravity-Related Force Recovery Mode

With respect to embodiments, variations, and examples of system 300 described above, method 400 may include using a gravity-related force recovery mode to recover a target material from a capture region of a chip.

In particular, block 430 refers to forming an assembly with an adapter configured to couple to a substrate. Preferably, block 430 is implemented via an embodiment, variant, or example of an adapter described above, whereby the adapter functions to define an interior cavity into which a target material may come together upon application of a force applied to the assembly. includes The forming step of the assembly may be achieved through the use of guides or other support structures to maintain the relative orientation between the chip and the adapter during the process of transferring the captured target material from the capture area of the chip for retrieval and through the use of a structural structure of the chip and/or adapter. This can be facilitated through features.

Block 440 refers to releasing a series of particles of target material into the adapter by transmitting a force to the adapter and capture region. Block 440 functions to transmit a force in a controlled manner to the capture region of the chip to facilitate release of the target material from the chip and to the adapter for retrieval. With respect to the gravity-related force recovery mode, the force is a centrifugal force generated through the use of a centrifugal separation device, but additionally or alternatively the force may include other suitable forces.

Block 450 refers to ejecting a series of particles of target material to the adapter. Block 450 serves to facilitate target agent delivery towards the adapter to facilitate target agent retrieval from the adapter in an efficient manner. The target material can then be extracted for further subsequent processing.

Variations and examples of centrifugation-related recovery modes associated with blocks 420 through 450 are further described in Section 4.3.1 below.

4.3.1 Modifications and Examples of Recovery Methods Related to Centrifugation

In particular, as shown in FIG. 19 , a variant of the centrifugal retrieval method 900 (eg, by filling wells and blocking the inlet and outlet ports of the sample processing chip with plugs) removes retrieval buffer from the capture region of the chip. accepting (910); forming (920) an assembly with an adapter having a vent for removing trapped air within an interior cavity defined between the adapter and the capture region (eg, by attaching the adapter and applying pressure to remove the trapped air); ; sonifying the assembly 930 (eg, at 47 kHz, at a different frequency) to facilitate target material separation from the well surface of the capture region; coupling (940) the assembly to a guide and support structure for positioning the assembly in the centrifugal separation device; centrifuging 950 the assembly (eg, in a relative centrifugation field of 1000) such that an applied force drives the target material to the adapter; and recovering (eg, using a recovery buffer delivered through the fluid network of the sample processing chip) a mass (eg, pellet) of the target material separated from the adapter ( 960 ).

The centrifugation-based recovery method can rapidly recover the target material in a manual operation time of 2-3 minutes (and a total time of ~15 minutes) with ~85% to 95% recovery efficiencies.

5. Conclusion

The drawings illustrate the architecture, functionality and operation of possible implementations of systems, methods and computer program products according to preferred embodiments, exemplary configurations, and variations thereof. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of code comprising one or more executable instructions for implementing the specified logical function(s). It should also be noted that, in some alternative implementations, the functions recited in the blocks may occur out of the order recited in the figures. For example, two blocks shown in succession may actually be executed substantially simultaneously, or sometimes in reverse order depending on the functionality involved. It is also noted that each block in the block diagrams and/or flowcharts, and combinations of blocks in the block diagrams and/or flowcharts, may be implemented by special-purpose hardware-based systems, or combinations of special-purpose hardware and computer instructions, that perform specified functions or operations. Be careful.

Those skilled in the art will recognize from the preceding detailed description and drawings and claims that modifications and variations can be made to the preferred embodiment of the present invention without departing from the scope of the invention as defined in the appended claims.

Claims (20)

A method for recovering and processing a material from a sample, said method comprising:
coupling with an adapter;
establishing a transfer means between a capture region of a capture substrate comprising a material derived from the sample and the adapter;
transferring a quantity of target material from the capture substrate to the adapter by applying a force to at least one of the adapter and the capture substrate;
removing the adapter from the capture substrate to recover the large amount of the target material from the capture substrate; and
and transferring said quantity of said target material from said adapter to a process vessel. 2. The method of claim 1, wherein said capture region defines a series of microwells, said method comprising capturing a series of cells in said series of microwells in a single cell format and comprising: A method for recovering and processing a material in a sample comprising the step of co-capturing a series of functionalized particles. 3. The method of claim 2, further comprising the step of lysing the series of cells and releasing the target agent from the series of cells for association with the series of functionalized particles. . 4. The method of claim 3, wherein the target material comprises at least one of a protein content and a nucleic acid content derived from lysed cells of the series of cells. 2. The method of claim 1, wherein engaging the adapter comprises interfacing a pipette interface with a first magnet and transferring the first magnet into a cavity of the adapter, wherein the target material is captured in the capture region. and cell-derived contents bound to functionalized magnetic particles, wherein said applying a force comprises applying a magnetic force to attract said target substance to an outer surface of said adapter for recovering and processing a substance from a sample. method. 6. The method of claim 5, wherein transferring the quantity of the target material from the adapter to the process vessel comprises placing the adapter coupled to the first magnet within the process vessel. and how to deal with it. 7. The method of claim 6, further comprising bringing a second magnet proximate to the wall of the process vessel to attract the target material from the adapter to the wall of the process vessel. way for. 8. The method of claim 7, wherein transferring the quantity of the target material from the adapter to the process vessel moves the first magnet away from the adapter such that the process vessel faces the wall adjacent to the second magnet. A method for recovering and processing a material from a sample, comprising enabling release of the target material from an adapter. 9. The method of claim 8, further comprising: moving the second magnet away from the wall of the process vessel; and extracting the quantity of the target material from the process vessel using a pipette tip coupled to the pipette interface. Further comprising a method for recovering and processing a material from a sample. 10. The method of claim 9, wherein said wall of said process vessel comprises a plane of said wall and comprises a curved surface away from said wall, said plane and said curved surface tapering toward a base of said process vessel, said quantity of target material wherein extracting comprises passing the pipette tip along the curved surface towards the base of the process vessel. The method of claim 1 , further comprising generating the mass of target material when processing the sample in a series of operations, wherein the series of operations comprises:
lysing the series of cells in the sample to release the mRNA content from the series of cells;
capturing mRNA content in the first set of functionalized particles;
performing reverse transcription with the mRNA content to produce a set of target molecules;
coupling a second set of magnetic particles with a first set of functionalized particles; and
and applying a magnetic force to the second set of magnetic particles to deliver the series of target molecules to the adapter. The method of claim 1 , further comprising generating the mass of target material when processing the sample in a series of operations, wherein the series of operations comprises:
lysing the series of cells in the sample to release the mRNA content from the series of cells;
capturing mRNA content in the first set of functionalized particles;
performing reverse transcription with the mRNA content to produce a set of target molecules;
cleaving the series of target molecules from the first population of functionalized particles;
associating the second set of magnetic particles with the series of target molecules; and
and applying a magnetic force to the second set of magnetic particles to deliver the series of target molecules to the adapter. A system for recovering and processing a material from a sample, the system comprising:
a first region configured to interface with a capture region of a capture substrate for capturing particles in a single particle format within a series of wells, a second region configured to interface with the capture region, and a second region in the first region an adapter comprising a cavity that extends into
a magnet complementary to said cavity of said adapter; and
and a support structure coupled to the magnet and providing a set of modes of operation for movement of the adapter relative to the capture substrate. 14. The system of claim 13, wherein the adapter defines a sleeve for the magnet and includes a surface, the surface preventing entrapment of magnetic particles within features of the surface. . 14. The method of claim 13, wherein the adapter comprises a protrusion extending from the second region of the adapter, and wherein the series of modes of operation of the support structure comprises a mode of detachment operation for detaching the adapter from the magnet. A system for recovering and processing material from a sample. 16. The system of claim 15, wherein the support structure includes a plunger in communication with the protrusion, the plunger configured to apply a displacement force upon activation of the plunger. 16. The system of claim 15, wherein the support structure comprises a coupling interface to a pipette component of an automated pipetting subsystem. A system for recovering and processing a material from a sample, the system comprising:
a first region configured to interface with a capture region of a capture substrate for capturing particles in a single particle format within a series of wells, a second region configured to interface with the capture region, and a second region in the first region an adapter comprising a cavity extending into; and
and a support structure coupled to the adapter and comprising a support structure that provides a set of modes of operation for movement of the adapter relative to the capture substrate. 19. The method of claim 18, further comprising a magnet coupled to the support structure, wherein the adapter defines a sleeve for the magnet and includes a surface, the surface preventing entrapment of magnetic particles within features of the surface. A system for recovering and processing a substance from a sample. 19. The system of claim 18, wherein the adapter comprises a vent configured to prevent gas entrapment between the adapter and the capture region.

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