Electrophoresis cassette and instrument

Abstract

Provide various implementations of electrophoresis systems and instruments to improve electrophoresis and ease of use. The various electrophoresis systems and instruments utilize different electrode designs, which can result in uniform electro-migration of analytes. The electrophoresis systems and instruments optimize the size and / or position of the electrodes relative to the sample, thereby increasing the uniformity of the electric field and reducing or minimizing the drift of analyte migration. In addition, the electrophoresis systems and instruments provide a solution to conveniently check the electrical connections in the systems and instruments before performing electrophoresis and alert the user to potential inappropriate electrical connections.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Patent Application Serial No. 63 / 026,975, filed May 19, 2020, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Due to different analyte levels (e.g., gene and / or protein expression) within different cells, cells within a tissue differ in cell morphology and / or function. The specific location of a cell within a tissue (e.g., the position of the cell relative to adjacent cells or the position of the cell relative to the tissue microenvironment) can affect, for example, the morphology, differentiation, fate, viability, proliferation, behavior, signaling, and crosstalk with other cells in the tissue.

[0004] Spatial heterogeneity has been studied using techniques that typically provide data on a few analytes in the context of a whole tissue or a part of a tissue (e.g., a tissue section), or that provide significant analyte data from individual, isolated cells, but fail to provide information on the location of individual cells from a native biological sample (e.g., a tissue).

[0005] Various methods have been used to prepare biological samples for analyzing analyte data in the sample. In some applications, the biological sample can be permeabilized to facilitate the transfer of analytes out of the sample and / or to facilitate the transfer of species (such as capture probes) into the sample. Summary of the Invention

[0006] This document generally relates to electrophoretic devices, apparatuses, systems, instruments, and methods for analyte migration.

[0007] Generally, electrophoresis can be used to migrate analytes from a sample towards a substrate including capture probes for spatial transcriptomics applications to enhance sensitivity and / or resolution. The present disclosure provides various implementations of electrophoresis devices, systems, and instruments to improve electrophoresis and ease of use. The electrophoresis systems and instruments described herein provide different designs for electrode geometries and / or placements and further provide different methods for electrical interfaces between components. For example, various configurations of the electrophoresis systems and instruments described herein utilize different electrode designs that can result in uniform electro-migration of analytes. In addition, the electrophoresis systems described herein take into account that in the case where the sample tissue is very thin, a relatively small electric field angle has no or little effect on the drift of analyte migration. In addition, various implementations of the electrophoresis systems and instruments optimize the size and / or position (in the x, y, and z directions) of the electrodes (e.g., the cathode) relative to the sample, thereby increasing the uniformity of the electric field and reducing or minimizing the drift of analyte migration. In addition, the electrophoresis systems and instruments described herein provide a solution to conveniently check the electrical connections in the systems and instruments before performing electrophoresis and alert the user to potential incorrect electrical connections.

[0008] Certain implementations of the present disclosure described herein provide an electrophoresis system for analyte migration. The system includes a substrate, a substrate cassette, a cathode assembly, and a power supply. The substrate may include multiple regions. Each region may be conductive and further configured to include one or more capture probes and a biological sample including analytes. The substrate may include a first electrode contact. The first electrode contact may be electrically connected to at least one of the multiple regions. The substrate cassette may be disposed at the substrate and include multiple holes corresponding to the multiple regions of the substrate. The multiple holes may define multiple buffer chambers including the multiple regions of the substrate. The substrate cassette may include a connection interface electrically coupled to the first electrode contact of the substrate. In some implementations, the substrate cassette may include a connection interface that exposes the first electrode contact of the substrate. The cathode assembly may include multiple electrodes respectively positioned in the multiple buffer chambers of the substrate cassette. The cathode assembly may include a second electrode contact. The second electrode contact may be electrically connected to at least one of the multiple electrodes. The power supply may be electrically connected to the first electrode contact of the substrate at the connection interface of the substrate cassette and electrically connected to the second electrode contact of the cathode assembly. The power supply may generate an electric field between the multiple regions and the multiple electrodes respectively, such that the analytes in the biological sample migrate towards the capture probes on the substrate.

[0009] In some implementations, the systems described herein may include one or more of the following features. Capture probes may be coated, spotted, or printed on each of a plurality of regions. A biological sample may be placed in contact with one or more of the plurality of regions on a substrate. A substrate cartridge may include: a substrate holder that includes a substrate mount for securing the substrate; and a gasket that includes a plurality of gasket holes configured to align with the plurality of regions when the substrate is secured by the substrate holder. The plurality of holes may include the plurality of gasket holes. The substrate holder may include a plurality of holder holes configured to align with the plurality of gasket holes when the substrate is secured by the substrate holder. The plurality of holes may include the plurality of gasket holes and the plurality of holder holes. The substrate may include a slide coated with a conductive material. The conductive material may include at least one of tin oxide (TO), indium tin oxide (ITO), transparent conductive oxide (TCO), aluminum-doped zinc oxide (AZO), fluorine-doped tin oxide (FTO), or any combination thereof. An electrophoresis system may include: a first wire that connects a power source to a first electrode contact; and a second wire that connects the power source to a second electrode contact. A plurality of buffer chambers may receive a buffer. The buffer may include a permeabilization reagent. The electrophoresis system may include a light source configured to illuminate the plurality of regions to permeabilize the biological sample on the plurality of regions. A detergent may be used to permeabilize the sample before, after, or during enzyme treatment. The sample may be permeabilized by a solubilizing reagent added to the sample. The sample may be permeabilized by exposing the sample to a protease.

[0010] The substrate cartridge may include a body that defines a cavity configured to partially receive the substrate. The connection interface of the substrate cartridge may include a slit defined in the body and configured to allow the substrate to extend partially out of the body such that a first electrode contact of the substrate is positioned outside the body. The body may include a main face and a lateral face extending from the periphery of the main face. The plurality of holes may be defined in the main face of the body. The slit may be defined in the lateral face of the body.

[0011] The substrate cartridge may include a body that defines a cavity configured to receive the substrate. The connection interface of the substrate cartridge may include a contact opening defined in the body and configured to expose a first electrode contact of the substrate therethrough. The body may include a main face and a lateral face extending from the periphery of the main face. The plurality of holes may be defined in the main face of the body. The contact opening may be defined in the main face adjacent to the lateral face. The contact opening may be further defined in the lateral face of the body to extend from the main face to the lateral face.

[0012] The electrophoresis system may include a first contact pin that is electrically attached to a substrate and provides a first electrode contact. The substrate cassette may include a body that defines a cavity configured to receive a substrate. The connection interface of the substrate cassette may include a contact hole defined in the body and configured to expose the first contact pin therethrough. The body may include a main face and a lateral face extending from the periphery of the main face. A plurality of holes may be defined in the main face of the body. The contact hole may be defined in the main face.

[0013] The substrate cassette may include a body that defines a cavity configured to receive a substrate; and a contact pin that extends through a wall of the body and has a first end and a second end opposite the first end. The first end may be disposed outside the body, and the second end may be disposed inside the body and configured to make electrical contact with a first electrode contact of the substrate. The electrode system may include a contact bracket that is electrically engaged with the substrate and provides a first electrode contact of the substrate. The second end of the contact pin may make electrical contact with the contact bracket.

[0014] The cathode assembly may include a cathode body configured to be at least partially positioned on the substrate cassette. A plurality of electrodes may project from the cathode body. Each of the plurality of electrodes may include at least one of a conductive wire, a conductive rod, or an array of conductive wires. Each of the plurality of electrodes may include a conductive plate at a distal end of at least one of a conductive wire, a conductive rod, or an array of conductive wires. The conductive plate may be configured as at least one of a circular plate, a square plate, or an annulus. The cathode body may include a second electrode contact.

[0015] The substrate cassette may include a second hole corresponding to the first electrode contact of the substrate and configured to define a second buffer chamber over the first electrode contact of the substrate. A plurality of buffer chambers may contain a first buffer. The second chamber may contain a second buffer different from the first buffer. The second buffer may have an electrolyte strength greater than that of the first buffer. The second buffer may include a sodium chloride solution. The cathode assembly may include a cathode body configured to be at least partially positioned on the substrate cassette. A plurality of electrodes may project from the cathode body and be configured to be respectively positioned in the plurality of buffer chambers. The cathode assembly may include a second electrode that projects from the cathode body and is configured to be positioned in the second chamber. The cathode body may include an anode contact electrically connected to the second electrode. A power supply may be electrically connected to the anode contact of the cathode body such that the power supply is electrically connected to the first electrode contact of the substrate via the second electrode, and the second electrode is positioned in the second chamber containing the second buffer over the first electrode contact of the substrate.

[0016] The substrate cassette may include a cassette body that defines a cavity configured to receive a substrate. The cassette body may include a cathode assembly such that a plurality of electrodes are configured to be positioned within a plurality of buffer chambers. The substrate cassette may include a plurality of cathode contact pins that extend through the wall of the cassette body and make electrical contact with a second electrode contact such that the cathode contact pins are electrically connected to at least one of the plurality of electrodes. Each of the plurality of electrodes may include at least one of a conductive wire, a conductive rod, or an array of conductive wires. Each of the plurality of electrodes may include a conductive plate at a distal end of at least one of a conductive wire, a conductive rod, or an array of conductive wires. The cassette body may include an anode contact pin that extends through the wall of the body and has a first end and a second end opposite the first end. The first end may be disposed outside the cassette body, and the second end may be disposed inside the cassette body and be configured to make electrical contact with a first electrode contact of the substrate. The electrode system may include a contact carriage that is electrically engaged with the substrate and provides the first electrode contact of the substrate. The second end of the anode pin may be in electrical contact with the contact carriage.

[0017] The body may include a cathode assembly such that a plurality of electrodes are configured to be positioned within a plurality of buffer chambers. The substrate cassette may include cathode contact pins that extend through the wall of the body and make electrical contact with a second electrode contact such that the cathode contact pins are electrically connected to at least one of the plurality of electrodes. The substrate cassette may include: an anode contact pin that extends from the body; and a conductive wire that has a first end and an opposite second end. The first end may be electrically connected to the anode contact pin, and the second end may be electrically connected to a first electrode contact positioned outside the body.

[0018] The electrophoresis system may include an anode cassette body and an anode contact pin. The anode cassette body may define a cavity configured to receive a first electrode contact of the substrate that is positioned outside the body of the substrate cassette. The anode contact pin may extend through the wall of the anode cassette body and have a first end and a second end opposite the first end. The first end may be disposed outside the anode cassette body, and the second end may be disposed inside the anode cassette body and be configured to make electrical contact with a first electrode contact of the substrate. The electrode system may include an anode contact carriage that is electrically engaged with the substrate and provides the first electrode contact of the substrate. The second end of the anode contact pin may be in electrical contact with the anode contact carriage.

[0019] The electrophoresis system may include a system housing that includes a power supply; a cassette tray that extends from the system housing and is configured to receive a substrate cassette thereon; and a cassette cover that extends from the system housing above the cassette tray.

[0020] An electrophoresis system can include a system housing that includes a power supply; a cassette tray that extends from the system housing and is configured to receive a substrate cassette thereon; and a cassette cover that extends from the system housing above the cassette tray. The cassette cover can include a cathode connector and an anode connector. The cathode connector can be electrically engaged with a cathode contact pin of the substrate cassette. The anode connector can be electrically engaged with an anode contact pin of the substrate cassette.

[0021] An electrophoresis system can include a system housing that includes a power supply; a cassette tray that extends from the system housing and is configured to receive a substrate cassette thereon; and a cassette cover that extends from the system housing above the cassette tray and includes a cathode assembly. A plurality of electrodes can project from the cassette cover toward a plurality of chambers of the substrate cassette. The cassette cover can include an anode connector that is configured to be electrically engaged with a contact pin of the substrate cassette.

[0022] Certain implementations of the present disclosure described herein provide a method for analyte migration. The method can include loading a substrate including capture probes and a substrate cassette into an electrophoresis instrument, the substrate cassette including a plurality of buffer chambers, the substrate including a plurality of regions, each region including one or more capture probes and a biological sample containing an analyte; arranging a cathode to place a plurality of electrodes respectively within the plurality of buffer chambers; electrically connecting a power supply to the substrate; electrically connecting the power supply to the cathode; applying a first voltage between the substrate and the cathode; detecting an output parameter in response to the first voltage; determining whether the output parameter meets a threshold; and based on the output parameter meeting the threshold, applying a second voltage to generate an electric field between the plurality of regions of the substrate and the plurality of electrodes through the plurality of buffer chambers to cause the analyte in the biological sample to move toward the capture probes on the substrate.

[0023] In some implementations, the method described herein can optionally include one or more of the following features. The output parameter can be impedance. The threshold can indicate the adequacy of at least one of the electrical connection of the power supply to the substrate, the electrical connection of the power supply to the cathode, or the electrical properties of the buffer. The method can include: based on the output parameter not meeting the threshold, generating a notification to notify that the electrical connection is inadequate; and stopping the application of the second voltage for generating the electric field. The threshold can be a value within a predetermined range. The method can include: before loading the substrate and the substrate cassette, placing the biological sample in contact with the capture probes on the substrate; arranging the substrate cassette onto the substrate to align a plurality of holes of the substrate cassette with the plurality of regions of the substrate and defining a plurality of buffer chambers over the plurality of regions; and supplying a buffer into the plurality of buffer chambers.

[0024] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, or patent application or item of information were specifically and individually incorporated by reference. If the publications, patents, patent applications, and items of information incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0025] When values are described in a range, it is to be understood that the description includes all possible sub-ranges within such range, as well as specific values falling within such range, whether or not the specific values or specific sub-ranges are expressly stated.

[0026] The term "each", when used to refer to a collection of items, is intended to identify a single item in the collection, but not necessarily every item in the collection, unless expressly stated otherwise or unless the context in which it is used otherwise clearly indicates.

[0027] Various embodiments of the features of the present disclosure are described herein. However, it is to be understood that these embodiments are provided only as examples, and that many variations, alterations, and substitutions may be envisioned by those skilled in the art without departing from the scope of the present disclosure. It is also to be understood that various alternatives to the specific embodiments described herein are also within the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended in any way to limit the scope of the appended claims. Like reference numerals in the drawings indicate like elements.

[0029] Figures 1A to 1B are a schematic side view and a top view of an exemplary electrophoresis system for analyte migration.

[0030] Figure 2 is a schematic perspective view of an exemplary substrate.

[0031] Figure 3 illustrates the use of Figures 1A to 1B an exemplary configuration of a substrate and a sample for electrophoresis processing using the electrophoresis system.

[0032] Figure 4A is a schematic top view of an exemplary substrate cassette.

[0033] Figure 4B is for use with Figure 4A a schematic top view of an exemplary substrate for use with the substrate cassette.

[0034] Figure 4C is for receiving Figure 4B the substrate of Figure 4ASchematic top view of a substrate cassette.

[0035] Figure 4D is a schematic perspective view of an example substrate for use with a Figure 4A substrate cassette.

[0036] Figure 5 Shows an Figure 4A example of a substrate cassette.

[0037] Figure 6 is a bottom cross-sectional perspective view of the substrate cassette taken along line A-A of Figure 5 the substrate cassette.

[0038] Figure 7A Shows a top view of an example substrate cassette in an open position.

[0039] Figure 7B Shows an Figure 7A example side view of the lateral side of the substrate cassette.

[0040] Figure 7C Shows an Figure 7B example cross-sectional view of the lateral side of the substrate cassette.

[0041] Figure 8 Shows an example of placing a substrate into a Figure 7A substrate cassette.

[0042] Figure 9 is a schematic top view of another example substrate cassette.

[0043] Figure 10A is a schematic top view of yet another example substrate cassette.

[0044] Figure 10B is for use with a Figure 10A substrate cassette and is a schematic top view of yet another example substrate.

[0045] Figure 10C is a schematic top view of a substrate cassette that receives a Figure 10B substrate. Figure 10A substrate cassette.

[0046] Figure 10D is a schematic cross-sectional side view of a substrate cassette that receives a Figure 10B substrate. Figure 10A substrate cassette.

[0047] Figure 11A is a schematic top view of yet another example substrate cassette.

[0048] Figure 11B is for use with a Figure 11A substrate cassette and is a schematic top view of yet another example substrate.

[0049] Figure 11C is a schematic top view of a substrate cassette that receives Figure 11B the substrate of Figure 11A

[0050] Figure 11D is a schematic top view of a substrate cassette that receives Figure 11B the substrate of Figure 11A

[0051] Figure 12A is a schematic top view of an exemplary cathode assembly.

[0052] Figure 12B is a schematic top view of an exemplary substrate cassette that receives an exemplary substrate.

[0053] Figure 12C is provided on Figure 12B the substrate cassette and the substrate of Figure 12A

[0054] Figure 12D is Figure 12C a schematic cross-sectional side view of the cathode assembly, the substrate cassette, and the substrate.

[0055] Figure 12E is Figure 12C a schematic cross-sectional side view of the cathode assembly, the substrate cassette, and the substrate, wherein the cathode assembly has another exemplary electrode.

[0056] Figure 12F is Figure 12C a schematic cross-sectional side view of the cathode assembly, the substrate cassette, and the substrate, wherein the cathode assembly has yet another exemplary electrode.

[0057] Figure 12G is Figure 12C a schematic cross-sectional side view of the cathode assembly, the substrate cassette, and the substrate, wherein the cathode assembly has yet another exemplary electrode.

[0058] Figure 13A is a schematic top view of another exemplary cathode assembly.

[0059] Figure 13B is a schematic top view of an exemplary substrate.

[0060] Figure 13C is a schematic top view of an exemplary substrate cassette.

[0061] Figure 13D is provided on Figure 13B the substrate cassette and Figure 13C the substrate of Figure 13A ​​​​

[0062] Figure 13E is Figure 13D A schematic cross-sectional side view of a cathode assembly, a substrate cassette, and a substrate, showing an example buffer chamber.

[0063] Figure 13F is Figure 13D A schematic cross-sectional side view of a cathode assembly, a substrate cassette, and a substrate, showing an example anode chamber.

[0064] Figure 14A A schematic top view of yet another example substrate cassette for receiving a substrate.

[0065] Figure 14B is Figure 14A A schematic cross-sectional side view of a substrate cassette and a substrate.

[0066] Figure 14C is Figure 14A A schematic cross-sectional side view of a variant of a substrate cassette and a substrate.

[0067] Figure 15A A schematic top view of yet another example substrate cassette for receiving a substrate.

[0068] Figure 15B is Figure 15A A schematic cross-sectional side view of a substrate cassette and a substrate.

[0069] Figure 16A A schematic top view of an example electrical connection configuration between a substrate cassette, a substrate, and a control system.

[0070] Figure 16B A schematic top view of another example electrical connection configuration between a substrate cassette, a substrate, and a control system.

[0071] Figure 16C is Figure 16B A schematic cross-sectional side view of a part of an electrical connection configuration.

[0072] Figure 17A A schematic perspective view of an example instrument for electrophoresis.

[0073] Figure 17B A schematic cross-sectional side view of an example lid of an instrument for receiving an example set of substrate cassettes and substrates.

[0074] Figure 17C A schematic cross-sectional side view of another example lid of an instrument for receiving an example set of substrate cassettes and substrates.

[0075] Figure 18 A flowchart of an example process for analyte migration.

[0076] Figure 19 is a block diagram of a computing device that can be used to implement the systems and methods described in this document as a client or server or multiple servers.

[0077] Figure 20A Shows a schematic diagram of an exemplary analysis workflow, where electrophoretic migration of the analyte is performed after permeabilization.

[0078] Figure 20B Shows a schematic diagram of an exemplary analysis workflow, where electrophoretic migration and permeabilization of the analyte are performed simultaneously.

[0079] Figure 21A Shows an exemplary vertical, single-slide configuration for use during electrophoresis.

[0080] Figure 21B Shows an exemplary parallel, single-slide configuration for use during electrophoresis.

[0081] Figure 21C Shows an exemplary multi-slide configuration for use during electrophoresis.

[0082] Figures 22A to 22B is an exploded view ( Figure 22A ) and a side view ( Figure 22B ) of an electrophoretic transfer system configured to direct a transcript analyte to a spatially barcoded capture probe array.

[0083] Figure 23 is a schematic diagram showing an exemplary workflow protocol using the electrophoretic transfer system. Detailed Description

[0084] As described herein, electrophoresis can be used to migrate analytes from a sample towards a substrate comprising capture probes for spatial transcriptomics applications to enhance sensitivity and / or resolution. The present disclosure provides various implementations of electrophoresis systems and instruments to improve electrophoresis and ease of use. The electrophoresis systems and instruments described herein provide different designs for electrode geometries and / or placements and further provide different methods for electrical interfaces between components. For example, various configurations of the electrophoresis systems and instruments described herein utilize different electrode designs that can result in uniform electro-migration of analytes. In addition, the electrophoresis systems described herein recognize that in the case where the sample tissue is very thin, a relatively small electric field angle has no or little effect on the drift of analyte migration. In addition, various implementations of the electrophoresis systems and instruments optimize the size and / or position (x, y, z directions) of the electrodes (e.g., the cathode) relative to the sample, thereby increasing the uniformity of the electric field and reducing or minimizing the drift of analyte migration. In addition, the electrophoresis systems and instruments described herein provide a solution to conveniently check the electrical connections in the systems and instruments before performing electrophoresis and alert the user to potential improper electrical connections.

[0085] Figures 1A to 1B FIGS. 4A and 4B are schematic side and top views of an exemplary electrophoresis system 100 for analyte migration. In some implementations, system 100 is configured similar to the electrophoresis systems described herein, e.g., with reference to Figures 20A to 20B and Figures 21A to 21C . System 100 can be used to actively migrate analytes (e.g., nucleic acids, proteins, charged molecules, etc.) described herein in a biological sample towards capture probes on a substrate. In some implementations, system 100 can include a substrate 102 as a first electrode (e.g., an anode), a second electrode 104 (e.g., a cathode), and a spacer 110 disposed between substrate 102 and second electrode 104.

[0086] Also referring to Figure 2 , substrate 102 is configured for placement of a sample containing one or more analytes 314 (not shown in Figure 2 ; see Figure 3) of the biological sample 112. For example, the biological sample 112 can be one or more cells or a tissue sample comprising one or more cells. The substrate 102 can include one or more regions 116 for placing or containing the sample 112 thereon. In some implementations, the substrate 102 can further include one or more capture probes 118 on the substrate region 116. The capture probes 118 can be placed on the substrate region 116 in various ways described herein. For example, the capture probes 118 can be attached (e.g., directly or indirectly, reversibly or irreversibly) to regions on an array. Alternatively or additionally, the capture probes 118 can be immobilized on the substrate region 116 of the substrate 102. The sample 112 can be placed and manipulated on the substrate 102 in various ways described herein. In some implementations, the biological sample can be placed in contact with the capture probes on each region. Alternatively, the biological sample does not contact the capture probes. For example, an intermediate layer (e.g., a gel) can be provided between the substrate and the sample, which is compatible with electrophoresis.

[0087] In some implementations, the substrate 102 is configured to be used as the first electrode in the system 100. For example, the substrate 102 can be used as an anode. In another example, the substrate 102 can be used as a cathode. The substrate 102 can be configured to be conductive at least in the substrate region 116. In some implementations, the substrate 102 can be configured as the conductive substrate described herein. For example, the substrate 102 can include one or more conductive materials that allow the substrate 102 to be used as an electrode (e.g., an anode). Examples of such conductive materials include tin oxide (TO), indium tin oxide (ITO), transparent conductive oxide (TCO), aluminum-doped zinc oxide (AZO), fluorine-doped tin oxide (FTO), or any combination thereof. Alternatively or additionally, other materials can be used to provide the desired conductivity for the substrate 102. In some implementations, the substrate 102 can be coated with a conductive material. For example, the substrate 102 can include a conductive coating 109 ( Figure 1A ) on the surface of the substrate 102, and the sample 112 is provided on the coating 109 of the substrate 102. In other implementations, the substrate 102 can be made partially or entirely of a conductive material. For example, a sample (e.g., a tissue section) can be placed directly on a conductive material (e.g., graphite, silicon, other semiconductor materials, etc.). For imaging purposes, the substrate 102 can be made of glass or other transparent materials.

[0088] Although in Figure 2 the substrate 102 is shown as including a single substrate region 116, other implementations of the substrate 102 can include multiple substrate regions configured to respectively place multiple samples thereon (e.g., Figures 1A to 1Btwo substrate regions in [reference], eight substrate regions in FIG. 4, etc.). Each of such multiple substrate regions can be configured similarly to the first substrate region 116 described herein.

[0089] Reference Figures 1A to 1B , where the substrate 102 is used as the anode and the second electrode 104 is configured as the cathode. Thus, in this example, the second electrode 104 can also be referred to as the cathode 104. As described herein, the cathode 104 can be provided in various configurations.

[0090] In some implementations, the substrate 102 and the cathode 104 can be disposed within a container (e.g., the substrate cassette described herein), which defines a buffer chamber 122 between the substrate 102 and the cathode 104. The buffer chamber 122 is configured to contain a buffer 124. In some implementations, the substrate 102 and the cathode 104 can be fully immersed in the buffer 124. In alternative implementations, one or both of the substrate 102 and the cathode 104 can be partially inserted into the buffer 124 located within the container.

[0091] The buffer 124 can be of various types. In some implementations, the buffer 124 includes a permeabilization reagent. In some implementations, the buffer 124 is contained within the buffer chamber 122 throughout the electrophoresis process. The permeabilization reagent can permeabilize the sample before and / or during electrophoresis. Additionally or alternatively, other methods described herein can be used, either independently or in combination with the permeabilization by the permeabilization reagent, to permeabilize the sample.

[0092] A spacer 110 can be disposed between the substrate 102 and the cathode 104 to space the substrate 102 and the cathode 104 apart by a distance D. The spacer 110 can include a non-conductive material such as plastic, glass, ceramic, rubber, silicone, etc. The distance D can be determined based on several factors to provide a desired level of spatial resolution, such as the intensity and / or duration of the electric field generated between the substrate 102 and the cathode 104, and other parameters described herein. The spacer 110 can define at least a portion of the buffer chamber 122 between the substrate 102 and the cathode 104.

[0093] The control system 130 can generate an electric field (-E) between the substrate 102 and the cathode 104. The control system 130 can include a controller 132 configured to apply a voltage between the substrate 102 and the cathode 104 using a power supply 134. The power supply 134 can include a high-voltage power supply. The controller 132 can be electrically connected to the substrate 102 and the cathode 104, for example, using wires. The control system 130 can include a user interface 136 configured to receive user input to start or stop the electrophoresis process. The user interface 136 can include various types of input devices, such as a graphical user interface, physical or virtual buttons, switches, keypads, keyboards, etc., which are configured to receive user input for adjusting the operating parameters of the system 100 or for accessing other information (e.g., instructions) associated with the system 100. Examples of operating parameters can include, but are not limited to, the applied voltage, the duration of voltage application, etc. In some implementations, the input device can be used to select a subset of the substrate regions 116 on the sample 112 such that the subset of the substrate regions 116 is electrically activated to generate an electric field between the subset of the substrate regions 116 and the cathode 104. Additionally, the user interface 136 can include output devices, such as a display, lights, etc., which are configured to output the operating parameters of the system 100 or other information associated with the system 100.

[0094] Figure 3 An example configuration of a substrate and a sample for performing electrophoresis using the electrophoresis system 100 is shown. Figure 3 The configuration and process shown therein can be similar to the electrophoresis process described herein, for example, with reference to Figures 21A to 21C . With reference to Figure 3 , the application of the electric field (-E) causes the analyte 314 (e.g., a negatively charged analyte) to move in the direction of the arrow shown towards the capture probe 318 (e.g., a positively charged analyte). In some implementations, the analyte 314 includes a protein or a nucleic acid. In some embodiments, the analyte 314 is a negatively charged protein or nucleic acid. In some implementations, the analyte 314 includes a positively charged protein or nucleic acid. In some embodiments, the analyte 314 includes a negatively charged transcript. For example, the analyte 314 includes a poly-A transcript. In some implementations, a detergent or other reagent can be added to change the charge of the analyte. For example, SDS, as used in SDS-PAGE, can coat the protein and provide substantially a uniform negative charge on the protein. Other implementations can be used to change the charge of a molecule by covalently linking another charged molecule. Yet another option is to change the pH value of the solution.

[0095] In some embodiments, the capture probe 318 is configured as a coating that contacts the substrate 302 (e.g., a coating provided on the surface of the substrate 302). In some embodiments, the capture probe 318 may include a feature array or may be replaced by a feature array. In some implementations, the feature array may be the same as the substrate regions described herein. In alternative embodiments, the feature array may be different from the substrate regions described herein. In some embodiments, the analyte 314 moves a distance (h) toward the capture probe 318. In some embodiments, the buffer 324 (e.g., a permeabilization reagent) may contact the sample 312, the substrate 302, the cathode 304, or any combination thereof. The buffer 324 may include any of the disclosed permeabilization reagents, including but not limited to dried permeabilization reagents, permeabilization buffers, buffers without permeabilization reagents, permeabilization gels, and permeabilization solutions.

[0096] Referring to FIGS. 4 to 16, various configurations of the electrophoresis system 100 are described. Generally, as described herein, the system 100 may include a substrate, a substrate cassette, a cathode assembly (e.g., FIGS. 12 to 13), and a control system. The substrate may be configured similar to the substrate 102 or other substrates described herein. The substrate cassette may be configured to be disposed on the substrate and define a buffer chamber (e.g., buffer chamber 122) on the substrate. The cathode assembly is configured to implement the cathode 104 described herein. For example, the cathode assembly may include a plurality of electrodes configured to be located in respective buffer chambers of the substrate cassette. The control system may be configured similar to the control system 130. For example, the control system includes a power supply electrically connected to the substrate and the cathode assembly and may be powered to generate an electric field between the substrate (e.g., its corresponding substrate regions) and the cathode assembly (e.g., its plurality of electrodes) such that analytes in a biological sample migrate toward capture probes on the substrate.

[0097] Referring Figures 4A to 4D , an example of the substrate cassette 404 is described. The substrate cassette 404 includes a body 410 and a plurality of holes 412. The body 410 may be configured to be mounted on the substrate 402 or receive the substrate 402. For example, the body 410 may be configured to define a cavity that can receive at least a portion of the substrate 402. The plurality of holes 412 are configured and arranged in the body 410 such that when the substrate cassette 404 is disposed on the substrate 402 or receives the substrate 402, the plurality of holes 412 are aligned with a plurality of substrate regions 416 ( Figure 4B ) of the substrate 402 and define a plurality of buffer chambers on the plurality of substrate regions 416 of the substrate 402.

[0098] Also referring Figure 4D, the substrate 402 can be configured similarly to the substrate 102. The substrate 402 is configured to hold a plurality of biological samples 413 containing one or more analytes. For example, each biological sample 413 can be one or more cells or a tissue sample comprising one or more cells. The substrate 402 can include a plurality of substrate regions 416 for placing or containing the samples 413 thereon. In some implementations, the substrate 402 can further include one or more capture probes 418 on each substrate region 416. The capture probes 418 can be placed on the substrate regions 416 in various ways described herein. For example, the capture probes 418 can be directly attached (e.g., reversibly or irreversibly) to features on the array. In another example, the capture probes 418 can be indirectly attached (e.g., reversibly or irreversibly) to features on the array. Alternatively or additionally, the capture probes 418 can be immobilized on the substrate regions 416 of the substrate 402. The samples 413 can be prepared on the substrate 102 in various ways described herein.

[0099] In some implementations, the substrate 402 is configured to be used as the first electrode in the electrophoresis system 100. For example, the substrate 402 can be used as the anode. In another example, the substrate 402 can be used as the cathode. The substrate 402 can be configured as the conductive substrate described herein. For example, the substrate 402 can include one or more conductive materials that allow the substrate 402 to function as an electrode (e.g., the anode). Examples of such conductive materials include tin oxide (TO), indium tin oxide (ITO), transparent conductive oxide (TCO), aluminum-doped zinc oxide (AZO), fluorine-doped tin oxide (FTO), and any combination thereof. Alternatively or additionally, other materials can be used to provide the desired conductivity to the substrate 402. In some implementations, the substrate 402 can be coated with a conductive material. For example, the substrate 402 can include a conductive coating on the surface of the substrate or on each substrate region of the substrate, and the sample 413 is provided on the coating of the substrate 402 or on each of its substrate regions.

[0100] Return reference Figure 4B , the substrate 402 includes a first electrode contact 420 for electrically connecting to a control system 408 (e.g., its power supply). For example, the first electrode contact 420 can be electrically connected to a first wire 422 extending from the control system 408 ( Figure 4C ). The first electrode contact 420 is electrically connected to the plurality of substrate regions 416.

[0101] The substrate cartridge 404 further includes a connection interface 414 configured to expose the first electrode contact 420 of the substrate 402 for electrical connection to the control system 408. For example, the substrate cartridge 404 can include a slit 417 as the connection interface 414. The slit 417 can be defined at the body 410. AsFigure 4C As shown, substrate 402 is received in substrate cassette 404. The slit 417 of substrate cassette 404 may allow substrate 402 to partially extend out of the main body 410 such that the first electrode contact 420 of substrate 402 is positioned outside the main body 410. The control system 408 may be connected to substrate 402 via the first electrode contact 420 exposed from substrate cassette 404. For example, the first wire 422 extending from the control system 408 may be electrically connected to the first electrode contact 420 outside substrate cassette 404. The first wire 422 may include a connection pin or clip 424 that may be removably attached to the first electrode contact 420 of substrate 402. Other types of fastening mechanisms may be used to connect the first wire 422 to the first electrode contact 420 of substrate 402.

[0102] Reference Figures 5 to 8 shows various examples of substrate cassettes 204. The substrate cassette 204 may also be referred to as a substrate holder.

[0103] Figure 5 An example of a substrate cassette 504 is shown. This embodiment of the substrate holder 504 may advantageously provide a single-piece component that may be arranged in an open configuration or a closed configuration as needed. Specifically, Figure 5 The top surface 593 of the substrate holder 504 in the closed position is shown. The substrate holder 504 includes a plurality of holes 512. For example, the substrate holder 504 includes a main body 510 having a main side 503 (e.g., a main surface or face), opposite longitudinal sides 505, and opposite transverse sides 507. The longitudinal sides 505 and the transverse sides 507 may form lateral sides (e.g., lateral surfaces or faces) extending from the periphery of the main side 503. The plurality of holes 512 may be provided at the main side 503 of the main body 510.

[0104] The substrate holder 504 may include a substrate loading mechanism for loading and holding substrates. For example, the substrate loading structure may include a first tab 550a and a second tab 550b. The first tab 550a and the second tab 550b may project from one of the longitudinal sides 505 of the substrate holder 504. In some embodiments, any type of fastener or engagement feature allowing releasable engagement may be used instead of the first tab 550a and the second tab 550b, e.g., screws and press-fit type connectors. In some embodiments, the substrate holder 504 includes 5 or fewer tabs (e.g., 4 or fewer tabs, 3 or fewer tabs, 2 or fewer tabs, or 1 tab). In some embodiments, the substrate holder 504 is a single molded unit. Any suitable plastic or polymer may be used as the suitable molding material.

[0105] Figure 6 is a bottom cross-sectional perspective view of a substrate holder 504 taken along line A-A Figure 5 The substrate holder 504 includes a gasket 654 and defines a cavity 652 configured to receive a substrate 502. In some embodiments, the substrate holder 504 is a single molded unit that includes the gasket 654. That is, in some embodiments, the substrate holder 504 and the gasket 654 are one piece. In some embodiments, the substrate holder 504 is overmolded with the gasket 654. For example, the substrate holder 504 is a first injection-molded plastic part, and a second part (e.g., a flexible material) is molded thereon to create the gasket 654. In some embodiments, the flexible material is an elastomer. In some embodiments, the flexible material is silicone rubber. In some embodiments, the gasket 654 is a separate part that is not molded with the substrate holder 504.

[0106] Referring Figures 5 to 6 , the substrate holder 504 includes a slit 516 defined at a lateral face of the body 510. As described herein, the slit 516 serves as an example of a connection interface 514 for exposing a first electrode contact 420 of the substrate 502. In some implementations, the slit 516 is defined at one of the lateral sides 507 and is configured to allow the substrate 502 to partially extend out of the body 510 at one of the lateral sides 507 such that the first electrode contact 620 of the substrate 502 is positioned outside the body 510. In other implementations, the slit 516 can be defined at a different side of the body 510, such as any suitable one of the main side 503, the longitudinal side 505, and the lateral side 507.

[0107] Figure 7A shows a top view of a substrate holder 704 in an open position. The opening and closing mechanism of the substrate holder 704 is a hinge mechanism. The bottom member 762 of the substrate holder 704 can be hinged to the top member 764 of the substrate holder 704 via a hinge 760. In some embodiments, the hinge 760 can be a living hinge. In some embodiments, the substrate holder 704 includes 10 or fewer hinges (e.g., 9 or fewer hinges, 8 or fewer hinges, 7 or fewer hinges, 6 or fewer hinges, 5 or fewer hinges, 4 or fewer hinges, 3 or fewer hinges, 2 or fewer hinges, or 1 hinge). Non-limiting examples of hinges that the substrate holder 704 can include include straight or flat living hinges, butterfly living hinges, child safety hinges, double living hinges, and triple living hinges.

[0108] The substrate holder 704 further includes one or more engaging features, such as a first notch 758a and a second notch 758b. When pressed together, the first notch 758a and the second notch 758b can engage the first protrusion 750a and the second protrusion 750b, respectively. The first notch 758a and the second notch 758b can protrude from a longitudinal side 705 of a top member 764 of the substrate holder 704. In some embodiments, the substrate holder 704 includes three, four, five, six, seven, eight, nine, ten or more notches. In some embodiments, the notches protrude from a longitudinal side 707 of the substrate holder 704. In some embodiments, the first notch 758a and the second notch 758b are rigid and do not bend when engaging the first protrusion 750a and the second protrusion 750b, respectively. In some embodiments, the first notch 758a and the second notch 758b can be flexible, respectively.

[0109] Figure 7B A side view of the lateral side 707 is shown. As Figure 7C shown, the first notch 758a and the second notch 758b can protrude upward and include a notch flange 766 that engages a protrusion ledge 768. Alternatively, in some embodiments, the substrate holder 704 includes a snap-fit locking mechanism for releasably receiving and releasably securing the substrate. Non-limiting examples of other types of fasteners in the locking mechanism for the substrate holder 704 include snaps, protrusions, male connectors, and female connectors.

[0110] Figure 7A and Figure 8 A view shows the substrate 702 being placed into the substrate holder 704. In some embodiments, the substrate 702 can be "loaded" onto an inner edge or inner rim of a bottom member 762 of the substrate holder 704, while a portion of the substrate 702 including the first electrode contact 720 is positioned outside the substrate holder 704. Once loaded, the top member 764 is closed by pressing the first notch 758a and the second notch 758b against the first protrusion 750a and the second protrusion 750b, respectively, thereby forming a tight seal with the substrate 702. In some embodiments, the substrate does not have to be tilted under the protrusion 750 or any other protrusion. In some embodiments, the substrate holder 704 includes one or more protrusions to assist in loading the slide onto an inner edge or inner rim of the bottom member 762 of the substrate holder 704. When the substrate 702 is loaded in the substrate holder 703, the first electrode contact 720 of the substrate 702 is exposed from the substrate holder 704 through a slit 716.

[0111] In other implementations, the hinge 760 can be provided at one of the lateral sides 707. An example of such a hinge arrangement is described in PCT / US20 / 79843, titled "IMAGING SUPPORT DEVICES", filed on April 24, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0112] Reference Figure 9 , in addition to the connection interface 214, another example of the substrate cassette 904 can be configured similar to the substrate cassettes 204, 504, and 704 described herein. Unlike Figure 3 and Figures 5 to 8 the substrate cassettes 204, 504, and 704, in this example, the substrate cassette 904 can be configured to fully receive the substrate 902. Further, instead of a slit, the substrate cassette 904 includes a contact opening 900 as the connection interface 914. The contact opening 900 is defined at the body 910 and is configured to expose the first electrode contact 920 of the substrate 902.

[0113] The contact opening 900 can be defined at the main side 903 of the body 910. In some implementations, the contact opening 900 is defined at the main side 903 adjacent to the lateral side of the body 910. As Figure 9 shown, for example, the contact opening 900 is defined at the main side 903 adjacent to one of the lateral sides 907. The contact opening 900 can be defined at both the main side 903 and the lateral side 907 so as to extend from the main side 903 of the body 910 to the lateral side 907.

[0114] Reference Figures 10A - 10D , yet another example of the substrate cassette 1004 is described, which includes a body 1010 and a plurality of holes 1012 as described herein. In addition to the connection interface 1014, the substrate cassette 1004 in this example is configured similar to the substrate cassettes described in FIGS. 4 to Figure 8 Unlike Figure 3 and Figures 5 to 8 the substrate cassettes 204, 504, and 704, in this example, the substrate cassette 1004 can be configured to fully receive the substrate 1002.

[0115] In this example, as Figure 10B shown, the substrate 1002 includes substrate contact pins 1015 that provide the first electrode contact 1020. The substrate contact pins 1015 can extend from the surface of the substrate 1002 that includes a plurality of substrate regions 1026. The substrate contact pins 1015 are electrically connected to the substrate 1002. In embodiments where the substrate 1002 includes a conductive coating 1009, the substrate contact pins 1015 can be electrically connected to the conductive coating 1009, asFigure 10D as shown.

[0116] The substrate cassette 1004 further includes contact holes 1010 as a connection interface 1014. The contact holes 1000 may be defined at the body 1010 and configured to expose the substrate contact pins 1015 of the substrate 1002. The contact holes 1000 may be defined at the main side 1003 of the body 1010. When the substrate 1002 is received in or held by the substrate cassette 1004, the contact holes 1000 may be positioned to align with the substrate contact pins 1015.

[0117] Referring Figures 11A - 11D , a fourth example of a substrate cassette 1104 is described, which includes a body 1110 and a plurality of holes 1112 as described herein. Except for the connection interface 1114, the substrate cassette 1104 in this example is configured similar to the substrate cassettes described in FIGS. 4 to Figure 8 as described. Different from the substrate cassettes 1104, 504, and 704 of Figure 3 and Figures 5 to 8 , in this example, the substrate cassette 1104 may be configured to fully receive the substrate 1102.

[0118] The substrate cassette 1104 includes cassette contact pins 1130 as a connection interface 1114. The cassette contact pins 1130 may extend through the body 1110 (e.g., the main side 1103 of the body 1110) such that a first end 1132 of the cassette contact pins 1130 is placed outside the body 1110 and a second end 1134 opposite to the cassette contact pins 1130 is placed inside the body 1110. The cassette contact pins 1130 are configured to be electrically connected to the substrate 1102 received in the substrate cassette 1104. In an embodiment where the substrate 1102 includes a conductive coating 1109, the cassette contact pins 1130 may be electrically connected to the conductive coating 1109. For example, the second end 1134 of the cassette contact pins 1130 is configured to be electrically connected to the conductive coating 1109, as Figure 11D shown. In some implementations, the system 100 may include a substrate contact bracket 1136 configured to engage with and be electrically connected to the substrate 1102. For example, the substrate contact bracket 1136 may engage with a portion of the substrate 1102 and be in electrical contact with the conductive coating 1109 of the substrate 1102, as Figure 11DAs shown. The cartridge contact pin 1130 can be configured to electrically connect to the substrate contact bracket 1136 when the substrate 1102 is received in the substrate cartridge 1104. For example, the second end 1134 of the cartridge contact pin 1130 is configured to contact the substrate contact bracket 1136 based on the substrate 1102 held in the substrate cartridge 1104. Thus, the cartridge contact pin 1130 can be electrically connected to the substrate 1102 and further provide a connection interface 1114 outside the substrate cartridge 1104. The first wire 1122 extending from the control system 1108 can be simply connected to the cartridge contact pin 1130 (e.g., its first end 1132) such that the control system 1108 can be electrically connected to the substrate 1102 that can be used as one of the electrodes (e.g., the anode) in electrophoresis.

[0119] Reference Figures 12A to 12D , an example of the cathode assembly 1206 is described. The cathode assembly 1206 can include a cathode body 1250 configured to be at least partially positioned on the substrate cartridge 1204. The cathode assembly 1206 can include a plurality of electrodes 1252 protruding from the cathode body 1250. When the cathode body 1250 is positioned on the substrate cartridge 1204, the plurality of electrodes 1252 are configured to be respectively positioned within the plurality of holes 1212 of the substrate cartridge 1204. Thus, the electrodes 1252 are disposed in respective buffer chambers defined by the holes 1212 of the substrate cartridge 1204 on the substrate region 1216 of the substrate 1202. Each of the electrodes 1252 serves as the cathode of each of the buffer chambers in the buffer chamber.

[0120] The cathode assembly 1206 can include a cathode contact 1254 configured to be electrically connected to the control system 1208. For example, the second wire 1226 extending from the control system 1208 is electrically connected to the cathode contact 1254. The second wire 1226 can include a connection pin or clip 1228 that can be removably attached to the cathode contact 1254 of the cathode assembly 1206. Other types of fastening mechanisms can be used to connect the second wire 1226 to the cathode contact 1254 of the cathode assembly 1206. The cathode contact 1254 is electrically connected to the plurality of electrodes 1252. In some implementations, as Figure 12D shown, the cathode assembly 1206 includes a conductive layer 1258 that is electrically connected to both the cathode contact 1254 and the plurality of electrodes 1252. The cathode contact 1254 can be exposed at the cathode body 1250 for easy access to the outside of the cathode body 1250.

[0121] As Figure 12CAs shown, the cathode assembly 1206 is disposed on the substrate 1204 that receives or holds the substrate 1202. The cathode assembly 1206 is oriented and arranged such that a plurality of electrodes 1252 are inserted into respective holes 1212 of the substrate cassette 1204 and are thus disposed on respective substrate regions 1216 of the substrate 1202. The cathode assembly 1206 can be used with substrates and substrate cassettes in various configurations. In Figure 12C the illustrated example shown, the cathode assembly 1206 is used with the substrate 1202 and the substrate cassette 1204 described in FIGS. 4 through Figure 8 . In other examples, the cathode assembly 1206 can be used with Figure 9 the substrates 1202 and substrate cassettes described in FIGS.

[0122] The electrodes 1252 can be of various shapes. For example, the electrodes 1252 can be configured as conductive wires 1255 ( Figure 12D ), conductive rods 1256 ( Figure 12E ), or arrays of conductive wires 1257 ( Figure 12F ). Alternatively, as Figure 12G shown, the electrodes 1252 can include conductive extensions 1261 and conductive plates 1263 located at the distal ends (i.e., free ends) of the conductive extensions 1261. The conductive extensions 1261 can be of various shapes, such as conductive wires 1255, conductive rods 1256, arrays of conductive wires 1257, or other suitable shapes. The conductive plates 1263 can be of various shapes, such as circular plates 1263A, rings 1263B, square plates 1263C, or other suitable shapes. Referring to Figures 13A - 13E , other examples of substrates 1302, substrate cassettes 1304, and cathode assemblies 1306 are described. Referring to Figure 13B , the substrate 1302 in this example is configured similarly to the substrate 1302 described herein. In this example, the substrate 1302 further includes an electrode region 1303 that serves as a first electrode contact 320. The electrode region 1302 is configured to be electrophoretically connected to the control system 1308 such that the substrate 1302 can be used as an electrode (e.g., an anode) in the system 100. In embodiments where the substrate 1302 includes a conductive coating 1309, the electrode region 1303 is electrically connected to the conductive coating 1309. For example, the electrode region 1303 can be a hole that exposes a portion of the conductive coating 1309 of the substrate 1302 to the second buffer 1307, as Figure 13F shown.

[0123] Referring to Figure 13C, the substrate cassette 1304 in this example is configured similarly to the substrate cassette 1304 described herein. The substrate cassette 1304 may include electrode holes 1301 and a plurality of holes 1312 described herein. The electrode holes 1301 are configured to align with the electrode regions 1303 of the substrate 1302 when the substrate cassette 1304 receives the substrate 1302. As Figure 13C shown, the substrate cassette 1304 is configured to fully receive the substrate 1302. Additionally, as Figures 13E to 13F shown, the plurality of holes 1312 and the electrode holes 1301 of the substrate cassette 1304 are respectively disposed on the plurality of substrate regions 1316 and the electrode regions 1303 of the substrate 1302. As described herein, the plurality of holes 1312 (either independently or in cooperation with Figure 13E the washer 1354 shown) may define a plurality of first buffer chambers (e.g., buffer chamber 122) on the plurality of substrate regions 1316. The first buffer chambers are configured to receive a first buffer 1305 (e.g., buffer 124) for analyte migration under electrophoresis. Additionally, the electrode holes 1301 (either independently or in cooperation with Figure 13F the washer 1354 shown) may define a second buffer chamber on the electrode region 1303. The second buffer chamber is configured to receive a second buffer 1307. In some implementations, the second buffer 1307 may be different from the first buffer 1305. The second buffer 1307 may have an electrolyte strength greater than that of the first buffer. For example, the second buffer 1307 may be selected to have an ionic strength of 1 to 1300 mmol / L. An example of the second buffer 1307 is a sodium chloride solution. Other examples of the second buffer 1307 include sodium hydroxide, potassium chloride, or any buffer with a high ionic concentration, which may include a higher concentration of the first buffer 1305. Further examples of the first buffer and the second buffer can be found in Reijenga et al., "Buffer Capacity, Ionic Strength and Heat Dissipation in Capillary Electrophoresis", Journal of Chromatography A, 744 (1996) 1347-153, the disclosure of which is incorporated herein by reference in its entirety.

[0124] Refer to Figure 13A, the cathode assembly 1306 includes a cathode body 1350, which is configured to be at least partially positioned on the substrate cassette 1304. The cathode assembly 1306 may include a plurality of electrodes 1350 protruding from the cathode body 1352. When the cathode body 1352 is positioned on the substrate cassette 1312, the plurality of electrodes 1350 are configured to be respectively positioned within the plurality of holes 1304 of the substrate cassette 1304. Thus, the electrodes 1352 are disposed in respective buffer chambers defined by the holes 1304 of the substrate cassette 1312 on the substrate region 316 of the substrate 1302. Each of the electrodes 1352 serves as the cathode for each of the buffer chambers in the buffer chamber.

[0125] The cathode assembly 1306 may include a second electrode contact 1354 configured to be electrically connected to the control system 1308. The second electrode contact 1354 may be used for electrode contact. For example, a second wire 1326 extending from the control system 1308 is electrically connected to the cathode contact 1354. The second wire 326 may include a connection pin or clip 1328, and the connection pin or clip 1328 may be removably attached to the cathode contact 1354 of the cathode assembly 1306. Other types of fastening mechanisms may be used to connect the second wire 1326 to the cathode contact 1354 of the cathode assembly 1306. The cathode contact 1354 is electrically connected to the plurality of electrodes 1352. In some implementations, as Figure 13E shown, the cathode assembly 1306 includes a conductive layer 1358, and the conductive layer 1358 is electrically connected to both the cathode contact 1354 and the plurality of electrodes 1352. The cathode contact 1354 may be exposed at the cathode body 1350 to facilitate access to the outside of the cathode body 1350.

[0126] In this example, the cathode assembly 1306 further includes a second electrode 1310 protruding from the cathode body 1350. As Figure 13F shown, the second electrode 1310 is configured to be positioned within the electrode hole 1301 of the substrate cassette 1304 when the cathode body 1350 is positioned on the substrate cassette 1304. Thus, the second electrode 1310 is disposed in a second buffer chamber defined by the electrode hole 1301 of the substrate cassette 1304 (either independently or in cooperation with the gasket 354), and the second buffer chamber is located on the electrode region 1303 of the substrate 1302. The second electrode 1310 may be used as part of the anode in electrophoresis.

[0127] The cathode assembly 1306 can include an anode contact 1313 configured to be electrically connected to a control system 1308. For example, a first wire 1322 extending from the control system 1308 is electrically connected to the anode contact 1313. The first wire 1322 can include a connection pin or clip 1328 that can be removably attached to the anode contact 1313 of the cathode assembly 1306. Other types of fastening mechanisms can be used to connect the first wire 1322 to the anode contact 1313 of the cathode assembly 1306. The anode contact 1313 is electrically connected to a second electrode 1310. In some implementations, as Figure 13F shown, the cathode assembly 1306 includes a conductive layer 1314 that is electrically connected to both the anode contact 1313 and the second electrode 1310. As Figure 13D shown, the anode contact 1313 can be exposed at the cathode body 1350 to facilitate access to the exterior of the cathode body 1350.

[0128] As Figure 13F shown, as the control system 1308 is electrically connected to the anode contact 1313 of the cathode assembly 1306, the control system 1308 is electrophoretically connected to an electrode region 1303 (i.e., a first electrode contact 320) of the substrate 1302 via the second electrode 1310, and the second electrode 1310 is positioned in a second chamber containing a second buffer 1307 on the electrode region 1303 of the substrate 1302.

[0129] Referring Figures 14A to 14C , yet another example of a substrate cassette 1404 is described. As described herein, the substrate cassette 1404 includes a body 1410 and a plurality of holes 1412. The substrate cassette 1404 in this example is configured similarly to the substrate cassette 404 described in FIGS. 4 through Figure 8 . Additionally, the substrate cassette 1404 is configured to incorporate at least a portion of the cathode assembly 1406 described herein. For example, as Figure 14B shown, the substrate cassette 1404 includes a plurality of electrodes 1403 that serve as the electrodes 1452 described herein. The plurality of electrodes 1403 are configured to be positioned within a buffer chamber 1405 (similar to the buffer chamber 122 described herein) defined at least by the holes 1412 of the substrate cassette 1404. Similar to the electrodes 1452, the electrodes 1403 can be of various configurations.

[0130] Referring Figure 14B, the substrate cartridge 1404 may include a plurality of cathode contact pins 1406 that extend through the wall of the cartridge body 1410 and are electrically connected to the plurality of electrodes 1403. The plurality of cathode contact pins 1406 are electrically connected to a second electrode contact or cathode contact 1454 that is exposed at the cartridge body 1410. Alternatively, one of the cathode contact pins 1456 may be exposed at the exterior of the cartridge body 1410 and serve as the cathode contact 1454. As described herein, the cathode contact 1454 is used to connect to the control system 1408, such as via a second wire 1426 extending from the control system 1408.

[0131] In this example, the substrate cartridge 1404 is used with the substrate 202 described in FIGS. 4 through Figure 8 In this example, the substrate cartridge 1404 may be used with the Figure 9 substrate 902 described in FIGS. 9 through 13.

[0132] Similar to the electrode 1452, the electrode 1403 may be of various configurations. For example, the electrode 1403 may be configured as a conductive wire (similar to the conductive wire 1255 in Figure 12D ), a conductive rod (similar to the conductive rod 1256 in Figure 12E ), or an array of conductive wires (similar to the array of conductive wires 1257 in Figure 12F ). Alternatively, the electrode 1403 may be configured similar to the motor 1252 shown in Figure 12G (e.g., having extensions with various tips, such as a circular plate, an annulus, a square plate, or other suitable shapes). Referring to Figure 14C , each buffer chamber 1405 may be provided with two or more electrodes 1403.

[0133] Referring to Figures 15A to 15B , yet another example of a substrate cartridge 1504 is described. The substrate cartridge 1504 in this example is configured similar to the Figures 14A to 14C substrate cartridge 1504. In addition, the substrate cartridge 1504 includes the Figures 11A to 11D connection interface 1514 shown in FIG. 29. Specifically, the substrate cartridge 1504 in this example further includes cartridge contact pins 1530 as the connection interface 1514. The cartridge contact pins 1530 may extend through the body 1510 such that a first end 1532 of the cartridge contact pins 1530 is placed outside the body 1510 while an opposite second end 1534 of the cartridge contact pins 1530 is placed inside the body 1510. The cartridge contact pins 1530 are configured to be electrically connected to the substrate 1502 received in the substrate cartridge 1504. In an embodiment where the substrate 1502 includes a conductive coating 1509, the cartridge contact pins 1530 may be electrically connected to the conductive coating 1509. For example, the second end 1534 of the cartridge contact pins 1530 is configured to be electrically connected to the conductive coating 1509, asFigure 15B as shown.

[0134] In some implementations, system 1500 may include a substrate contact bracket 1536 configured to engage with and be electrically connected to substrate 1502. For example, substrate contact bracket 1536 may engage with a portion of substrate 1502 and make electrical contact with conductive coating 1509 of substrate 1502, as Figure 15B shown. Cartridge contact pin 1530 may be configured to be electrically connected to substrate contact bracket 1536 when substrate 1502 is received in substrate cartridge 1504. For example, second end 1534 of cartridge contact pin 1530 is configured to contact substrate contact bracket 1536 based on substrate 1502 held in substrate cartridge 1504. Thus, cartridge contact pin 1530 may be electrically connected to substrate 1502 and further provide a connection interface 1514 external to substrate cartridge 1504.

[0135] Accordingly, control system 1508 may be easily connected to system 1500. For example, first wire 1522 extending from control system 1508 may simply be connected to cartridge contact pin 1530 (e.g., its first end 1532) such that control system 1508 may be electrically connected to substrate 1502 which may be used as one of the electrodes (e.g., anode) in electrophoresis. Additionally, second wire 1526 extending from control system 1508 may simply be connected to cathode contact 1554 such that control system 1508 may be electrically connected to electrode 602 (e.g., cathode) in the electrophoresis system.

[0136] Refer to Figures 16A - 16C , an example connection scheme is described. Refer to Figure 16A , substrate cartridge 1604 and substrate 1602 are configured similar to Figures 14A to 14C substrate cartridge 1404 and substrate 1402 shown. Additionally, substrate cartridge 1604 includes an anode contact pin 1630 extending from cartridge body 1610. Further, a wire line 1632 is provided to electrically connect anode contact pin 1630 to first electrode contact 1620 of substrate 1602. For example, wire line 1632 includes a first end 1634 and an opposite second end 1635. First end 1634 may be electrically connected to anode contact pin 1630, and second end 1635 may be electrically connected to first electrode contact 1620 positioned external to body 1610. Connection pins, clips or other suitable fastening mechanisms may be provided at first end 1634 and second end 1635 to connect wire line 1632 to anode contact pin 1630 and first electrode contact 1620 respectively.

[0137] Refer to Figure 16B , substrate cartridge 1604 and substrate 1602 are similar to Figures 14A to 14CThe substrate cassette 1404 and the substrate 1402 shown are configured. In addition, the anode cassette 1604 includes an anode cassette body 1650. The anode cassette body 1650 may define a cavity configured to receive a portion of the substrate 1602. For example, the anode cassette body 1650 may be configured to receive at least the first electrode contact 1620 of the substrate 1602 that is positioned outside the cassette body 1610 of the substrate cassette 1604.

[0138] The anode cassette body 1650 may include a connection interface 1614 similar to Figures 11A to 11D the connection interface 1114 described in Figure 16C . Specifically, as shown in Figure 16C , the anode cassette body 1650 includes cassette contact pins 1630 as the connection interface 1614. The cassette contact pins 1630 may extend through the anode cassette body 1650 such that a first end 1630 of the cassette contact pins 1630 is placed outside the anode cassette body 1650, while an opposite second end of the cassette contact pins 1640 is placed inside the anode cassette body 1650. The cassette contact pins 1630 are configured to be electrically connected to the substrate 1602 received in the anode cassette body 1650. In an embodiment where the substrate 1602 includes a conductive coating 1609, the cassette contact pins 1630 may be electrically connected to the conductive coating 1609. For example, the second end 1634 of the cassette contact pins 1630 is configured to be electrically connected to the conductive coating 1609, as shown in Figure 16C .

[0139] In some implementations, the system 100 may include a substrate contact bracket 1636 configured to engage with the substrate 1602 and be electrically connected to the substrate 1602. For example, the substrate contact bracket 1636 may engage with a portion of the substrate 1602 and be in electrical contact with the conductive coating 1609 of the substrate 1602, as shown in Figure 16C . The cassette contact pins 1630 may be configured to be electrically connected to the substrate contact bracket 1636 when the substrate 1602 is received in the anode cassette body 1650. For example, the second end 1634 of the cassette contact pins 1630 is configured to contact the substrate contact bracket 1636 based on the substrate 1602 held in the anode cassette body 1650. Thus, the cassette contact pins 1630 may be electrically connected to the substrate 1602 and further provide a connection interface 1614 outside the anode cassette body 1650. A first wire 1622 extending from the control system 1608 may simply be connected to the cassette contact pins 1630 (e.g., its first end 1631) such that the control system 1608 may be electrically connected to the substrate 1602 that can be used as one of the electrodes (e.g., anode) in electrophoresis.

[0140] Refer to Figures 17A - 17C, an example instrument 1700 for electrophoresis is described. Instrument 1700 can be used with one or more components of electrophoresis system 100 described herein. In some implementations, instrument 1700 is configured to automate at least a portion of an electrophoresis process using substrate 1702, substrate cassette 1704, cathode assembly 1707, and control system 1708.

[0141] Reference Figure 17A , instrument 1700 has instrument housing 1703, cassette tray 1704, and cassette lid 1706. Instrument housing 1703 is configured to receive at least one or all components of the components of electrophoresis system 100. For example, instrument housing 1703 is configured to include control system 1708. In some implementations, instrument housing 1703 may include controller 132 and power supply 134. Power supply 134 can be connected to an electrical outlet external to instrument housing 1703. Alternatively or additionally, power supply 134 can include a battery received within instrument housing 1703. Instrument housing 1703 may further include user interface 136 of control system 108. User interface 136 may be adapted for a user to control the position of instrument 1700 (e.g., adjacent to cassette tray 1705).

[0142] Cassette tray 1705 is configured to receive substrate cassette 1704. For example, substrate cassette 1704 that receives substrate 1702 can be placed on cassette tray 1705. In some implementations, cassette tray 1705 extends from instrument housing 1703 such that substrate cassette 1704 can be conveniently placed on cassette tray 1705. In some implementations, cassette tray 1705 can be retracted into instrument housing 1703 such that electrophoresis can be performed within instrument housing 1703. Once electrophoresis is completed, cassette tray 1705 extends from instrument housing 1703 such that substrate cassette 1704 can be removed from cassette tray 1705. Alternatively, cassette tray 1705 can remain external to instrument housing 1703 before, during, and after performing electrophoresis.

[0143] Cassette tray 1705 can be configured to receive various implementations of substrate cassette 1704 described herein. Reference Figure 17B , in one example, Figures 15A to 15B the substrate cassette 1704 described in Figure 17B , in Figures 15A to 15BIn the embodiment where the substrate cassette 1704 described in [[ ]] is placed on the cassette tray 1705, the cassette lid 1706 may include a cathode connector 1710 and an anode connector 1711. The cathode connector 1710 is configured to be electrically engaged with the cathode contact 1754 (or one end of the cathode contact pin 606) of the substrate cassette 1704. The cathode connector 1710 is further electrically connected to the control system 1708. For example, the cathode connector 1710 is electrically connected to a second wire 1726 extending from the control system 1708. The anode connector 1711 is configured to be electrically engaged with the anode contact of the substrate cassette 1704 (i.e., the first end 1732 of the cassette contact pin 1730). The anode connector 1711 is further electrically connected to the control system 1708. For example, the anode connector 1711 is electrically connected to a first wire 1722 extending from the control system 1708.

[0144] In some implementations, the cassette lid 1706 may be movable between a raised position and a lowered position. In the raised position, the cassette lid 1706 is positioned at a first predetermined distance from the cassette tray 1705 such that the substrate cassette 1704 can be placed on the cassette tray 1705 without interfering with the cassette lid 1706. Once the substrate cassette 1704 is placed on the cassette tray 1705, the cassette lid 1706 can be moved to the lowered position, in which the cassette lid 1706 is positioned closer to the cassette tray 1705 and is positioned at a second predetermined distance (shorter than the first predetermined distance) from the cassette tray 1705. In the lowered position, the cathode connector 1710 and the anode connector 1711 are respectively engaged with the cathode contact 1754 and the anode contact (i.e., the first end 1732 of the cassette contact pin 1730) of the substrate cassette 1704. The cathode connector 1710 and the anode connector 1711 may be positioned to align with the cathode contact 1754 and the anode contact (i.e., the first end 1732 of the cassette contact pin 1730) of the substrate cassette 1704 when the cassette lid 1706 is positioned in the lowered position relative to the substrate cassette 1704. In some implementations, the cassette lid 1706 can be manually operated between the raised position and the lowered position. Alternatively or additionally, the cassette lid 1706 is automatically actuated using an actuator (e.g., a motor) controlled by a controller (e.g., the control system 1708). For example, a user can place the substrate cassette 1704 on the cassette tray 1705 and select a menu via a user interface (e.g., the user interface 136 (FIG. 1)) to lower the cassette lid 1706, and the control system 1708 operates the actuator to move the cassette lid 1706 such that the cassette lid 1706 engages with the substrate cassette 1704 and makes the necessary electrical connections with the substrate cassette 1704 as described above.

[0145] Referring to [[ ]] Figure 17C , in another example, Figures 11A to 11DThe substrate cassette 1704 described in [description] is placed on the cassette tray 1705. In this example, the cassette lid 1706 is configured to be located above the cassette tray 1705 and provide at least a portion of the cathode assembly 1707. For example, the cassette lid 1706 may include a plurality of electrodes 1720 that project from the cassette lid 1706 and are configured to extend into a plurality of buffer chambers 1723 (defined at least by a plurality of holes 1712 of the substrate cassette 1704). The cassette lid 1706 may further include a cathode connector 1724 that is electrically connected to the plurality of electrodes 1720 and is further configured to be electrically connected to the control system 1708 via, for example, a second wire 1726 extending from the control system 1708.

[0146] In addition, the cassette lid 1706 may include an anode connector 1727. The anode connector 1727 is configured to be electrically engaged with the anode contact of the substrate cassette 1704 (i.e., the first end 1732 of the cassette contact pin 1730). The anode connector 1727 is further electrically connected to the control system 1708. For example, the anode connector 726 is electrically connected to a first wire 1722 extending from the control system 1708.

[0147] As described above, the cassette lid 1706 may be movable between a raised position and a lowered position. Once the substrate cassette 1704 is placed on the cassette tray 1705 and the cassette lid 1706 is in the raised position, the cassette lid 1706 may be moved to the lowered position. In the lowered position, the plurality of electrodes 1720 are respectively positioned within the buffer chambers 1723. In addition, in the lowered position, the anode connector 1711 engages the anode contact of the substrate cassette 1704 (i.e., the first end 1732 of the cassette contact pin 1730).

[0148] Figure 18 is a flowchart of an example process 1800 for analyte migration. The process 1800 may be performed using the electrophoresis system 100 (including the instrument 1700) described herein. The process 1800 may be used to check the electrical connections in the system 100 before performing electrophoresis.

[0149] In some implementations, process 1800 may include loading a substrate (e.g., substrate 1002) into an electrophoresis instrument (e.g., instrument 1700) (1802). For example, as described herein, substrate 1002 includes capture probes on substrate region 1016, and one or more biological samples may be placed on substrate 1002 such that the biological samples contact the capture probes. Alternatively, the biological samples do not contact the capture probes. For example, an intermediate layer (e.g., a gel) may be disposed between the substrate and the samples, which is compatible with electrophoresis. A substrate cassette (e.g., substrate cassette 1004) may be arranged on substrate 1002 such that a plurality of holes 1012 of substrate cassette 1004 are aligned with substrate region 1016 of substrate 1002, and a plurality of buffer chambers (e.g., buffer chambers 122, 1723) are defined on substrate region 1016 of substrate 1002. Then, a buffer (e.g., buffer 124) is supplied into the buffer chambers.

[0150] Process 1800 may include arranging a cathode relative to the substrate (1804). For example, the cathode may include a plurality of electrodes (e.g., electrodes 1252, 1720), and the plurality of electrodes may be respectively placed in a plurality of chambers.

[0151] Process 1800 may include electrically connecting a power supply to the substrate and the cathode (1806). As described herein, the power supply may be included in control system 1008 and is controlled by control system 1008. The power supply (or the control system) may be electrically connected to the substrate (as the anode) and the cathode (e.g., the plurality of electrodes) in various ways described herein, such as using various connection interfaces implemented in substrate 1002, substrate cassette 1004, cathode assembly 1006, lid 1706, etc.

[0152] Process 1800 may include applying a test voltage between the substrate (i.e., the anode) and the cathode (1808). The test voltage may be lower than the voltage required to perform electrophoresis in instrument 1700. The test voltage may be applied to ensure electrical connections in electrophoresis system 100 (including instrument 1700) before performing electrophoresis. Such electrical connections may include at least one of the electrical connection between the power supply and the substrate, the electrical connection between the power supply and the cathode, or the electrical properties of the buffer.

[0153] Process 1800 may include detecting output parameters in response to the test voltage (1810). In some implementations, the output parameter may be impedance. For example, the test voltage may allow current to flow through system 100 (including instrument 1700), and process 1800 may measure the current in system 100 and calculate the impedance based on the measured current and the applied test voltage. In other implementations, other parameters in system 100 may be used.

[0154] Procedure 1800 may include determining whether an output parameter meets a threshold (1812). The threshold may be a value indicating a proper electrical connection in system 100 (including instrument 1700). For example, when a proper electrical connection is made in system 100 (such as an electrical connection between a power supply and a substrate, an electrical connection between a power supply and a cathode, electrical properties of a buffer, etc.), the threshold may be the impedance of system 100. The threshold may be a single value. Alternatively, the threshold may be a range of values.

[0155] Procedure 1800 may include applying a voltage for analyte migration (1814) after determining that the output parameter meets the threshold. For example, when the output parameter meets the threshold, the electrical connection in the system may be considered proper. Thus, the actual voltage for electrophoresis may be applied to generate an electric field between a substrate (e.g., multiple substrate regions of the substrate) and a cathode (e.g., multiple electrodes) through one or more buffer chambers. The electric field may cause analytes in the biological sample to migrate towards the capture probes on the substrate.

[0156] Procedure 1800 may include stopping the application of the voltage for analyte migration (1816) after determining that the output parameter does not meet the threshold. For example, when the output parameter does not meet the threshold, the electrical connection in the system may be considered improper. Thus, if instrument 1700 has started applying the actual voltage, instrument 1700 may stop applying the actual voltage, or if instrument 1700 has not started applying the actual voltage, instrument 1700 does not continue to apply the actual voltage.

[0157] Procedure 1800 may include generating a notification of a potential electrical connection problem (1818) after determining that the output parameter does not meet the threshold. The notification may be output to instrument 1700 or other devices accessible to the user. For example, the notification may be output via user interface 136. The notification may include information alerting the user to an improper electrical connection in system 100 (including instrument 1700). The notification may be output before subsequent electrophoresis steps (such as applying the actual voltage for electrophoresis) so that the user can decide to take any action to investigate and / or remedy such an improper connection. For example, based on the notification, the user may check the physical connection between the power supply and the substrate or between the power supply and the cathode, and check the buffer. The user may then adjust the physical connection, or replace / refill the buffer, to address any issues. Alternatively, the notification may be output during or after performing these subsequent electrophoresis steps (such as applying the actual voltage for electrophoresis). The user may consider the possibility of such a connection problem when analyzing the analytes captured by electrophoresis.

[0158] Figure 19FIG. 1900 is a block diagram of a computing device that can be used to implement the systems and methods described in this document as a client or server or multiple servers. Computing device 1900 is intended to represent various forms of digital computers, such as laptop computers, desktop computers, workstations, personal digital assistants, servers, blade servers, mainframes, and other suitable computers. Computing device 1950 is intended to represent various forms of mobile devices, such as personal digital assistants, cellular telephones, smart phones, and other similar computing devices. The components shown here, their connections and relationships, and their functions are only examples and are not meant to limit the implementations described and / or claimed in this document.

[0159] Computing device 1900 includes a processor 1902, a memory 1904, a storage device 1906, a high-speed interface 1904 that is connected to memory 1908 and a high-speed expansion port 1910, and a low-speed interface 1914 that is connected to a low-speed bus 1912 and the storage device 1906. Each of the components 1902, 1904, 1906, 1908, 1910, and 1912 is interconnected using various buses and can be mounted on a common motherboard or in other suitable manners. Processor 1902 can process instructions for execution within computing device 1900, including instructions stored in memory 1904 or on storage device 1906 that are for displaying graphical information of a graphical user interface (GUI) on an external input / output device, such as a display 1916 coupled to the high-speed interface 1908. In other implementations, multiple processors and / or multiple buses and multiple memories and memory types can be used, as appropriate. Additionally, multiple computing devices 1900 can be connected, where each device provides a portion of the necessary operations (e.g., as a server group, blade server cluster, or multi-processor system).

[0160] Memory 1904 stores information within computing device 1900. In one implementation, memory 1904 is one or more volatile memory units. In another implementation, memory 1904 is one or more non-volatile memory units. Memory 1904 can also be another form of computer-readable medium, such as a magnetic disk or optical disk.

[0161] The storage device 1906 can provide large-capacity storage for the computing device 1900. In one implementation, the storage device 1906 can be or include a computer-readable medium, such as a floppy disk device, a hard disk device, an optical disc device, or a magnetic tape device, a flash memory, or other similar solid-state memory devices, or an array of devices (including devices in a storage area network or other configurations). The computer program product can be tangibly embodied in an information carrier. The computer program product can also include instructions that, when executed, perform one or more methods, such as those described above. The information carrier is a computer or machine-readable medium, such as the memory 1904, the storage device 1906, or the memory on the processor 1902.

[0162] The high-speed controller 1908 manages the bandwidth-intensive operations of the computing device 1900, while the low-speed controller 1912 manages the less bandwidth-intensive operations. Such a functional assignment is only an example. In one implementation, the high-speed controller 1908 is coupled to the memory 1904, the display 1916 (e.g., via a graphics processor or accelerator), and to the high-speed expansion port 1910, which can accept various expansion cards (not shown). In this implementation, the low-speed controller 1912 is coupled to the storage device 1906 and the low-speed expansion port 1914. The low-speed expansion port can include various communication ports (e.g., USB, Bluetooth, Ethernet, wireless Ethernet), which can be coupled (e.g., via a network adapter) to one or more input / output devices, such as a keyboard, a pointing device, a scanner, or a network device (such as a switch or a router).

[0163] The computing device 1900 can be implemented in many different forms, as shown in the figure. For example, it can be implemented as a standard server 1920, or implemented multiple times in a group of such servers. It can also be implemented as part of a rack server system 1924. In addition, it can be implemented in a personal computer such as a notebook computer 1922. Alternatively, the components from the computing device 1900 can be combined with other components (not shown) in a mobile device. Each of such devices can include one or more of the computing device 1900, and the entire system can be composed of multiple computing devices 1900 that communicate with each other.

[0164] In addition, the computing device 1900 can include a Universal Serial Bus (USB) flash drive. The USB flash drive can store an operating system and other applications. The USB flash drive can include input / output components, such as a wireless transmitter or a USB connector that can be inserted into a USB port of another computing device.

[0165] Various implementations of the systems and techniques described herein can be implemented in digital electronic circuitry, integrated circuit systems, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and / or combinations thereof. These various implementations can include implementations in one or more computer programs that are executable and / or interpretable on a programmable system including at least one programmable processor, which may be special purpose or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0166] These computer programs (also referred to as programs, software, software applications, or code) include machine instructions for a programmable processor and can be implemented in high-level procedural and / or object-oriented programming languages, and / or in assembly / machine language. As used herein, the term “machine-readable medium,” “computer-readable medium” refers to any computer program product, apparatus, and / or device (e.g., a disk, optical disk, memory, programmable logic device (PLD)) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and / or data to a programmable processor.

[0167] For providing interaction with a user, the systems and techniques described herein can be implemented on a computer having a display device, keyboard, and pointing device, the display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying data to the user, and the keyboard and pointing device (e.g., a mouse or trackball) for providing input to the computer by the user. Other kinds of devices can also be used to provide interaction with the user; for example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input received from the user can be in any form, including acoustic, speech, or tactile input.

[0168] The systems and techniques described herein can be implemented in a computer system that includes a backend component (e.g., as a data server), or includes a middleware component (e.g., an application server), or includes a frontend component (e.g., a client computer having a graphical user interface or a web browser, through which a user can interact with an implementation of the systems and techniques described herein), or any combination of such backend, middleware, or frontend components. The components of the system can be interconnected by digital data communication in any form or medium (e.g., a communication network). Exemplary communication networks include local area networks (“LANs”), wide area networks (“WANs”), peer-to-peer networks (with ad-hoc or static members), grid computing infrastructures, and the Internet.

[0169] A computing system can include clients and servers. Clients and servers are typically located remotely from each other and typically interact through a communication network. The relationship of client and server arises from computer programs running on respective computers and having a client-server relationship to each other.

[0170] Active capture method

[0171] In some of the methods described herein, an analyte in a biological sample (e.g., in a cell or tissue section) can be transported (e.g., passively or actively) to a capture probe (e.g., a capture probe immobilized on a substrate (e.g., a substrate or a bead)).

[0172] For example, an electric field (e.g., using electrophoresis), pressure, fluid flow, gravity, temperature, and / or a magnetic field can be used to transport the analyte to the capture probe (e.g., an immobilized capture probe). For example, a pressure gradient, a chemical concentration gradient, a temperature gradient, and / or a pH gradient can be used to transport the analyte through, e.g., a gel (e.g., a hydrogel), a fluid, or a permeabilized cell to the capture probe (e.g., an immobilized capture probe). For example, the analyte can be transported through a gel (e.g., a hydrogel), a fluid, or a permeabilized cell to the capture probe (e.g., an immobilized capture probe).

[0173] In some examples, an electrophoresis field can be applied to the analyte to facilitate the migration of the analyte towards the capture probe. In some examples, a sample containing the analyte contacts a substrate (e.g., a slide, a coverslip, or a bead) having a capture probe immobilized on the substrate, and an electric current is applied to facilitate the directed migration of the charged analyte towards the capture probe on the substrate. An electrophoresis assembly (e.g., an electrophoresis chamber) can be used to apply the electric current, where the biological sample contacts the cathode and the capture probe (e.g., a capture probe immobilized on the substrate), and where the capture probe contacts the biological sample and the anode.

[0174] In some embodiments, methods utilizing active capture methods can employ a conductive substrate (e.g., any of the conductive substrates described herein). In some embodiments, the conductive substrate includes paper, a hydrogel film, or a glass slide with a conductive coating. In some embodiments, the conductive substrate (e.g., any of the conductive substrates described herein) includes one or more capture probes.

[0175] Figure 20A and Figure 20B illustrates different example analysis workflows of an active capture method using an electric field (e.g., using electrophoresis). In some examples, a biological sample 2002 (e.g., a tissue sample) can be brought into contact with a first substrate 2004. In some embodiments, the first substrate 2004 can have one or more coatings on its surface (e.g., any of the conductive substrates described herein). Non-limiting examples of the coatings include nucleic acids (e.g., RNA) and conductive oxides (e.g., indium tin oxide). In some embodiments, the first substrate 2004 can have a functionalized chemistry on its surface. In Figure 20A and Figure 20B the example shown, the first substrate 2004 is covered with a first coating 2006, and the first coating 2006 (e.g., a conductive coating) is further covered with a second coating 2008. In some embodiments, the first coating 2006 is an indium tin oxide (ITO) coating. In some embodiments, the second coating 2008 is a large sheet of capture probes (e.g., any of the capture probes described herein). In some embodiments, the substrate can include an ITO coating. In some embodiments, the substrate can include capture probes or capture probes attached to features of the substrate.

[0176] The biological sample 2002 and the second coating 2008 (e.g., a large sheet of capture probes) can be brought into contact with a permeabilization solution 2010. Non-limiting examples of the permeabilization solution include enzymes (e.g., proteinase K, pepsin, and collagenase), detergents (e.g., sodium dodecyl sulfate (SDS), Triton X-100, Tween 80, and Tween 20), ribonuclease inhibitors, buffers optimized for electrophoresis, buffers optimized for permeabilization, buffers optimized for hybridization, or combinations thereof. The permeabilization reagent can also include, but is not limited to, dry permeabilization reagents, permeabilization buffers, buffers without permeabilization reagents, permeabilization gels, and permeabilization solutions. In some examples, the biological sample (e.g., a tissue sample) can be permeabilized first and then electrophoresed.

[0177] Figure 20AAn exemplary analysis workflow including a first step 2012 is shown, in which a biological sample 2002 can be permeabilized prior to electrophoresis of the sample 2002. Any permeabilization method disclosed herein can be used during the first step 2012. The biological sample 2002 includes an analyte 2014. In some embodiments, the analyte 2014 is a negatively charged analyte. The first substrate 2004 can include capture probes 2016 that are immobilized or attached to the first substrate 2004 or to features (e.g., beads) 2018 on the substrate. In some embodiments, the capture probes 2016 can include any capture probe disclosed herein. In some embodiments, the capture probes are directly or indirectly attached to the first substrate 2004. In some embodiments, the capture probes 2016 are positively charged.

[0178] In step 2020, after the permeabilization of the biological sample 2002 is complete, the sample 2002 can be electrophoresed. During electrophoresis, the biological sample 2002 is subjected to an electric field that can be generated by sandwiching the biological sample 2002 between a first substrate 2004 and a second substrate 2022, connecting each substrate to a cathode and an anode, respectively, and passing a current through the substrates. The application of the electric field "-E" causes the analyte 2004 (e.g., a negatively charged analyte) to migrate along Figure 20A the direction of the arrow shown in the figure towards the substrate 2004 and the capture probes 2016 (e.g., positively charged capture probes). In some embodiments, the analyte 2014 migrates a distance "h" towards the capture probes 2016. In some embodiments, the analyte 2014 migrates through one or more permeabilized cells within the permeabilized biological sample towards the capture probes 2016 (e.g., from an original position within the permeabilized cell to a final position within or proximal to the capture probes 2016). The second substrate 2022 can include a first coating 2006 (e.g., a conductive coating) that allows the generation of the electric field "-E".

[0179] In some embodiments, the analyte 2014 is a protein or a nucleic acid. In some embodiments, the analyte 2014 is a negatively charged protein or nucleic acid. In some embodiments, the analyte 2014 is a positively charged protein or nucleic acid. In some embodiments, the capture probes 2016 are proteins or nucleic acids. In some embodiments, the capture probes 2016 are positively charged proteins or nucleic acids. In some embodiments, the capture probes 2016 are negatively charged proteins or nucleic acids. In some embodiments, the analyte 2014 is a negatively charged transcript. In some embodiments, the analyte 2014 is a poly(A) transcript. In some embodiments, the capture probes 2016 are attached to features in an array of features. In some embodiments, the permeabilization reagent 2010 can contact the sample 2002, the first substrate 2004, the second substrate 2022, or any combination thereof.

[0180] Figure 20B Shows an example analysis workflow in which the biological sample 2002 can be simultaneously permeabilized and electrophoresed. In some embodiments, the simultaneous permeabilization and electrophoresis of the biological sample 2002 can reduce the total duration of the analysis workflow, thereby translating into a more efficient workflow.

[0181] In some embodiments, the permeabilization reaction is carried out at a cooling temperature (e.g., about 4°C). In some embodiments, carrying out the permeabilization reaction at a cooling temperature controls the enzymatic activity of the permeabilization reaction. In some embodiments, the permeabilization reaction is carried out at a cooling temperature to minimize the drift and / or diffusion of the analyte 2014 from its original position (e.g., the position in the cells of the biological sample 2002) until the user is ready to initiate the permeabilization reaction. In some embodiments, the permeabilization reaction is carried out at a warm temperature (e.g., a temperature in the range of about 15°C to about 37°C or higher) to initiate and / or increase the rate of the permeabilization reaction. In some embodiments, once electrophoresis and / or heating permeabilization reaction is applied, the permeabilization reaction allows the analyte to migrate from its original position (e.g., the position in the cells of the biological sample 2002) to the capture probe 2016 on the first substrate 2004.

[0182] Reference Figures 21A - 21C , shows an example substrate configuration for use in the active migration of an analyte from a first position to a second position via electrophoresis. Figure 21A Shows an example substrate configuration for use in electrophoresis, in which the first substrate 2104 and the second substrate 2122 are aligned at approximately 90 degrees relative to each other. In this example, the first substrate 2104 including the biological sample 2102 is placed below the second substrate 2122. Both the first substrate 2104 and the second substrate 2122 can be connected to wires 2124 that direct an electric current from a power source to the substrates, thereby generating an electric field between the substrates. Figure 21B Shows an example substrate configuration for use during electrophoresis, in which the first substrate 2104 and the second substrate 2122 are parallel to each other. In this example, the first substrate 2104 including the biological sample 2102 is also placed below the second substrate 2122.

[0183] Figure 21CShows yet another example substrate configuration for use in electrophoresis, where the second substrate 2122 and the third substrate 2126 are aligned at approximately 90 degrees relative to the first substrate 2104. Thus, in this example, the first biological sample 2102a and the second biological sample 2102b can be electrophoresed simultaneously. In some embodiments, 3, 4, 5, 6, 7, 8, 9, 10 or more biological samples can be placed on the same substrate and electrophoresed simultaneously. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more top substrates can be placed above the same bottom substrate containing one or more samples for electrophoresing one or more samples simultaneously. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more top substrates can be placed vertically (e.g., at approximately 90 degrees) above the same bottom substrate containing one or more samples for electrophoresing one or more samples simultaneously. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more top substrates can be placed at a specific angle (e.g., at approximately 10, 20, 30, 40, 45, 50, 60, 70 or 80 degrees) relative to the same bottom substrate containing one or more samples for electrophoresing one or more samples simultaneously. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more top substrates can be placed in a parallel orientation above the same bottom substrate containing one or more biological samples for electrophoresing one or more samples simultaneously. In some embodiments, the configuration of the top substrates can be arranged above the same bottom substrate containing one or more biological samples for electrophoresing one or more samples simultaneously. In some embodiments, the first configuration of the top substrates can be arranged above the second array of bottom substrates containing one or more biological samples for electrophoresing one or more samples simultaneously. In some embodiments, electrophoresing two or more biological samples on the same substrate simultaneously can provide the advantage of a more efficient workflow. In some embodiments, one or more of the top substrates can contain biological samples.

[0184] In some embodiments, methods utilizing an active capture approach can include one or more solutions between the biological sample and the substrate (e.g., a substrate including capture probes). In some embodiments, one or more solutions between the biological sample and the substrate including capture probes can include a permeabilization buffer (e.g., any of the permeabilization buffers described herein). In some embodiments, one or more solutions between the biological sample and the substrate including capture probes can include an electrophoresis buffer.

[0185] In some embodiments, active capture of an analyte can include one or more porous materials between a biological sample and a substrate comprising capture probes. In some embodiments, the one or more porous materials between the biological sample and the substrate comprising capture probes can include paper or a blotting membrane. In some embodiments, the one or more porous materials between the biological sample and the substrate comprising capture probes can include a gel containing one or more solutions. For example, by way of non-limiting example, the gel can be an SDS-PAGE gel. In some embodiments, the one or more porous materials between the biological sample and the substrate comprising capture probes can contain a permeabilization buffer. In some embodiments, the one or more porous materials between the biological sample and the substrate comprising capture probes can contain an electrophoresis buffer. In some embodiments, active capture of an analyte can include one or more solutions and one or more porous materials between a biological sample and a substrate comprising capture probes.

[0186] In some embodiments, the one or more porous materials between a biological sample and a substrate comprising capture probes (e.g., an array) can act as a filter to separate an analyte (e.g., an analyte of interest) from other molecules or analytes present in the biological sample. In some embodiments, the analyte (e.g., an analyte of interest) is an RNA transcript. In some embodiments, the one or more porous materials between a biological sample and a substrate comprising capture probes can act as a filter to separate the RNA transcript from other molecules (e.g., analytes) such as proteins, lipids, and / or other nucleic acids. In some embodiments, the one or more porous materials between a biological sample and a substrate comprising capture probes can act as a filter to separate an analyte and other molecules based on physicochemical properties. For example, by way of non-limiting example, analytes can be separated according to properties such as charge, size (e.g., length, radius of gyration, hydrodynamic diameter, etc.), hydrophobicity, hydrophilicity, molecular binding (e.g., immunoaffinity), and combinations thereof. In some embodiments, the one or more porous materials between a biological sample and a substrate comprising capture probes can separate an analyte from other molecules to reduce non-specific binding near the capture probes and thus improve the binding between the analyte and the capture probes, thereby enhancing subsequent assay performance.

[0187] In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can act as a molecular sieve substrate for electrophoretic analyte separation. For example, by way of non-limiting illustration, the separation of analytes can be based on physicochemical properties such as charge, size (e.g., length, radius of gyration, hydrodynamic diameter, etc.), electrophoretic mobility, zeta potential, isoelectric point, hydrophobicity, hydrophilicity, molecular binding (e.g., immunological affinity), and combinations thereof. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can have a uniform pore size. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can have pore size discontinuities, as commonly used in different gel electrophoresis protocols. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can have a pore size gradient. For example, one or more porous materials (e.g., hydrogels) can have a pore size gradient such that when the analytes migrate towards the substrate comprising the capture probes (array), the gradient separates the analytes. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can separate analytes based on length. For example, in an electrophoretic setup, shorter analytes will have a higher electrophoretic mobility and thus migrate faster towards the capture probes relative to longer analytes. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes separate analytes based on length such that only the shorter analytes can migrate through the one or more porous materials to reach the capture probes while the longer analytes cannot reach the capture probes.

[0188] In some embodiments, a particular subset of analytes (e.g., a subset of transcripts) can be captured by applying an electrophoretic field for a certain amount of time. In some embodiments, a particular subset of analytes (e.g., a subset of transcripts) can be captured by selecting different porous materials (e.g., porous materials having different compositions) between the biological sample and the substrate comprising capture probes. In some embodiments, a particular subset of analytes (e.g., a subset of transcripts) can be captured by applying an electrophoretic field for a certain amount of time and selecting different porous materials between the biological sample and the substrate comprising the capture probes (e.g., array).

[0189] In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can have pore size discontinuities, which can result in an increased concentration of migrating analytes (e.g., “stacking”). For example, one or more porous materials (e.g., hydrogels) between the biological sample and the substrate comprising capture probes can have pore size discontinuities, which can result in an increased concentration of analytes near the capture probes, leading to favorable binding kinetics and increased sensitivity. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can have pore size discontinuities that enhance the separation between migrating analytes of different sizes and / or lengths. In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can include a first porous material and a second porous material, wherein the first porous material has a larger pore size than the second porous material. In some embodiments, the first porous material is located on or near the surface of the biological sample. In some embodiments, the second porous material (e.g., a second porous material having a smaller pore size than the first porous material) can be placed on or near the surface of the first porous material. In some embodiments, as analytes migrate sequentially from the biological sample (e.g., via electrophoretic migration) through the first porous material and the second porous material, the migrating analytes can be collected (e.g., “stacked”) at the interface between the first porous material and the second porous material.

[0190] In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can include a gradient of pore sizes for continuous stacking as analytes migrate through decreasing pore sizes (e.g., decreasing pore diameters). In some embodiments, one or more porous materials between the biological sample and the substrate comprising capture probes can include a gradient of pore sizes such that the pore diameter decreases as analytes migrate from the biological sample to the substrate comprising capture probes. In some embodiments, the pore size gradient can increase the resolution between analytes of different sizes. In some embodiments, the pore size gradient can increase the concentration of analytes near the capture probes. In some embodiments, as analytes migrate through a gradient of decreasing pore sizes (e.g., decreasing pore diameters), the pore size gradient can continuously decrease the rate of analyte migration and collection (e.g., “stacking”).

[0191] In some embodiments, one or more porous materials between the biological sample and the substrate including capture probes can include a gradient of a gel for continuous stacking as analytes migrate through the decreasing pore sizes (e.g., decreasing aperture diameters) of the gradient gel. In some embodiments, as the analytes migrate towards the capture probes, the gradient gel can have pores with decreasing diameters. In some embodiments, the gradient gel can increase the separation resolution between analytes of different sizes. In some embodiments, the gradient gel can increase the concentration of analytes near the capture probes. In some embodiments, as the analytes migrate through the gradient gel with decreasing pore sizes (e.g., decreasing diameters), the gradient gel can continuously reduce the rate of analyte migration and collection (e.g., stacking).

[0192] In some embodiments, the biological sample can be placed in a first substrate holder (e.g., the substrate holder described herein). In some embodiments, the spatial barcode capture probe array (e.g., capture probes, barcode array) can be placed on a second substrate holder (e.g., the substrate holder described herein). In some embodiments, the biological sample can be placed in a first substrate holder that also contains capture probes. In some embodiments, the first substrate holder, the second substrate holder, or both can be conductive (e.g., any conductive substrate described herein). In some embodiments, the first substrate holder including the biological sample, the second substrate holder including the capture probes, or both can be contacted with a permeabilization reagent (e.g., permeabilization buffer), and an electric field can be used to migrate analytes from the biological sample towards the barcode array.

[0193] In some embodiments, electrophoresis can be applied to the biological sample on the barcode array when the biological sample is in contact with the permeabilization buffer. In some embodiments, electrophoresis can be applied to the biological sample on the barcode array when the biological sample is in contact with an electrophoresis buffer (e.g., a buffer lacking a permeabilization reagent). In some embodiments, after a desired amount of time, the permeabilization buffer can be replaced with the electrophoresis buffer. In some embodiments, electrophoresis can be applied simultaneously with the permeabilization buffer or the electrophoresis buffer. In some embodiments, electrophoresis can be applied after a desired contact time amount between the biological sample and the permeabilization buffer or the electrophoresis buffer.

[0194] In some embodiments, a biological sample can be placed on a substrate (e.g., a porous membrane, hydrogel, paper, etc.). In some embodiments, the biological sample placed on the substrate can have a gap (e.g., a spacing) between the substrate and a substrate holder (e.g., a conductive substrate holder). In some embodiments, a barcode array can be placed on the substrate (e.g., a porous membrane, hydrogel, paper, etc.). In some embodiments, the barcode array can have a gap between the substrate and a substrate holder (e.g., a conductive substrate holder). In some embodiments, the barcode array can be placed in the immediate vicinity of the biological sample or at a desired distance from the biological sample. In some embodiments, a buffer reservoir can be used between the substrate holder (e.g., a conductive substrate holder) and the barcode array, between the substrate holder and the biological sample, or both. This setup allows analytes to migrate to the barcode array without getting close to the electrodes (e.g., the conductive substrate holder), resulting in a more stable electrophoresis.

[0195] In some embodiments, a combination of at least two buffers with different ionic compositions can be used to differentially migrate analytes based on the ionic mobility of the analytes (e.g., isotachophoresis (ITP)). For example, using two or more buffers with different ionic compositions can increase the concentration of analytes before they contact the barcode array. Isotachophoresis includes at least two buffers that contain a common counterion (e.g., an ion with a different charge sign than the analyte) and different coions (e.g., ions with the same charge sign as the analyte) (Smejkal P. et al., "Microfluidic isotachophoresis: A review", Electrophoresis, 34.111493 - 1509, (2013), which is incorporated herein by reference in its entirety). In some embodiments, one buffer can contain coions (e.g., a "leading" buffer) that have a higher ionic mobility (e.g., the speed at which they travel through the solution in an electric field) than the analyte. In some embodiments, a second buffer can contain coions that have a lower ionic mobility than the analyte (e.g., a "trailing" buffer). In some embodiments, a third buffer can contain coions with an ionic mobility between the electrophoretic mobilities of the analytes. In some embodiments, a biological sample can be placed on a first substrate holder (e.g., a conductive substrate holder), and the barcode array can be placed on a second substrate holder (e.g., a second conductive substrate holder) and in contact with a permeabilization buffer, and an electric field can be used to migrate analytes from the biological sample towards the barcode array. As the electric field is applied to the biological sample, the analytes can concentrate in the buffer as they migrate towards the capture probes. In some embodiments, isotachophoresis can be used in conjunction with gel-based separation (any gel-based separation described herein).

[0196] In some embodiments, a permeabilization buffer can be applied to a region of interest in a biological sample (e.g., a region of interest as described herein). In some embodiments, a permeabilization reagent (e.g., a hydrogel comprising the permeabilization reagent) can be applied to a region of interest in a biological sample. For example, the region of interest can be a region that is small in area relative to the total area of the biological sample. In some embodiments, the permeabilization buffer or permeabilization reagent can be contacted with the biological sample and a substrate comprising capture probes (e.g., an array). In some embodiments, the biological sample can have more than one region of interest (e.g., two, three). In some embodiments, the biological sample, the substrate comprising the capture probes, or both can be placed in a conductive substrate holder. In some embodiments, an analyte can be released from the region(s) of interest and migrate from the biological sample towards the capture probes using an electric field.

[0197] In some embodiments, electrophoretic transfer of the analyte can be performed while preserving the relative spatial position of the analyte in the biological sample and minimizing passive diffusion of the analyte from its position in the biological sample. In some embodiments, the analyte captured by the capture probes (e.g., capture probes on a substrate) retains the spatial position of the analyte present in the biological sample from which the analyte was obtained (e.g., when the analyte is actively migrated to the capture probes by electrophoretic transfer, the spatial position of the analyte captured by the capture probes on the substrate can be more accurate or more representative of the spatial position of the analyte in the biological sample than when the analyte is not actively migrated to the capture probes). In some embodiments, the electrophoretic delivery and binding process is described by the Damköhler number (Da), which is the ratio of the reaction rate and the mass transport rate. The fraction of bound analyte and the shape of the biological sample will depend on the parameters in Da. These parameters include the electrophoretic velocity U e (which depends on the electrophoretic mobility μ of the analyte e and the electric field strength E), the density p of the capture probes (e.g., barcode oligonucleotides) 0 、the binding rate k between the probe (e.g., barcode oligonucleotides) and the analyte on and the capture area thickness L.

[0198]

[0199] Rapid migration (e.g., electro-migration) can reduce assay time and can minimize molecular diffusion of the analyte.

[0200] In some embodiments, electrophoretic transfer of analytes can be performed while maintaining the relative spatial alignment of analytes in a sample. Thereby, analytes captured by capture probes (e.g., capture probes on a substrate) retain the spatial information of the cell or biological sample from which the analytes were obtained. Applying an electric field to the analytes can also cause a temperature (e.g., heat) increase. In some embodiments, the elevated temperature (e.g., heat) can facilitate the migration of analytes towards the capture probes.

[0201] In some examples, a spatially addressable microelectrode array is used for spatially constrained capture of at least one charged analyte of interest by capture probes. For example, a spatially addressable microelectrode array can permit discrete (e.g., local) application of an electric field rather than a uniform electric field. The spatially addressable microelectrode array can be independently addressable. In some embodiments, the electric field can be applied to one or more regions of interest in a biological sample. The electrodes can be adjacent to each other or spaced apart from each other. The microelectrode array can be configured to include a high density of discrete sites having a smaller area for applying an electric field to facilitate the migration of the charged analyte(s) of interest. For example, electrophoretic capture can be performed on a region of interest using a spatially addressable microelectrode array.

[0202] A high density of discrete sites on the microelectrode array can be used. The surface can include any suitable density of discrete sites (e.g., a density suitable for processing a sample on a conductive substrate within a given amount of time). In one embodiment, the surface has a density of discrete sites greater than or equal to about 500 sites per 1 mm 2 In some embodiments, the surface has about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 20000, about 40000, about 60000, about 80000, about 100000, or about 500000 sites per 1 mm 2 In some embodiments, the surface has about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 20000, about 40000, about 60000, about 80000, about 100000, or about 500000 sites per 1 mm 2The density of discrete sites of at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 10000, at least about 20000, at least about 40000, at least about 60000, at least about 80000, at least about 100000 or at least about 500000 sites.

[0203] Figure 22A and Figure 22B A schematic diagram showing an electrophoretic transfer system configured to direct a nucleic acid analyte (e.g., an mRNA transcript) to a spatially barcoded capture probe array is shown in. In this exemplary configuration of the electrophoretic system, sample 2202 is sandwiched between cathode 2201 and spatially barcoded capture probe arrays 2204, 2205, and spatially barcoded capture probe arrays 2204, 2205 are sandwiched between sample 2202 and anode 2203 such that sample 2202 is in contact with spatially barcoded capture probe 2207. When an electric field is applied to the electrophoretic transfer system, negatively charged nucleic acid analyte 2206 will be pulled towards positively charged anode 2203 and into spatially barcoded arrays 2204, 2205 containing spatially barcoded capture probe 2207. Spatially barcoded capture probe 2207 interacts with the nucleic acid analyte (e.g., an mRNA transcript hybridizes with a spatially barcoded nucleic acid capture probe to form a DNA / RNA hybrid) 2207 such that analyte capture is more efficient. The electrophoretic system setup can vary depending on the target analyte. For example, a protein can be positive, negative, neutral, or polar depending on the protein and other factors (e.g., isoelectric point, solubility, etc.). Those skilled in the art have the knowledge and experience to arrange an electrophoretic transfer system to facilitate the capture of a specific target analyte.

[0204] Figure 23FIG. is an illustration showing an exemplary workflow protocol using an electrophoretic transfer system. In the example, Panel A depicts a flexible array of spatially barcoded features in contact with a sample. The sample can be a flexible array, where the array is immobilized on a hydrogel, membrane, or other flexible substrate. Panel B depicts the contact of the array with the sample and imaging of the array-sample assembly. Images of the sample / array assembly can be used to verify sample placement, select regions of interest, or for any other reason for imaging samples on the array as described herein. Panel C depicts the application of an electric field using an electrophoretic transfer system to assist in the efficient capture of target analytes. Here, negatively charged mRNA target analytes migrate towards the positively charged anode. Panel D depicts the application of reverse transcription reagents and first-strand cDNA synthesis of the captured target analytes. Panel E depicts array removal and preparation for library construction (Panel F) and next-generation sequencing (Panel G).

[0205] The spatial analysis methods and compositions described herein can provide extensive analyte and / or expression data of various analytes within a biological sample at high spatial resolution while preserving the native spatial context. The spatial analysis methods and compositions can include, for example, using capture probes comprising spatially barcoded (e.g., nucleic acid sequences that provide information about the location or localization of an analyte within a cellular or tissue sample (e.g., a mammalian cell or mammalian tissue sample)) and capture domains capable of binding to analytes (e.g., proteins and / or nucleic acids) produced and / or present in cells. The spatial analysis methods and compositions can also include using capture probes having capture domains that capture intermediate reagents for the indirect detection of analytes. For example, the intermediate reagent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate reagent. Thus, detection of the intermediate reagent indicates the analyte in the cellular or tissue sample.

[0206] Non-limiting aspects of spatial analysis methods and compositions are described in the following documents: U.S. Patent Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198; U.S. Patent Application Publications Nos. 2020 / 239,946, 2020 / 080,136, 2020 / 277,663, 2020 / 024,641, 2019 / 330,617, 2019 / 264,268, 2020 / 256,867, 2020 / 022,444, 2019 / 194,709, 2019 / 161,796, 2019 / 085,383, 2019 / 055,594, 2018 / 216,161, 2018 / 051,322, 2018 / 245,142, 2017 / 241,911, 2017 / 089,811, 2017 / 067,096, 2017 / 029,875, 2017 / 016,053, 2016 / 108,458, 2015 / 000,0854, 2013 / 171,621; WO 2018 / 091,676, WO 2020 / 176,788; Rodriques et al., Science 363(6434):1463 - 1467, 2019; Lee et al., Nat. Protoc. 10(3):442 - 458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197 - 1202, 2018; the "Visium Spatial Gene Expression Reagent Kits User Guide" (e.g., version C, dated June 2020), and / or the "Visium Spatial Tissue Optimization Reagent Kits User Guide" (e.g., version C, dated July 2020), both of which are available on the 10x Genomics support document website and may be used in any combination herein.This document describes further non-limiting aspects of spatial analysis methods and compositions.

[0207] Some common terms that can be used in this disclosure can be found in WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Generally, a "barcode" is a label or identifier that is used to convey or is capable of conveying information (e.g., information about an analyte in a sample, bead, and / or capture probe). A barcode can be part of an analyte or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purposes of this disclosure, an "analyte" can include any biological substrate, structure, part, or component to be analyzed. The term "target" can similarly refer to an analyte of interest.

[0208] Analytes can be broadly classified into two categories: nucleic acid analytes and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specifically phosphorylated or acetylated variants of proteins, amidated variants of proteins, hydroxylated variants of proteins, methylated variants of proteins, ubiquitinated variants of proteins, sulfated variants of proteins, viral proteins (e.g., viral capsids, viral envelopes, viral coats, viral appendages, viral glycoproteins, viral spikes, etc.), extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the (multiple) analytes can be localized to (multiple) subcellular locations, including, for example, organelles such as mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, the (multiple) analytes can be peptides or proteins, including, but not limited to, antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. In some embodiments, analytes can be detected indirectly, such as by detecting an intermediate reagent, such as a ligation product or an analyte capture reagent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.

[0209] A "biological sample" is typically obtained from a subject and is used for analysis using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and typically includes cells and / or other biological materials of the subject. In some embodiments, the biological sample can be a tissue section. In some embodiments, the biological sample can be a fixed and / or stained biological sample (e.g., a fixed and / or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, the biological sample (e.g., a fixed and / or stained biological sample) can be imaged. The biological sample is also described in Section (I)(d) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0210] In some embodiments, the biological sample is permeabilized using one or more permeabilizing reagents. For example, permeabilization of the biological sample can facilitate analyte capture. Exemplary permeabilizing reagents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments section of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0211] Array-based spatial analysis methods involve transferring one or more analytes from a biological sample to a feature array on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of the analytes within the biological sample is determined based on the features to which the analytes bind (e.g., directly or indirectly) on the array and the relative spatial location of the features within the array.

[0212] A "capture probe" refers to any molecule that can capture (directly or indirectly) and / or label an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI)) and a capture domain. In some embodiments, the capture probe can include a cleavage domain and / or a functional domain (e.g., a primer binding site, such as for next-generation sequencing (NGS)). See Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Generation of the capture probe can be achieved by any suitable method, including those described in Section (II)(d)(ii) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0213] In some embodiments, any suitable multiplexing technique (such as those described in Section (IV) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663) can be used to detect (e.g., simultaneously or sequentially) more than one analyte type (e.g., nucleic acid and protein) from a biological sample.

[0214] In some embodiments, one or more analyte capture reagents can be used to detect one or more analytes (e.g., protein analytes). As used herein, an “analyte capture reagent” refers to a reagent that interacts with an analyte (e.g., an analyte in a biological sample) and a capture probe (e.g., a capture probe attached to a substrate or feature) to identify the analyte. In some embodiments, the analyte capture reagent includes: (i) an analyte binding moiety (e.g., a moiety that binds to the analyte), e.g., an antibody or an antigen-binding fragment thereof; (ii) an analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or portion that is configured to hybridize, bind, couple, or otherwise interact with a capture domain of a capture probe. In some cases, the analyte binding moiety barcode (or a portion thereof) can be capable of being removed (e.g., cleaved) from the analyte capture reagent. Additional descriptions of analyte capture reagents can be found in Section (II)(b)(ix) of WO 2020 / 176788 and / or Section (II)(b)(viii) of U.S. Patent Application Publication No. 2020 / 0277663.

[0215] There are at least two methods of associating a spatial barcode with one or more neighboring cells such that the spatial barcode identifies one or more cells and / or the contents of one or more cells as associated with a particular spatial location. One method facilitates the departure of an analyte or analyte proxy (e.g., an intermediate reagent) from the cell and towards a spatial barcode array (e.g., including a spatial barcode capture array). Another method is to cleave the spatial barcode capture probe from the array and facilitate the spatial barcode capture probe towards and / or into the biological sample or onto the biological sample.

[0216] In some cases, a capture probe can be configured to prime, replicate, and thereby produce an optional barcode extension product from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate reagent (e.g., a ligation product or an analyte capture reagent), or a portion thereof) or a derivative thereof (see, e.g., WO 2020 / 176788 and / or Section (II)(b)(vii) of U.S. Patent Application Publication No. 2020 / 0277663 regarding extended capture probes). In some cases, a capture probe can be configured to form a ligation product with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate reagent, or a portion thereof), thereby creating a ligation product that serves as a template proxy.

[0217] As used herein, an "extended capture probe" refers to a capture probe having additional nucleotides added to the end (e.g., the 3' or 5' end) of the capture probe to extend the total length of the capture probe. For example, an "extended 3' end" indicates that additional nucleotides are added up to the 3' nucleotide of the capture probe to extend the length of the capture probe, e.g., by a polymerization reaction for extending nucleotide molecules, including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, the extended capture probe includes adding a nucleic acid sequence to the 3' end of the capture probe that is complementary to the nucleic acid sequence of an analyte or an intermediate reagent that specifically binds to the capture domain of the capture probe. In some embodiments, the capture probe is extended using a reverse transcriptase. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probe includes the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.

[0218] In some embodiments, the extended capture probe is amplified (e.g., in a bulk solution or on an array) to produce an amount sufficient for downstream analysis (e.g., via DNA sequencing). In some embodiments, the extended capture probe (e.g., a DNA molecule) serves as a template for an amplification reaction (e.g., a polymerase chain reaction).

[0219] Other variants of the spatial analysis method are described in section (II)(a) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, including an imaging step in some embodiments. The analysis of capturing an analyte (and / or an intermediate reagent or a part thereof), for example, includes sample removal, extension of a capture probe, sequencing (e.g., sequencing of a cleaved and extended capture probe and / or a cDNA molecule complementary to the extended capture probe), sequencing on an array (e.g., using in situ hybridization or in situ ligation methods, for example), temporal analysis, and / or proximity capture, which are described in section (II)(g) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Some quality control measures are described in section (II)(h) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0220] Spatial information can provide information of biological and / or medical significance. For example, the methods and compositions described herein can allow for: identifying one or more biomarkers of a disease or disorder (e.g., for diagnosis, prognosis, and / or for determining treatment efficacy); identifying candidate drug targets for treating a disease or disorder; identifying a subject (e.g., diagnosing) as having a disease or disorder; identifying the stage and / or prognosis of a disease or disorder in a subject; identifying a subject as having an increased likelihood of developing a disease or disorder; monitoring the progression of a disease or disorder in a subject; determining the treatment efficacy of a disease or disorder in a subject; identifying a subset of patients for whom a treatment is effective for a disease or disorder; modifying the treatment of a subject having a disease or disorder; selecting subjects to participate in a clinical trial; and / or selecting a treatment method for a subject having a disease or disorder.

[0221] Spatial information can provide information of biological significance. For example, the methods and compositions described herein can allow for: identifying transcriptome and / or proteome expression profiles (e.g., in healthy and / or diseased tissues); identifying multiple analyte types in close proximity (e.g., nearest neighbor analysis); determining upregulated and / or downregulated genes and / or proteins in diseased tissues; characterizing the tumor microenvironment; characterizing the tumor immune response; characterizing cell types and their co-localization in tissues; and identifying genetic variations within a tissue (e.g., based on gene and / or protein expression profiles associated with specific disease or disorder biomarkers).

[0222] Typically, for spatially array-based methods, a substrate serves as a support for attaching capture probes either directly or indirectly to array features. A "feature" is an entity that serves as a support or repository for the various molecular entities used in spatial analysis. In some embodiments, some or all of the features in the array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Exemplary features and geometric properties of the array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020 / 176788 and U.S. Patent Application Publication No. 2020 / 0277663.

[0223] Typically, when a biological sample contacts a substrate comprising capture probes (e.g., a substrate having capture probes embedded, spotted, printed, fabricated thereon, or a substrate having features (e.g., beads, wells) comprising capture probes), analytes and / or intermediate reagents (or portions thereof) can be captured. As used herein, "contact", "contacted", and / or "contacting" of a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that the capture probes can interact (e.g., covalently or non-covalently bind (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020 / 176788 and / or U.S. Application Publication No. 2020 / 0277663.

[0224] In some cases, spatial analysis can be performed by attaching and / or introducing molecules (e.g., peptides, lipids, or nucleic acid molecules) having barcodes (e.g., spatial barcodes) to a biological sample (e.g., to cells in a biological sample). In some embodiments, multiple molecules (e.g., multiple nucleic acid molecules) having multiple barcodes (e.g., multiple spatial barcodes) are introduced into a biological sample (e.g., into multiple cells in a biological sample) for spatial analysis. In some embodiments, the biological sample can be physically separated (e.g., dissociated) into individual cells or cell groups for analysis after attaching and introducing the molecules having barcodes to the biological sample. Some such spatial analysis methods are described in Section (III) of WO 2020 / 176788 and / or U.S. Application Publication No. 2020 / 0277663.

[0225] In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA template ligation (RTL). Methods for RTL have been previously described. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug 21;45(14):e128. Generally, RTL involves hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3' end and / or the other oligonucleotide includes phosphorylated nucleotides at the 5' end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymer sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) joins the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to each other. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNase H). The released ligation product can then be captured by capture probes on an array (e.g., instead of directly capturing the analyte), optionally amplified and sequenced, to determine the location and abundance (optionally) of the analyte in a biological sample.

[0226] During spatial information analysis, sequence information of a spatial barcode associated with an analyte is obtained, and this sequence information can be used to provide information about the spatial distribution of the analyte in a biological sample. Various methods can be used to obtain spatial information. In some embodiments, a specific capture probe and its captured analyte are associated with a specific location in a feature array on a substrate. For example, a specific spatial barcode can be associated with a specific array location prior to array fabrication, and the sequence of the spatial barcode can be stored together with the specific array location information (e.g., in a database) such that each spatial barcode uniquely maps to a specific array location.

[0227] Alternatively, a specific spatial barcode can be deposited at a predetermined location in the feature array during fabrication such that at each location, only one type of spatial barcode is present, such that the spatial barcode is uniquely associated with a single feature of the array. When necessary, the array can be decoded using any of the methods described herein so that the spatial barcode is uniquely associated with the array feature location, and this mapping can be stored as described above.

[0228] When sequence information of a capture probe and / or an analyte is obtained during spatial information analysis, the location of the capture probe and / or the analyte can be determined by referring to stored information that uniquely associates each spatial barcode with an array feature location. In this way, a specific capture probe and a captured analyte are associated with a specific location in the feature array. Each array feature location represents a location relative to a coordinate reference point of the array (e.g., an array position, a fiducial marker). Thus, each feature location has an "address" or position in the coordinate space of the array.

[0229] Some exemplary spatial analysis workflows are described in the exemplary embodiment sections of WO 2020 / 176788 and / or U.S. Application Publication No. 2020 / 0277663. See, for example, the exemplary embodiments beginning with "In some non-limiting examples of the workflows described herein, a sample can be immersed..." in WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Also see, for example, the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., version C, dated June 2020) and / or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., version C, dated July 2020).

[0230] In some embodiments, spatial analysis can be performed using dedicated hardware and / or software, such as any system described in Section (II)(e)(ii) and / or Section (V) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, or any device and method in one or more of the "Control Slide for Imaging", "Methods of Using a Control Slide and a Substrate", "Systems of Using a Control Slide and a Substrate for Imaging", and / or "Sample and Array Alignment Devices and Methods", "Information Tags" sections of WO 2020 / 123320.

[0231] A suitable system for performing spatial analysis can include components such as a chamber for containing a biological sample (e.g., a flow cell or a sealable, fluid-impermeable chamber). The biological sample can be mounted in, for example, a biological sample holder. One or more fluid chambers can be connected to the chamber and / or the sample holder via fluid conduits, and fluid can be delivered to the chamber and / or the sample holder via a fluid pump, a vacuum source, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to the fluid conduits to regulate the flow of reagents from a reservoir to the chamber and / or the sample holder.

[0232] The system can optionally include a control unit including one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid-state storage medium such as, but not limited to, a magnetic storage medium, an optical storage medium, or other solid-state storage media, a persistent storage medium, a writable and / or rewritable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and its components) can generally perform any of the steps and functions described herein. In the case where the system is connected to a remote device, the remote device (or remote devices) can perform any of the steps and features described herein. The system can optionally include one or more detectors for capturing images (e.g., CCD, CMOS). The system can also optionally include one or more light sources for illuminating the sample (e.g., LED-based, diode-based, laser), a substrate having features, an analyte from a biological sample captured on the substrate, and various control and calibration media.

[0233] The system can optionally include software instructions encoded and / or implemented in one or more of a tangible storage medium and hardware components (such as an application for an application-specific integrated circuit). When executed by the control unit (and specifically an electronic controller) or an integrated circuit, the software instructions can cause the control unit, the integrated circuit, or other components executing the software instructions to perform any of the method steps or functions described herein.

[0234] In some cases, the systems described herein can detect a biological sample on an array (e.g., a tiled image). Exemplary methods for detecting a biological sample on an array are described in PCT Application No. 2020 / 061064 and / or U.S. Patent Application Serial No. 16 / 951,854.

[0235] Prior to transferring an analyte from a biological sample to a feature array on a substrate, the biological sample can be aligned with the array. Alignment of the biological sample and the feature array including capture probes can facilitate spatial analysis, which can be used to detect differences in the presence and / or level of analytes within different locations in the biological sample, e.g., to generate a three-dimensional map of the presence and / or level of analytes. Exemplary methods for generating two-dimensional and / or three-dimensional maps of the presence and / or level of analytes are described in PCT Application No. 2020 / 053655, and spatial analysis methods are generally described in WO 2020 / 061108 and / or U.S. Patent Application Serial No. 16 / 951,864.

[0236] In some cases, one or more fiducial markers (e.g., objects that appear in the generated image and are placed within the field of view of an imaging system) can be used to align a map of the presence and / or level of analytes with an image of the biological sample, as described in WO 2020 / 123320, in the "Substrate Attributes" section and the "Control Slide for Imaging" section of PCT Application No. 2020 / 061066, and / or U.S. Patent Application Serial No. 16 / 951,843. The fiducial markers can be used as reference points or measurement scales for alignment (e.g., for aligning the sample and the array, for aligning two substrates, for determining the position of the sample or the array on the substrate relative to the fiducial markers) and / or for quantitative measurements of dimensions and / or distances.

[0237] Although this specification contains many specific implementation details, these specific implementation details should not be construed as limitations on the scope of any invention or of any claimed rights, but rather as descriptions of features specific to particular implementations of particular inventions. Certain features described in the context of separate implementations in this specification can also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation can also be implemented separately or in any suitable sub-combination in multiple implementations. Moreover, although the features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be deleted from the combination, and the claimed combination can be directed to a sub-combination or a variant of a sub-combination.

[0238] Similarly, although multiple operations are depicted in the drawings in a particular order, this should not be construed as requiring that the operations be performed in the particular order shown or in sequential order, or that all of the illustrated operations be performed to achieve the desired result. In some cases, multitasking and parallel processing may be advantageous. Additionally, the various system components described above in the various implementations should not be construed as requiring such separation in all implementations, and it should be understood that the described program components and systems can generally be integrated together in a single software product or packaged into multiple software products.

[0239] Accordingly, particular implementations of the subject matter have been described. Other implementations are within the scope of the appended claims. In some cases, the recited actions in the claims can be performed in a different order and still achieve the desired result. Additionally, the processes depicted in the drawings do not necessarily need to be in the particular order shown or in sequential order to achieve the desired result. In some implementations, multitasking and parallel processing may be advantageous.

Claims

1. An electrophoresis system for analyte migration, the system comprising: a substrate including a plurality of regions, each region configured to be conductive and including a biological sample and one or more capture probes, the biological sample including an analyte, wherein the substrate includes a first electrode contact electrically connected to at least one of the plurality of regions, and wherein the one or more capture probes include a capture domain; a substrate cassette configured to be disposed at the substrate and including a plurality of holes corresponding to the plurality of regions of the substrate, the plurality of holes configured to define a plurality of buffer chambers including the plurality of regions of the substrate, wherein the substrate cassette includes a connection interface electrically coupled to the first electrode contact of the substrate; a cathode assembly including a plurality of electrodes respectively positioned within the plurality of buffer chambers of the substrate cassette, wherein the cathode assembly includes a second electrode contact electrically connected to at least one of the plurality of electrodes; and a power supply electrically connected to the first electrode contact of the substrate at the connection interface of the substrate cassette and electrically connected to the second electrode contact of the cathode assembly, the power supply configured to generate an electric field between the plurality of regions and the plurality of electrodes respectively, such that the analyte in the biological sample moves towards the capture probes on the substrate.

2. The electrophoresis system according to claim 1, wherein, the one or more capture probes are coated, spotted or printed on each of the plurality of regions.

3. The electrophoresis system according to claim 1, wherein, the biological sample is placed in contact with one or more of the plurality of regions on the substrate.

4. The electrophoresis system according to any one of claims 1 to 3, wherein, the substrate cassette includes: a substrate holder including a substrate mount for fixing the substrate; and a gasket including a plurality of gasket holes configured to be aligned with the plurality of regions when the substrate is fixed by the substrate holder, wherein the plurality of holes include the plurality of gasket holes.

5. The electrophoresis system according to claim 4, wherein, the substrate holder includes: a plurality of holder holes configured to be aligned with the plurality of gasket holes when the substrate is fixed by the substrate holder, wherein the plurality of holes include the plurality of gasket holes and the plurality of holder holes.

6. The electrophoresis system according to any one of claims 1 to 3, wherein, the substrate includes a glass slide coated with a conductive material.

7. The electrophoresis system according to claim 6, wherein, the conductive material includes at least one of tin oxide, indium tin oxide, transparent conductive oxide, aluminum-doped zinc oxide, fluorine-doped tin oxide, or any combination thereof.

8. The electrophoresis system according to any one of claims 1 to 3, further comprising: a first wire connecting the power supply to the first electrode contact; and A second wire that connects the power supply to the second electrode contact.

9. The electrophoresis system according to any one of claims 1 to 3, wherein, the plurality of buffer chambers are configured to receive a buffer.

10. The electrophoresis system according to claim 9, wherein, the buffer includes a permeabilizing reagent.

11. The electrophoresis system according to any one of claims 1 to 3, further comprising: a light source configured to irradiate the plurality of regions to permeabilize the biological samples on the plurality of regions.

12. The electrophoresis system according to any one of claims 1 to 3, wherein, a detergent is used to permeabilize the sample before, after or during enzyme treatment.

13. The electrophoresis system according to any one of claims 1 to 3, wherein, the sample is permeabilized by a lysis reagent added to the sample.

14. The electrophoresis system according to any one of claims 1 to 3, wherein, the sample is permeabilized by exposing the sample to a protease.

15. The electrophoresis system according to any one of claims 1 to 3, wherein, the substrate cassette includes: a body that defines a cavity configured to partially receive the substrate, wherein the connection interface of the substrate cassette includes a slit defined at the body and configured to allow the substrate to partially extend out of the body such that the first electrode contact of the substrate is positioned outside the body.

16. The electrophoresis system according to claim 15, wherein, the body includes a main surface and side surfaces extending from the periphery of the main surface, wherein the plurality of holes are defined at the main surface of the body, and wherein the slit is defined at the side surface of the body.

17. The electrophoresis system according to any one of claims 1 to 3, wherein, the substrate cassette includes: a body that defines a cavity configured to receive the substrate, wherein the connection interface of the substrate cassette includes a contact opening defined at the body and configured to expose the first electrode contact of the substrate therethrough.

18. The electrophoresis system according to claim 17, wherein, the body includes a main surface and side surfaces extending from the periphery of the main surface, wherein the plurality of holes are defined at the main surface of the body, and wherein the contact opening is defined at the main surface adjacent to the side surface.

19. The electrophoresis system according to claim 18, wherein, the contact opening is further defined at the side surface of the body to extend from the main surface to the side surface.

20. The electrophoresis system according to any one of claims 1 to 3, further comprising: a first contact pin electrically attached to the substrate and providing the first electrode contact, wherein the substrate cassette includes: a body that defines a cavity configured to receive the substrate, Wherein the connection interface of the substrate cassette includes contact holes defined at the body and configured to expose the first contact pins therethrough.

21. The electrophoresis system according to claim 20, wherein, the body includes a main surface and side surfaces extending from the periphery of the main surface, wherein the plurality of holes are defined at the main surface of the body, and wherein the contact holes are defined at the main surface.

22. The electrophoresis system according to any one of claims 1 to 3, wherein, the substrate cassette includes: a body defining a cavity configured to receive the substrate; and contact pins extending through the wall of the body and having a first end and a second end opposite the first end, the first end being disposed outside the body and the second end being disposed inside the body and configured to make electrical contact with the first electrode contact of the substrate.

23. The electrophoresis system according to claim 22, further including: a contact bracket electrically engaged with the substrate and providing the first electrode contact of the substrate, wherein the second end of the contact pin is configured to make electrical contact with the contact bracket.

24. The electrophoresis system according to any one of claims 1 to 3, wherein, the cathode assembly includes: a cathode body configured to be at least partially positioned on the substrate cassette, wherein the plurality of electrodes protrude from the cathode body.

25. The electrophoresis system according to claim 24, wherein, each of the plurality of electrodes includes at least one of a conductive wire, a conductive rod, or an array of conductive wires.

26. The electrophoresis system according to claim 25, wherein, each of the plurality of electrodes includes a conductive plate at a distal end of at least one of the conductive wire, the conductive rod, or the array of conductive wires.

27. The electrophoresis system according to claim 26, wherein, the conductive plate is configured as at least one of a circular plate, a square plate, or an annulus.

28. The electrophoresis system according to claim 24, wherein, the cathode body includes the second electrode contact.

29. The electrophoresis system according to any one of claims 1 to 3, wherein, the substrate cassette includes a second hole corresponding to the first electrode contact of the substrate and is configured to define a second buffer chamber on the first electrode contact of the substrate, wherein the plurality of buffer chambers are configured to contain a first buffer, and wherein the second buffer chamber is configured to contain a second buffer different from the first buffer.

30. The electrophoresis system according to claim 29, wherein, the second buffer has a greater electrolyte strength than the first buffer.

31. The electrophoresis system according to claim 30, wherein, the second buffer includes a sodium chloride solution.

32. The electrophoresis system according to claim 29, wherein, the cathode assembly includes: A cathode body configured to be at least partially positioned on the substrate cassette, with a plurality of electrodes protruding from the cathode body and configured to be respectively positioned in the plurality of buffer chambers; and A second electrode protruding from the cathode body and configured to be positioned in the second buffer chamber.

33. The electrophoresis system according to claim 32,[[]]END]] wherein,[[]]END]] the cathode body comprises:[[]]END]] an anode contact electrically connected to the second electrode,[[]]END]] wherein the power supply is electrically connected to the anode contact of the cathode body such that the power supply is in electrical contact with the first electrode of the substrate via the second electrode, and the second electrode is positioned in the second buffer chamber containing the second buffer solution on the first electrode contact of the substrate.

34. The electrophoresis system according to any one of claims 1 to 3,[[]]END]] wherein,[[]]END]] the substrate cassette comprises:[[]]END]] a cassette body defining a cavity configured to receive the substrate, wherein the cassette body comprises the cathode assembly such that the plurality of electrodes are configured to be positioned in the plurality of buffer chambers; and a plurality of cathode contact pins extending through the wall of the cassette body and making electrical contact with the second electrode such that the cathode contact pins are electrically connected to at least one of the plurality of electrodes.

35. The electrophoresis system according to claim 34,[[]]END]] wherein,[[]]END]] each of the plurality of electrodes comprises at least one of a wire, a conductive rod, or an array of wires.

36. The electrophoresis system according to claim 35,[[]]END]] wherein,[[]]END]] each of the plurality of electrodes comprises a conductive plate at the distal end of at least one of the wire, the conductive rod, or the array of wires.

37. The electrophoresis system according to claim 34,[[]]END]] wherein,[[]]END]] the cassette body comprises:[[]]END]] an anode contact pin extending through the wall of the cassette body and having a first end and a second end opposite the first end, the first end being disposed outside the cassette body, and the second end being disposed inside the cassette body and configured to make electrical contact with the first electrode contact of the substrate.

38. The electrophoresis system according to claim 37, further comprises:[[]]END]] a contact bracket electrically engaged with the substrate and providing the first electrode contact of the substrate,[[]]END]] wherein the second end of the anode contact pin is configured to make electrical contact with the contact bracket.

39. The electrophoresis system according to claim 15,[[]]END]] wherein,[[]]END]] the body comprises the cathode assembly such that the plurality of electrodes are configured to be positioned in the plurality of buffer chambers,[[]]END]] wherein the substrate cassette further comprises:[[]]END]] a cathode contact pin extending through the wall of the body and making electrical contact with the second electrode such that the cathode contact pin is electrically connected to at least one of the plurality of electrodes; an anode contact pin extending from the body; and A conductive wire having a first end and an opposite second end, the first end being electrically connected to the anode contact pin and the second end being electrically connected to the first electrode contact positioned outside the body.

40. The electrophoresis system according to claim 15, further comprising: An anode cassette body defining a cavity configured to receive the first electrode contact positioned outside the body of the substrate cassette for the substrate; and An anode contact pin extending through a wall of the anode cassette body and having a first end and a second end opposite the first end, the first end being disposed outside the anode cassette body and the second end being disposed inside the anode cassette body and configured to be in electrical contact with the first electrode contact of the substrate.

41. The electrophoresis system according to claim 40, further comprising: An anode contact bracket electrically engaging with the substrate and providing the first electrode contact of the substrate, wherein the second end of the anode contact pin is configured to be in electrical contact with the anode contact bracket.

42. The electrophoresis system according to any one of claims 1 to 3, further comprising: A system housing including the power supply; A cassette tray extending from the system housing and configured to receive the substrate cassette thereon; and A cassette cover extending from the system housing above the cassette tray.

43. The electrophoresis system according to claim 37 or 38, further comprising: A system housing including the power supply; A cassette tray extending from the system housing and configured to receive the substrate cassette thereon; and A cassette cover extending from the system housing above the cassette tray, the cassette cover including a cathode connector and an anode connector, the cathode connector being configured to be in electrical engagement with the cathode contact pin of the substrate cassette and the anode connector being configured to be in electrical engagement with the anode contact pin of the substrate cassette.

44. The electrophoresis system according to claim 22, further comprising: A system housing including the power supply; A cassette tray extending from the system housing and configured to receive the substrate cassette thereon; and A cassette cover extending from the system housing above the cassette tray and including the cathode assembly, wherein the plurality of electrodes project from the cassette cover towards the plurality of buffer chambers of the substrate cassette, the cassette cover further comprising: An anode connector configured to be in electrical engagement with the contact pin of the substrate cassette.

45. A method for analyte migration, the method comprising: Loading a substrate including capture probes and a substrate cassette into an electrophoresis instrument, the substrate cassette including a plurality of buffer chambers, the substrate including a plurality of regions, each region including a biological sample and one or more capture probes, the biological sample containing an analyte; Arranging a cathode to place a plurality of electrodes in the plurality of buffer chambers respectively; Electrically connecting a power supply to the substrate; Electrically connect the power supply to the cathode; Apply a first voltage between the substrate and the cathode; Detect an output parameter in response to the first voltage; Determine whether the output parameter meets a threshold; And Based on the output parameter meeting the threshold, apply a second voltage to generate an electric field between the multiple regions of the substrate and the multiple electrodes through the multiple buffer chambers, so that the analyte in the biological sample moves towards the capture probes on the substrate.

46. The method according to claim 45, wherein, The output parameter is impedance.

47. The method according to claim 45, wherein, The threshold indicates the adequacy of at least one of the following: the electrical connection between the power supply and the substrate, the electrical connection between the power supply and the cathode, or the electrical properties of one or more buffers in the multiple buffer chambers.

48. The method according to any one of claims 45 to 47, further comprising: Based on the output parameter not meeting the threshold, generate a notification to notify that the electrical connection is inappropriate; And Stop applying the second voltage for generating the electric field.

49. The method according to any one of claims 45 to 47, wherein, The threshold is a value within a predetermined range.

50. The method according to any one of claims 45 to 47, further comprising: Before loading the substrate into the substrate cassette, place the biological sample in contact with the capture probes on the substrate; Arrange the substrate cassette on the substrate to align the multiple holes of the substrate cassette with the multiple regions of the substrate and define the multiple buffer chambers on the multiple regions; And Supply buffer in the multiple buffer chambers.

51. The electrophoresis system according to any one of claims 1 to 3, wherein, The substrate cassette includes a connection interface that exposes the first electrode contact of the substrate.

Images