HK1237709B

HK1237709B - Multi-well separation apparatus and reagent delivery device

Info

Publication number: HK1237709B
Application number: HK17111630.4A
Authority: HK
Inventors: N.陈; R．K．沙纳汉; D.滕; D．J．布劳恩
Original assignee: 卓格瑞公司
Priority date: 2014-02-18
Filing date: 2015-02-18
Publication date: 2021-01-15

Abstract

Described herein are multi-well separation devices configured to allow a composition comprising a target agent to be separated into multiple wells, subdivided, recombined into a single well, and/or re-separated into the same or a different configuration of wells. Also described herein are reagent loading devices configured to simultaneously deliver one or more test agents to a plurality of volumes without having to individually deliver the test agents. Together, these devices allow high throughput parallel processes without repetitive pipetting or liquid handling robotics, though they may also be used separately. Also described herein are kits and systems for chemical or biological assays, as well as methods for using the multi-well separation devices and reagent loading devices described herein.

Description

Porous separation devices and reagent delivery devices

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to provisional application serial number 61/941,368, filed February 18, 2014, which is incorporated herein by reference in its entirety.

Technical Field

Described herein are devices, kits, systems, and methods for efficiently and reversibly separating a composition comprising a target agent into multiple volumes without requiring the composition to be separated individually into each of the multiple volumes, so that each of the multiple volumes, as well as the composition as a whole, can be efficiently processed. Also described herein are devices, kits, systems, and methods for efficiently delivering a test agent to multiple volumes without requiring the test agent to be delivered individually into each of the multiple volumes.

Background Art

High-throughput screening (High-throughput screen) allows researchers to quickly carry out a large amount of chemical, biological or pharmacological tests in parallel and is an important aspect of biological and chemical research, for example, in the development of new drugs. Such screening can be carried out manually or automatically by utilizing robotics or liquid handling devices to process samples of interest. Multiwell plates, also referred to as microtiter or microwell plates, are commonly used to keep samples in the case of assessment. Such multiwell plates, which are typically disposable and made of plastic and/or glass, generally include a grid of small, open holes (divot) or holes. The need for screening a large amount of chemical or biological samples in parallel has led to the development of multiwell plates with a large amount of identical holes, such as 96, 384, 1536 or 3456 individual holes.

When performing a screening procedure, a target agent—e.g., a cell or compound—is typically placed in each well of a multi-well plate. Subsequently, different reagents or test agents can be added to different wells of the plate to screen for their effects on the target agent. After all reaction components have been placed in the wells of the multi-well plate and the necessary reaction conditions have been met, the reaction results can be analyzed manually or by mechanically traversing all the wells of the plate.

Although robotic equipment has emerged for these processes, such equipment is typically expensive and can be slow if the number of pipette channels on the liquid handler is less than the number of wells to be transferred. On the other hand, manual methods are slow, laborious, and prone to error. Moreover, traditional methods of mixing reagents or test agents into wells containing target agents have limitations. Traditional mixing involves shaking or vibrating an oscillator that shakes or vibrates the entire multi-well plate. For small volume wells, shaking or vibrating the entire multi-well plate does not produce sufficient mixing in the individual wells; the greater the viscosity of the liquid and the smaller the wells, the less efficient the shaking or vibration will be. The traditional alternative is to pipette a portion of the liquid in each individual well up and down, in and out of the well, using the pipette tip. Using pipetting to mix the well contents generates large shear stresses at the bottom of the well, especially directly below the pipette opening. These fluid shear stresses can separate the target agent (e.g., cells), damage the target agent, or apply excessive mechanical stimulation to the target agent, and uneven shear forces and flows can cause non-uniform distribution of the target agent on the bottom surface of the well. Therefore, more efficient and accurate methods of manipulating target and test agents for use in screening and similar processes are desired.

SUMMARY OF THE INVENTION

The porous separation devices described herein allow a composition comprising a target agent to be separated into multiple wells; to be subdivided and recombined into a single well; and/or to be re-separated into wells of the same or a different configuration. This is achieved by having a separating wall structure, unlike current multi-well plates having fixed walls, that can be reversibly removed from a volume containing a composition comprising a target agent. In this way, the device can allow for reversible and repeatable separation and combination of volumes without the need to transfer the composition from one well to another via a pipette or the like.

Reagent loading device is described herein equally, and it is configured to test agent be delivered to each independent hole of porous separation device simultaneously.Reagent loading device described herein allows one or more test agents to be delivered to multiple volumes simultaneously, without the need for one or more test agents to be delivered to each of multiple volumes separately.This is realized by the reagent loading device with multiple protrusions (protrusion), and each described protrusion comprises rod and the closed tip that is suitable for keeping reagent.According to this, closed tip all can be loaded with reagent separately in the configuration corresponding to the desired delivery configuration of multiple volumes.Reagent loading device can be configured to promote the mixing of reagent and multiple volumes, as by being configured to vibrate, and can comprise containment element (containment element) or other design that is configured to protect the reagent that is loaded on the closed tip.

Together, these devices allow high-throughput parallel processing without the need for repeated pipetting or liquid handling robots. However, it should be understood that in some variations, the porous separation device described herein can be used separately from the reagent loading device described herein. Similarly, it should be understood that in some variations, the reagent loading device described herein can be used separately from the porous separation device described herein. This paper also describes test kits and systems for chemical or biological analysis, as well as methods using the porous separation device and reagent loading device described herein.

Generally, the porous separation devices described herein may include a substrate and a removable separation pore structure coupled to the substrate. In some of these variations, the separation pore structure may include a plurality of walls defining a plurality of openings. In some of these variations, the substrate and the separation pore structure may form a plurality of pores.

In some variations, the porous separation devices described herein may include a substrate, a boundary wall, and a removable separation pore structure coupled to the substrate. In some of these variations, the separation pore structure may include a plurality of walls defining a plurality of openings. In some of these variations, the substrate and the separation pore structure may form a plurality of pores, and the boundary wall and the substrate may form a cavity.

In some variations, a porous separation device described herein may include a substrate, a boundary wall, a boundary seal, and a removable separation pore structure coupled to the substrate. In some of these variations, the separation pore structure may include a plurality of walls defining a plurality of openings, the substrate and the separation pore structure may form a plurality of pores, and the boundary seal may form a leak-proof seal with the boundary wall and the substrate to form a cavity.

In some variations, the porous separation devices described herein may include a substrate, a removable separation pore structure coupled to the substrate, and a separation seal. In some of these variations, the separation pore structure may include a plurality of walls defining a plurality of openings. In some of these variations, the separation seal may form a leak-proof seal between the substrate and the separation pore structure to form a plurality of pores.

In some variations, the porous separation device described herein may include a substrate, a boundary wall, a boundary seal, a removable separation hole structure coupled to the substrate, and a separation seal. In some of these variations, the separation hole structure may include a plurality of walls defining a plurality of openings. In some of these variations, the separation seal may form a leak-proof seal between the substrate and the separation hole structure to form a plurality of holes. In some of these variations, the boundary seal may form a leak-proof seal between the boundary wall and the substrate to form a cavity. In some of these variations, the boundary seal may be located between a substrate support and the substrate.

In some variations, the porous separation device described herein may include a substrate, a removable separation pore structure coupled to the substrate, and a concentrating pore structure located between the substrate and the separation pore structure. In some of these variations, the separation pore structure may include a plurality of walls defining a plurality of openings. In some of these variations, the substrate and the separation pore structure may form a plurality of pores. In some of these variations, the concentrating pore structure may include a plurality of openings, and each of the plurality of openings of the concentrating pore structure may correspond to one of the plurality of openings defined by the separation pore structure. In some of these variations, each of the plurality of openings of the concentrating pore structure may have a proximal cross-sectional area and a distal cross-sectional area, and the proximal cross-sectional area may be greater than the distal cross-sectional area.

In some of these variations, the separation pore structure can be reversibly removably coupled to the substrate. In some of these variations, a plurality of walls can define at least about 96 openings. In some of these variations, a plurality of walls can define at least about 480 openings. In other variations, a plurality of walls can define at least about 6 openings, at least about 12 openings, at least about 24 openings, at least about 48 openings, at least about 384 openings, at least about 1536 openings, at least about 3456 openings or more than 3456 openings. In some of these variations, the porous separation device may further include a boundary wall coupled to the substrate. In some of these variations, the substrate and the boundary wall can define at least one cavity. In some of these variations, the substrate and the boundary wall can further define at least two separated regions in the cavity. In some of these variations, the boundary wall can be removably coupled to the substrate. In some of these variations, the porous separation device may further include a substrate support configured to couple the substrate to the boundary wall. In some of these variations, the substrate support may include at least one clip, wherein the at least one clip may be configured to attach to a portion of the boundary wall. In some of these variations, the boundary wall may be reversibly movably coupled to the substrate. In some of these variations, the boundary wall may be fixedly coupled to the substrate. In some of these variations, the boundary wall may be integral with the substrate.

In some of these variations, the separation hole structure may be coupled to the substrate by attachment to a boundary wall. In some of these variations, the separation hole structure may include at least one clip, wherein the at least one clip may be configured to attach to a portion of the boundary wall. In some of these variations, the porous separation device may further include a second removable separation hole structure coupled to the substrate. In some of these variations, the second separation hole structure may include a second plurality of walls defining a second plurality of openings. In some of these variations, the substrate and the second separation hole structure may form a second plurality of holes. In some of these variations, the second separation hole structure may fit within one of the plurality of openings defined by the separation hole structure. In some of these variations, each of the second plurality of holes may have a smaller volume than each of the plurality of holes defined by the first separation hole structure.

In some of these variations, the porous separation device may further include a concentrating pore structure located between the separation pore structure and the substrate. In some of these variations, the concentrating pore structure may include a plurality of openings, wherein each of the plurality of openings of the concentrating pore structure may correspond to one of the plurality of openings defined by the separation pore structure. In some of these variations, each of the plurality of openings may have a proximal cross-sectional area and a distal cross-sectional area, and the proximal cross-sectional area may be greater than the distal cross-sectional area. In some of these variations, the distal cross-sectional area may be zero. In some of these variations, each of the plurality of openings defined by the separation pore structure may have a first cross-sectional area, and each of the plurality of openings of the concentrating pore structure may have a second cross-sectional area at the distal end, and the first cross-sectional area may be greater than the second cross-sectional area. In some of these variations, each of the plurality of openings of the concentrating pore structure may contain a protein. In some of these variations, each of the plurality of openings of the concentrating pore structure may contain a polymer. In some of these variations, each of the plurality of openings of the concentrating pore structure may contain a hydrogel. In some of these variations, each of the plurality of openings in the concentrating pore structure may contain a chemical coating.

In some of these variations, the concentrating pore structure may be fixedly attached to the substrate. In some of these variations, the concentrating pore structure may be fixedly attached to the boundary wall. In some of these variations, the porous separation device may further include a seal located between the boundary wall and the substrate. In some of these variations, the seal may be fixedly attached to the boundary wall. In some of these variations, the seal may be fixedly attached to the substrate. In some of these variations, the seal may comprise rubber. In some of these variations, the seal may comprise plastic. In some of these variations, the seal may comprise a polymer. In some of these variations, the porous separation device may further include a separation seal located between the separation pore structure and the substrate. In some of these variations, the separation seal may be fixedly attached to the boundary wall. In some of these variations, the separation seal may be fixedly attached to the substrate. In some of these variations, the separation seal may be fixedly attached to the separation pore structure. In some of these variations, the separation seal may comprise rubber. In some of these variations, the separation seal may comprise plastic. In some of these variations, the separation seal may comprise a polymer.

In some of these variations, each of the multiple openings of the separation pore structure may have a hexagonal cross-sectional shape. In some of these variations, each of the multiple openings of the separation pore structure may have a rectangular cross-sectional shape. In some of these variations, each of the multiple openings of the separation pore structure may have a circular cross-sectional shape. In some of these variations, the separation pore structure may include rubber. In some of these variations, the separation pore structure may include plastic. In some of these variations, the separation pore structure may include silicon. In some of these variations, the separation pore structure may include metal. In some of these variations, the separation pore structure may include a polymer. In some of these variations, the separation pore structure may include glass. In some of these variations, the separation pore structure may include rubber. In some of these variations, each of the multiple pores may have a volume of approximately 100 μL to 100 mL. In some of these variations, the volume of each of the plurality of wells may be less than about 100 μL, about 100 μL to about 200 μL, about 200 μL to about 400 μL, about 400 μL to about 600 μL, about 600 μL to about 800 μL, about 800 μL to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about 20 mL, about 20 mL to about 40 mL, about 40 mL to about 60 mL, about 60 mL to about 80 mL, about 80 mL to about 100 mL, or greater than about 100 mL. In some of these variations, the depth of each of the plurality of wells may be about 1 mm to about 40 mm. In some of these variations, the depth of each of the plurality of wells may be about 5 mm to about 15 mm, about 10 mm to about 20 mm, about 15 mm to about 25 mm, about 20 mm to about 30 mm, about 25 mm to about 35 mm, about 30 mm to about 40 mm, or greater than about 40 mm.

In some of these variations, the substrate may be a planar structure. In some of these variations, the substrate may comprise glass. In some of these variations, the substrate may comprise plastic. In some of these variations, the substrate may comprise silicon. In some of these variations, the substrate may comprise ceramic. In some of these variations, the substrate may comprise metal. In some of these variations, the substrate may comprise a combination of one or more materials selected from glass, plastic, silicon, ceramic, and metal. In some of these variations, the substrate may be adapted to hold the targeting agent in a fixed position. In some of these variations, the substrate may be coated with a protein. In some of these variations, the substrate may be coated with a hydrogel. In some of these variations, the substrate may be coated with a polymer. In some of these variations, the substrate may be immobilized with a compound. In some of these variations, the substrate may be immobilized with a protein. In some of these variations, the substrate may be immobilized with immobilized cells. In some of these variations, the substrate may be immobilized with microorganisms. In some of these variations, the substrate may have a maximum dimension of less than about 13 cm. In some of these variations, the substrate may have a maximum dimension of about 11 cm to about 15 cm, a maximum dimension of about 7.5 cm, a maximum dimension of about 1 cm to about 30 cm, a maximum dimension of about 5 cm to about 25 cm, a maximum dimension of about 10 cm to about 20 cm, or a maximum dimension greater than about 30 cm.

In some of these variations, the target agent may include cells. In some of these variations, the target agent may include proteins. In some of these variations, the target agent may include compounds. In some of these variations, the target agent may include polymers. In some of these variations, the porous separation device may further include a cover. In some of these variations, the porous separation device may be configured for single use.

This paper also describes a test kit for chemical or biological analysis. Generally, the test kit for chemical or biological analysis may include a substrate and a separation hole structure configured to reversibly and removably couple with the substrate. In some of these variations, the separation hole structure may include a plurality of walls that limit a plurality of openings. In some of these variations, the substrate and the separation hole structure may be configured to form a plurality of holes when coupled. In some of these variations, the separation hole structure may be configured to reversibly and removably couple with the substrate. In some of these variations, a plurality of walls may limit at least about 96 openings. In some of these variations, a plurality of walls may limit at least about 480 openings. In some of these variations, a plurality of walls may limit at least about 6 openings, at least about 12 openings, at least about 24 openings, at least about 48 openings, at least about 384 openings, at least about 1536 openings, at least about 3456 openings or more than 3456 openings.

In some of these variations, the test kit for chemical or biological analysis may further include a boundary wall configured to couple with the substrate. In some of these variations, the substrate and boundary wall may be configured to define at least one cavity when coupled. In some of these variations, the substrate and boundary wall may further be configured to define at least two separate areas within the cavity when coupled. In some of these variations, the boundary wall may be configured to removably couple with the substrate.

In some of these variations, the test kit for chemical or biological analysis may further include a substrate support configured to couple substrate to a boundary wall. In some of these variations, the substrate support may include at least one buckle, wherein at least one buckle may be configured to be attached to a part of the boundary wall. In some of these variations, the boundary wall may be configured to reversibly and removably couple with the substrate. In some of these variations, the boundary wall may be fixedly coupled to the substrate. In some of these variations, the boundary wall may be integrated with the substrate. In some of these variations, the separation hole structure may be configured to be coupled to the substrate by being attached to the boundary wall. In some of these variations, the separation hole structure may include at least one buckle, and at least one buckle may be configured to be attached to a part of the boundary wall.

In some of these variations, the test kit for chemical or biological analysis may further include a second removable separation hole structure coupled to the substrate, wherein the second separation hole structure may include a second plurality of walls defining a second plurality of openings. In some of these variations, the substrate and the second separation hole structure may be configured to form a second plurality of holes when coupled. In some of these variations, the second separation hole structure may be configured to fit in one of the plurality of openings defined by the first separation hole structure. In some of these variations, each of the second plurality of holes may have a smaller volume than each of the plurality of holes defined by the first separation hole structure.

In some of these variations, the kit for chemical or biological analysis may further include a concentrating pore structure configured to be located between the separation pore structure and the substrate. In some of these variations, the concentrating pore structure may include a plurality of openings, wherein each of the plurality of openings may correspond to one of the plurality of openings defined by the separation pore structure. In some of these variations, each of the plurality of openings of the concentrating pore structure may have a proximal cross-sectional area and a distal cross-sectional area, wherein the proximal cross-sectional area may be greater than the distal cross-sectional area. In some of these variations, the distal cross-sectional area may be zero. In some of these variations, each of the plurality of openings defined by the separation pore structure may have a first cross-sectional area, and each of the plurality of openings of the concentrating pore structure may have a second cross-sectional area at the distal end, wherein the first cross-sectional area may be greater than the second cross-sectional area. In some of these variations, each of the plurality of openings of the concentrating pore structure may contain a protein. In some of these variations, the concentrating pore structure may be fixedly attached to the substrate. In some of these variations, the concentrating pore structure may be fixedly attached to the boundary wall.

In some of these variations, the test kit for chemical or biological analysis may further include a seal configured to be located between the boundary wall and the substrate. In some of these variations, the seal may be fixedly attached to the boundary wall. In some of these variations, the seal may be fixedly attached to the substrate. In some of these variations, the seal may include rubber. In some of these variations, the seal may include plastic. In some of these variations, the seal may include a polymer. In some of these variations, the test kit for chemical or biological analysis may further include a separation seal configured to be located between the separation hole structure and the substrate. In some of these variations, the separation seal may be fixedly attached to the boundary wall. In some of these variations, the separation seal may be fixedly attached to the substrate. In some of these variations, the separation seal may be fixedly attached to the separation hole structure. In some of these variations, the separation seal may include rubber. In some of these variations, the separation seal may include plastic. In some of these variations, the separation seal may include a polymer.

In some of these variations, each of the multiple openings of the separation pore structure may have a hexagonal cross-sectional shape. In some of these variations, each of the multiple openings of the separation pore structure may have a rectangular cross-sectional shape. In some of these variations, each of the multiple openings of the separation pore structure may have a circular cross-sectional shape. In some of these variations, the separation pore structure may include rubber. In some of these variations, the separation pore structure may include plastic. In some of these variations, the separation pore structure may include silicon. In some of these variations, the separation pore structure may include metal. In some of these variations, the separation pore structure may include a polymer. In some of these variations, the separation pore structure may include glass. In some of these variations, the separation pore structure may include rubber. In some of these variations, the volume of each of the multiple pores may be approximately 100 μL to 100 mL. In some of these variations, the volume of each of the plurality of wells may be less than about 100 μL, about 100 μL to about 200 μL, about 200 μL to about 400 μL, about 400 μL to about 600 μL, about 600 μL to about 800 μL, about 800 μL to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about 20 mL, about 20 mL to about 40 mL, about 40 mL to about 60 mL, about 60 mL to about 80 mL, about 80 mL to about 100 mL, or greater than about 100 mL. In some of these variations, the depth of each of the plurality of wells may be about 1 mm to about 40 mm. In some of these variations, the depth of each of the plurality of wells may be about 5 mm to about 15 mm, about 10 mm to about 20 mm, about 15 mm to about 25 mm, about 20 mm to about 30 mm, about 25 mm to about 35 mm, about 30 mm to about 40 mm, or greater than about 40 mm.

In some of these variations, the target agent may include cells. In some of these variations, the target agent may include proteins. In some of these variations, the target agent may include compounds. In some of these variations, the target agent may include polymers. In some of these variations, the test kit for chemical or biological analysis may further include a lid. In some of these variations, the test kit for chemical or biological analysis may be configured for single use.

This paper also describes the system for chemical or biological analysis.Usually, the system for chemical or biological analysis may include a reagent loading device and a separator.In some variations, the separator may be any of the porous separation device described herein.In some variations, the reagent loading device may be any of the reagent loading device described herein.In some variations, the reagent loading device may include a plurality of projections, wherein each projection may include rod and a closed tip that is suitable for keeping reagent.In some of these variations, each of a plurality of projections may be configured to fit in one of any of a plurality of holes of the porous separation device described herein.

Also described herein are methods for performing a chemical or biological analysis. Generally, the method for performing a chemical or biological analysis can include applying a target agent to a substrate; coupling a first separation pore structure to the substrate, wherein the separation pore structure can include a plurality of walls defining a plurality of openings, and the substrate and the first separation pore structure can form a first plurality of pores, thereby separating the target agent into a first plurality of subpopulations; and applying a first plurality of test agents to the first plurality of subpopulations, wherein the effects of the first plurality of test agents on the target agent can be analyzed.

In some variations, a method for performing a chemical or biological analysis may include applying a cell suspension comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include a plurality of walls defining a plurality of openings, and the substrate and the separation pore structure may form a plurality of pores, thereby separating the cells into a plurality of subpopulations; and applying a plurality of drugs to the plurality of subpopulations, and wherein the effects of the plurality of drugs on the cells may be analyzed.

In some variations, a method for performing a chemical or biological analysis may include applying a cell suspension comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include multiple walls defining multiple openings, and the substrate and the separation pore structure may form multiple pores to separate the cells into multiple subpopulations; and simultaneously applying multiple drugs to the multiple subpopulations using a reagent loading device comprising multiple protrusions, wherein the effects of the multiple drugs on the cells can be analyzed.

In some variations, a method for performing a chemical or biological analysis may include applying a hydrogel comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include a plurality of walls defining a plurality of openings, and the substrate and the separation pore structure may form a plurality of pores, thereby separating the hydrogel into a plurality of subpopulations and separating the cells into a plurality of subpopulations; and applying a plurality of drugs to the plurality of subpopulations, wherein the effects of the plurality of drugs on the cells may be analyzed.

In some variations, a method for performing a chemical or biological analysis may include applying a hydrogel comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include a plurality of walls defining a plurality of openings, and the substrate and the separation pore structure may form a plurality of pores, thereby separating the hydrogel into a plurality of subpopulations and separating the cells into a plurality of subpopulations; and simultaneously applying a plurality of drugs to the plurality of subpopulations, wherein the effects of the plurality of drugs on the cells may be analyzed.

In some variations, a method for performing a chemical or biological analysis may include applying a cell suspension comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include multiple walls defining multiple openings, and the substrate and the separation pore structure may form multiple pores, thereby separating the cells into multiple subpopulations; decoupling the separation pore structure from the substrate; applying a drug to the substrate; recoupling the separation pore structure to the substrate, thereby again separating the cells into multiple subpopulations; applying multiple first antibodies to the multiple subpopulations; and applying multiple second antibodies to the multiple subpopulations.

In some variations, a method for performing a chemical or biological analysis may include applying a cell suspension comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include multiple walls defining multiple openings, and the substrate and the separation pore structure may form multiple pores, thereby separating the cells into multiple subpopulations; decoupling the separation pore structure from the substrate; applying a drug to the substrate; recoupling the separation pore structure to the substrate; thereby again separating the cells into multiple subpopulations; applying multiple first antibodies to the multiple subpopulations; decoupling the separation pore structure from the substrate; and applying a second antibody to the substrate.

In some variations, a method for performing a chemical or biological analysis may include applying a cell suspension comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include a plurality of walls defining a plurality of openings, and the substrate and the separation pore structure may form a plurality of pores, thereby separating the cells into a plurality of subpopulations; applying a plurality of drugs to the plurality of subpopulations; applying a plurality of first antibodies to the plurality of subpopulations; decoupling the separation pore structure from the substrate; and applying a second antibody to the substrate.

In some variations, a method for performing a chemical or biological analysis may include applying a cell suspension comprising cells to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include multiple walls defining multiple openings, and the substrate and the separation pore structure may form multiple pores, thereby dividing the cells into multiple subpopulations; decoupling the separation pore structure from the substrate; applying a drug to the substrate; re-coupling the separation pore structure to the substrate, thereby again dividing the cells into multiple subpopulations, applying multiple first antibodies to the multiple subpopulations simultaneously using a reagent loading device including multiple protrusions, and applying multiple second antibodies to the multiple subpopulations simultaneously using a reagent loading device including multiple protrusions.

In some variations, a method for performing a chemical or biological analysis may include applying a composition comprising a drug to a substrate; coupling a separation pore structure to the substrate, wherein the separation pore structure may include a plurality of walls defining a plurality of openings, and the substrate and the separation pore structure may form a plurality of pores to separate the composition into a plurality of subpopulations; and applying one of the cell libraries to each of the plurality of pores, wherein the effect of the drug on the cell type may be analyzed.

In some variations, a method for performing a chemical or biological analysis may include applying a composition comprising a drug to a substrate; coupling a separation well structure to the substrate, wherein the separation well structure may include a plurality of walls defining a plurality of openings, and the substrate and the separation well structure may form a plurality of wells to separate the composition into a plurality of subpopulations, and simultaneously applying one of a cell library to each of the plurality of wells using a reagent loading device comprising a plurality of protrusions, and wherein the effect of the drug on the cell type can be analyzed.

In some of these variations, the method for performing a chemical or biological analysis may further include removing the first separation pore structure from the substrate. In some of these variations, the method for performing a chemical or biological analysis may further include treating the target agent with a universal reagent. In some of these variations, the method for performing a chemical or biological analysis may further include coupling the first separation pore structure to the substrate again. In some of these variations, the method for performing a chemical or biological analysis may further include coupling a second separation pore structure to the substrate, wherein the second separation pore structure may include a plurality of walls defining a plurality of openings, and wherein the substrate and the second separation pore structure may form a second plurality of holes, thereby dividing the target agent into a second plurality of subpopulations, wherein the second plurality of subpopulations may be different from the first plurality of subpopulations; and applying a second plurality of test agents to the second plurality of subpopulations. In some of these variations, the method for performing a chemical or biological analysis may further include coupling a second separation pore structure to the substrate, wherein the second separation pore structure may include a plurality of walls defining a plurality of openings, wherein the second separation pore structure may be configured to fit within one of the plurality of openings defined by the first separation pore structure. In some of these variations, the method for performing a chemical or biological analysis may further include removing the second separation pore structure from the substrate. In some of these variations, the method for performing a chemical or biological analysis may further include recoupling the second separation pore structure to the substrate. In some of these variations, the method for performing a chemical or biological analysis may include reversibly coupling the substrate to the separation pore structure to form a plurality of pores.

In some of these variations, the first plurality of subpopulations may include at least a first subpopulation and a second subpopulation, and the first test agent may be applied to the first subpopulation and the second test agent may be applied to the second subpopulation. In some of these variations, the first plurality of test agents may be applied by a reagent loading device including a plurality of protrusions, each of which may include a rod and a closed tip suitable for holding the reagent. In some of these variations, each of the plurality of protrusions may be configured to fit within one of the first plurality of holes. In some of these variations, the method for performing a chemical or biological analysis may further include loading the reagent loading device with the first plurality of test agents. In some of these variations, the reagent loading device may be pre-loaded with the first plurality of test agents.

Independent of porous separation device described above, on the other hand, the present invention provides a reagent loading device.In some variations, porous separation device described herein and reagent loading device described herein can be used or configured to use together.In other variations, porous separation device described herein can be used or configured to use independently of the reagent loading device.In also having other variations, the reagent loading device can be used or configured to use independently of the porous separation device.

Typically, the reagent loading device may include a plurality of projections. In some variations, each projection may include a rod and a closed tip that is suitable for keeping reagent. In some of these variations, each closed tip may be loaded with reagent. In some of these variations, at least two of the closed tip may be loaded with different reagents. In some of these variations, each of the closed tip may be loaded with different reagents. In some of these variations, a plurality of projections may be configured to vibrate. In some variations, the reagent loading device may include a plate to which a plurality of projections may be attached.

In some of these variations, the plurality of protrusions may comprise plastic. In some of these variations, the plurality of protrusions may comprise silicon. In some of these variations, the plurality of protrusions may comprise metal. In some of these variations, the plurality of protrusions may comprise a polymer. In some of these variations, each of the plurality of protrusions may be at least about 1 mm long. In some of these variations, each of the plurality of protrusions may be at least about 5 mm long. In some of these variations, each of the plurality of protrusions may be at least about 1 cm long. In some of these variations, the length of the protrusions may be from about 1 mm to about 2 mm, from about 2 mm to about 4 mm, from about 4 mm to about 6 mm, from about 6 mm to about 8 mm, from about 8 mm to about 1 cm, from about 1 cm to about 2 cm, from about 2 cm to about 4 cm, from about 4 cm to about 6 cm, or longer than 6 cm. In some of these variations, the maximum cross-sectional dimension of the tip may be about 100 microns. In some of these variations, the maximum cross-sectional dimension of the protrusion may be about 1 μm to about 10 μm, about 10 μm to about 100 μm, about 100 μm to about 1 mm, about 1 mm to about 5 mm, about 5 mm to about 1 cm, about 1 cm to about 2 cm, or longer than about 2 cm.

In some of these variations, the closed tip may have a square cross-section. In some of these variations, the closed tip may have a circular cross-section. In some of these variations, the closed tip may have a pointed shape. In some of these variations, the closed tip may include a depression. In some of these variations, the depression may be linear. In some of these variations, the closed tip may include two intersecting linear depressions. In some of these variations, the depression may be hemispherical. In some of these variations, the depression may be cylindrical. In some of these variations, the tip may include a hydrogel or sol-gel. In some of these variations, the tip may include a polymer. In some of these variations, the tip may include plastic. In some of these variations, the tip may be substantially smooth. In some of these variations, the tip may include an uneven surface. In some of these variations, the tip may be dissolvable. In some of these variations, the plurality of protrusions may be configured to vibrate. In some of these variations, each of the plurality of protrusions may be coupled to a motor. In some of these variations, the plurality of protrusions may be coupled to a motor. In some of these variations, each of the plurality of needles may be configured to emit ultrasonic frequency waves.

In some of these variations, the reagent loading device may further include a plate to which a plurality of projections can attach. In some of these variations, the plate may further include an indicator configured to indicate the direction of the reagent loading device. In some of these variations, the indicator may further be configured to provide a handle for operating the array, for example, an arrow-shaped handle.

In some of these variations, the closed tips of the multiple protrusions may be protected by a containment element. In some of these variations, the containment element may include a separate hole configured to isolate each protrusion. In some of these variations, each of the closed tips of the multiple protrusions may be enclosed in one of the multiple caps. In some of these variations, the reagent loading device may include legs that extend beyond the closed tips to protect the closed tips from resting on a flat surface. In some of these variations, each of the tips may be loaded with a reagent. In some of these variations, the reagent may be in solid form. In some of these variations, the reagent may be in pure liquid form. In some of these variations, the reagent may be in gel form. In some of these variations, the reagent may be a liquid solution. In some of these variations, each of the closed tips may be capable of loading at least approximately 1 pL of liquid solution. In some of these variations, each of the closed tips may be capable of loading at least approximately 1 nL of liquid solution. In some of these variations, each of the closed tips may be capable of loading at least approximately 1 μL of liquid solution. In some of these variations, each of the closed tip can be capable of loading a liquid solution of about 1 pL to about 10 pL, about 10 pL to about 100 pL, about 100 pL to about 1 nL, about 1 nL to about 10 nL, about 10 nL to about 100 nL, about 100 nL to about 1 μ L, about 1 μ L to about 10 μ L or more than about 10 μ L. In some of these variations, at least two of the closed tip can be pre-loaded with different reagents. In some of these variations, each of the closed tip can be pre-loaded with different reagents. In some of these variations, reagent can be selected from protein, nucleic acid or compound. In some of these variations, reagent can be selected from cell, microorganism, or plant.

This paper also describes the test kit for reagent being loaded on the reagent loading device. Usually, the test kit for reagent being loaded on the reagent loading device may include a plurality of projections, wherein each of a plurality of projections may have a closed tip, containment element, chamber and at least one lid that are suitable for keeping reagent. In some variations, the chamber may include a plurality of compartments.

This paper also describes the method of loading the liquid solution comprising a reagent into any one of the above-mentioned reagent loading devices. Generally, the method of loading a liquid solution may include dipping a reagent loading device in a chamber comprising a liquid solution, and lifting the reagent loading device to leave the chamber. In some variations, the chamber may include a plurality of compartments. In some of these variations, at least two of the plurality of compartments may contain different reagents. In some of these variations, each of the plurality of compartments may contain different reagents. In some of these variations, reagent may be selected from protein, nucleic acid or compound. In some of these variations, reagent may be selected from cell, microorganism or plant. In some of these variations, the method of loading a liquid solution may further include loading a chamber with a liquid solution. In some of these variations, the method of loading a liquid solution may further include applying a liquid solution of a defined volume to the closed tip of each of a plurality of protrusions. In some of these variations, at least two of a plurality of protrusions may be loaded with a liquid solution comprising different reagents. In some of these variations, each of a plurality of protrusions may be loaded with a liquid solution comprising different reagents.

This article also describes methods for loading one or more reagents into multiple isolated areas on a substrate. Generally, the method for loading one or more reagents may include contacting each of the multiple isolated areas with one of the multiple closed tips and removing the multiple closed tips from the multiple isolated areas. In some variations, the multiple closed tips may be arranged in an array. In some variations, each of the multiple closed tips may be loaded with one of the one or more reagents. In some of these variations, the multiple isolated areas may include multiple points. In some of these variations, the multiple isolated areas may include multiple holes. In some of these variations, each of the multiple isolated areas may contain a target agent. In some of these variations, at least two of the multiple closed tips may be loaded with different reagents. In some of these variations, each of the multiple closed tips may be loaded with different reagents. In some of these variations, each of the multiple closed tips may contain a liquid, and the method for loading one or more reagents may further include mixing the liquid in each of the multiple isolated areas with a plurality of protrusions. In some of these variations, mixing may include stirring. In some of these variations, mixing may include sonication. In some of these variations, the method for loading one or more reagents may further include discarding the reagent loading device after removing the multiple protrusions from the multiple isolated areas. In some of these variations, the reagent loading device can be operated by a robotic device. In some of these variations, the substrate can be operated by a robotic device.

Also described herein is a test kit for loading reagents, wherein the test kit comprises a reagent loading device comprising a plurality of protrusions and a plate to which a plurality of protrusions are attached, wherein each protrusion comprises a rod and a closed tip suitable for keeping reagents; and an antibody library. In some variations, the antibody library can be pre-loaded on the reagent loading device. In some variations, the test kit further comprises an adapter, wherein the adapter corresponds to the reagent loading device and is configured to cooperate around the porous plate. In some of these variations, the adapter may comprise a wedge (key) corresponding to the notch of the reagent loading device. In some of these variations, the adapter may resist the vibration of the reagent loading device when the reagent loading device is partially loaded into the porous plate, but may allow the reagent loading device to vibrate when the reagent loading device is fully loaded into the porous plate.

Brief introduction of the attached figure

Figure 1A is a perspective view of one embodiment of an assembled porous separation device. Figure 1B is an exploded perspective view of a porous separation device.

2A-2B are perspective views of a boundary wall, a substrate, and a substrate support in a coupled configuration and a decoupled configuration, respectively.

3 is a perspective view of a substrate and substrate support.

4A-4B are perspective and side views, respectively, of a substrate support.

5 is a perspective view of a cavity of a porous separation device including boundary walls fixedly attached to a base.

Figures 6A-6B are top and bottom perspective views of a separation hole structure and separation seal. Figure 6C is a side view of the separation hole structure and separation seal of Figures 6A-6B.

7A-7B are perspective and top views of a concentrated hole structure.

8A-8B are close-up views from the top and side, respectively, of another embodiment of a concentrated hole structure.

9 is a perspective view of a boundary wall, a concentrating pore structure, a substrate, and a substrate support.

10A-10B are top and bottom perspective views, respectively, of an agent delivery device.

11 is a perspective view of the reagent delivery device of FIGS. 10A-10B being inserted into the porous separation device of FIGS. 1A-1B .

12A-12H are perspective views of a closed tip of a protrusion of an agent delivery device.

13 is a perspective view of a porous separation device.

14A-14B are perspective views of portions of a porous separation device.

15A-15B are side views of a porous separation device with a separation hole clip in a first configuration and a second configuration, respectively.

16A-16B are perspective views of different embodiments of porous separation devices in which a substrate support and separation wall structure are irreversibly coupled to a boundary wall.

17A-17B are side views of a porous separation device in a first configuration and a second configuration, respectively, wherein a substrate support is attached to a boundary wall by a hinge.

18 is a perspective view of an agent delivery device.

19A-19B are perspective views of the reagent delivery device of FIG. 18 partially and fully inserted into a porous separation device, respectively.

Figure 20A is a perspective view of a motor unit. Figure 20B is a perspective view of the motor unit of Figure 20A with the reagent delivery device of Figure 18. Figure 20C is a perspective view of the motor unit of Figure 20A with the reagent delivery device and porous separation device of Figure 18, respectively.

Figure 21A is a perspective view of a substrate holder and boundary seal. Figure 21B is a perspective view of the substrate holder and boundary seal of Figure 21A coupled to a boundary wall and substrate. Figure 21C is a bottom perspective view of the boundary wall of Figure 21B alone. Figure 21D is a bottom perspective view of the boundary wall and substrate of Figure 21B.

Figure 22 is a perspective view of a porous separation device and adapter.

23A and 23B are perspective and top views, respectively, of a reagent loading device corresponding to the porous separation device and adapter of FIG. 22 .

24A-24B are close-up perspective views of portions of a reagent loading device and a porous separation device and an adapter or separate adapters.

25A-25B are close-up top views of portions of the reagent loading device and adapter of FIG. 22 , with the reagent loading device correctly oriented ( FIG. 25A ) and incorrectly oriented ( FIG. 25B ).

26A-26B show examples of orientation wedges for adapters.

Figure 27 shows a top view of another porous separation device and adapter.

28A-28B are perspective views of the reagent loading device relative to the assembled porous separation device and adapter of FIG. 27 before being lowered and fully lowered, respectively.

29 is a top view of another porous separation device with an intact orienting wedge.

30A-30B are perspective views of the reagent loading device partially and fully lowered relative to the porous separation device of FIG. 29 , respectively.

Figure 31A shows a perspective view of a 96-well plate and an adapter. Figures 31B-31C show perspective views of a reagent loading device inserted into the plate and adapter of Figure 31A. Figure 31D shows a bottom view of a reagent loading device inserted into the plate and adapter.

32 shows a perspective view of another motor unit having the reagent delivery device of FIGS. 22 to 25A-25B and a porous separation device and adapter.

33A-33B show perspective views of the motor unit without the reagent delivery device and the porous separation device and adapter.

Detailed Description of the Invention

The porous separation devices described herein allow a composition comprising a target agent to be separated into multiple wells; to be redistributed and recombined into a single well; and/or to be re-separated into wells of the same or a different configuration. This is achieved by having a separation wall structure—unlike current multi-well plates having fixed walls—that is reversibly removable from a volume containing a composition comprising a target agent. As such, the device can allow for reversible and repeatable separation and combination of volumes without the need to transfer the composition from one well to another via a pipette or the like.

The reagent loading device described herein allows one or more test agents to be delivered to multiple volumes simultaneously, without the need for one or more test agents to be delivered to each of multiple volumes separately. This is achieved by the reagent loading device with a plurality of projections, each of which comprises a rod and a closed tip that is suitable for keeping reagent. As such, closed tip all can be loaded with reagent in the configuration corresponding to the desired delivery configuration of a plurality of volumes. The reagent loading device can be configured to promote the mixing of reagent and a plurality of volumes, as by configuring to vibrate, and can comprise a containment element that is configured to protect the reagent that is loaded on the closed tip.

The reagent loading device can be configured to deliver the test agent to each individual hole of the porous separation device simultaneously. Together, these devices allow high-throughput parallel processing without the need for repeated pipetting or liquid handling robots. However, it should also be understood that the device can be used alone. This article also describes test kits and systems for chemical or biological analysis, as well as methods using the porous separation device and reagent loading device described herein.

Porous separation device

FIG1A illustrates a perspective view of an embodiment of an assembled porous separation device 100. FIG1B illustrates an exploded perspective view of a porous separation device 100. Typically, the porous separation device 100 may include a cavity 450 and a removable separation pore structure 602, which may be matched in the cavity 450. The composition (e.g., a cell suspension) in the cavity 450 may be reversibly separated into different holes by arranging the separation pore structure 602 in the cavity 450, as described in more detail below. Typically, the cavity 450 may be formed by a boundary wall 202 and a substrate 302, which may be coupled together to form the cavity 450 by a substrate support 402 in some variations, or may be fixedly attached to each other or integrated with each other in other variations. Each of these elements will be described in more detail below.

Boundary wall

Boundary wall 202 can form a side portion of cavity 450. In the variation shown in Figures 2A and 2B, boundary wall 202 can include four orthogonal parts - a first part 202a, a second part 202b, a third part 202c, and a fourth part 202d. These four parts can define a rectangular area. In some variations, the four orthogonal parts can be an integral component. However, in other variations, the four orthogonal parts can include more than one component (e.g., two, three, four or more), which can be attached in any suitable manner (e.g., using an adhesive (glue, adhesive polymer, and the like), welding, mechanical fasteners, chemical bonding, a combination of these methods, or similar methods).

However, it should be understood that the boundary wall 202 need not define a rectangular area, and further, it need not include four sections. In some variations, for example, the boundary wall 202 may define any polygon (e.g., a triangle, a quadrilateral (e.g., a parallelogram, a trapezoid), a pentagon, a hexagon, etc.). It should be understood that the boundary wall 202 need not be generally planar but may be curved to define an area having a curved shape (e.g., a circle, an ellipse, an oval, a ring, a circular segment, etc.). In some variations, the boundary wall 202 may include fewer than four sections (e.g., one, two, or three sections) or more than four sections (e.g., five, six, seven, eight, or more sections). The boundary wall 202 may also define more than one area. For example, in some variations, the boundary wall 202 may include a fifth section that may be attached to an opposing section of the boundary wall (e.g., on the first end of the first section 202a and on the second end of the third section 202c). In such variations, the boundary wall 202 may define two rectangular areas.

base

The substrate 302 can form the bottom of the cavity 450. Therefore, the substrate 302 can serve as the bottom of the composition (e.g., cell suspension) placed in the cavity 450. In the case where the composition placed in the cavity 450 includes a fluid target agent, such as a cell, the substrate 302 can serve as a surface on which the target agent can be deposited. Once deposited on the near surface 304 of the substrate 302, the substrate 302 can be suitable for keeping the target agent in a fixed position, as described in more detail below. In some cases, the substrate 302 can be configured to allow the target agent to be located on the substrate 302 in a substantially uniform layer.

As shown in Figure 3, substrate 302 may include a substantially flat surface. The substrate may include any suitable material, such as, but not limited to, glass, plastic, silicon, ceramic, metal, a combination of these materials, or similar materials. In some variations, the substrate may include a suitable, generally available laboratory equipment, such as, but not limited to, a glass slide or a cover slip. The size of substrate 302 may be appropriately engaged with boundary wall 202. That is, substrate 302 may have a cross-sectional shape that is approximately the same size as the cross-sectional shape of boundary wall 202 or larger than the cross-sectional shape of boundary wall 202. In the variation shown in Figure 3, substrate 302 may include a substantially flat rectangle with the same cross-sectional shape as boundary wall 202, so that the outer edge of substrate 302 is flush with the outer surface of boundary wall 202 (as shown in Figure 2A).

In some variations, the substrate may include a coating on its proximal surface. For example, the substrate may include a coating, such as, but not limited to, a coating comprising one or more compounds, proteins, gels (e.g., hydrogels), polymers, copolymers, immobilized cells, microorganisms, conductive surfaces, or the like. As an example, the coating may include a gel containing a growth medium (e.g., an agar gel). In some variations where the coating comprises a gel, a liquid may be loaded into a cavity and subsequently solidified to polymerize into a gel. In some variations where the substrate comprises a coating, the coating may be covalently bound by a chemical crosslinker. For example, in variations where the substrate comprises glass or silicon, the substrate may be covalently bound to a silane, which in turn binds to a coating comprising one or more compounds, proteins, gels, or polymers. In some variations, a metal coating may be deposited by vaporization. In some variations, the coating pattern may be produced by microfabrication techniques such as microprinting and photolithography. In some variations, the coating may improve the suitability of the substrate for holding a target agent in a fixed position. In other variations, the coating may assist in detection, dielectrophoresis, migration studies, chemotaxis (through the passage between pores). In some variations, the substrate may include microfluidics or electrodes.

Boundary sealing

The porous separation device 100 can further include a boundary seal 204. When the boundary wall 202 and the base 302 are coupled, the boundary seal 204 can form a leak-proof seal between the boundary wall 202 and the base 302 (described in more detail below). The boundary seal 204 can include any material suitable for forming a seal, such as, but not limited to, rubber, plastic, or a polymer. The boundary seal 204 can include a thin strip of such material having a shape corresponding to the distal side 206 of the boundary wall 202.

In some variations, when the boundary wall 202 and the substrate 302 are coupled, the boundary seal 204 may be located between the boundary wall 202 and the substrate 302. In some of these variations, the boundary seal 204 may be fixed to the distal side 206 of the boundary wall 202. In these variations, the boundary seal 204 can be fixed to the distal side 206 in any suitable manner, such as, but not limited to, an adhesive (glue, adhesive polymer, and the like), a chemical bond, or the like. In these variations, the fixation of the boundary seal 204 to the distal side 206 of the boundary wall 202 can generate a leakproof seal between the boundary seal 204 and the boundary wall 202, while the compressive force (described below) between the boundary wall 202 and the substrate 302 can press the boundary seal 204 and the substrate 302 together to generate a leakproof seal. In other variations, the boundary seal 202 can also be fixed to the proximal surface 304 of the substrate 302 in any suitable manner. In these variations, the fixation of the boundary seal 204 to the proximal surface 304 of the substrate 302 can create a leak-proof seal between the boundary seal 204 and the substrate 302, while the compressive force between the boundary wall 202 and the substrate 302 (described below) can press the boundary seal 204 and the boundary wall 202 together, which can create a leak-proof seal. In still other variations, when the boundary wall 202 and the substrate 302 are coupled, the boundary seal 204 may not be fixed to the boundary wall 202 or the substrate 302, but instead may be sandwiched between the boundary wall 202 and the substrate 302 by the compressive force (described in more detail below). In still other variations in which the boundary wall 202 is fixedly attached to the substrate 302 (described in more detail below), the boundary seal 204 may be fixed to both the proximal surface 304 of the substrate 302 and the distal side 206 of the boundary wall 202.

In other variations, the boundary seal may be located between the substrate holder and the substrate. Examples of such variations are shown in Figure 21A, in which boundary seal 2104 may be located on the near side of substrate holder 2404. In these variations, boundary seal 2104 may be secured to the near side of substrate holder 2404 (e.g., by pre-fixing (pre-secured) or casting), but not necessarily so. In these variations in which boundary seal 2104 is located on the near side of substrate holder 2404, boundary wall 2202 and substrate 2304 may be placed on top of boundary seal 2014, as shown in Figure 21B. More specifically, boundary wall 2202 (as shown in the bottom perspective view in Figure 21C) may include a recessed area 2406 along the far side of each portion of boundary wall 2202. The recessed area 2406 can be configured to hold the substrate 2304 so that when the substrate 2304 is placed in the recessed area 2406, the distal surface of the substrate 2304 and the distal surface of the boundary wall 2202 are horizontal (as shown in the bottom perspective view in Figure 21D). As shown in Figure 21B, when the boundary wall 2202 and substrate 2304 are placed on the boundary seal 2104 and substrate support 2404, the boundary seal 2104 can seal any space between the substrate 2304 and the boundary wall 2202 by the compressive force between the boundary wall and substrate support when they are coupled (as described in more detail below). In these variations, the location of the boundary seal between the substrate support and the substrate (e.g., the distal end of the substrate) rather than between the substrate and the boundary wall can facilitate a larger working area on the near surface of the substrate (e.g., the surface located in the levitation chamber).

However, it should be understood that the porous separation device described herein does not need to include boundary seals. For example, in the absence of boundary seals, if the boundary wall and substrate are configured to form a cavity in which a composition (e.g., a cell suspension) can be appropriately maintained without leakage, boundary seals may be unnecessary. For example, in a variation in which the boundary wall is fixedly attached to the substrate or is integrated with the substrate, the porous separation device may not include boundary seals. As another example, in a variation in which the boundary wall and substrate are not fixedly attached or are not integrated but are configured to form a leakproof seal, the porous separation device may not include boundary seals. This may be the case, for example, if the boundary wall comprises a material such as rubber, plastic, or polymer that can form a seal with the material of the substrate. In these cases, the compression force that presses the boundary wall and substrate together can directly generate a leakproof seal between the boundary wall and the substrate. Still in other variations, if the intended cavity maintains a gel, solid, or the like that does not require a tight seal to prevent leakage, boundary seals may be unnecessary.

Substrate support

The substrate 302 can be coupled to the boundary wall 202 via a substrate support 402, which is illustrated in FIG3 with the substrate 302 and separately in FIG4A. The substrate support can also be configured to couple the separation pore structure 602 to the boundary wall 202, as described in more detail below. The substrate support 402 can have any design suitable for coupling the boundary wall 202 and the substrate 302. In some variations, the substrate support 402 can reversibly couple the boundary wall 202 and the substrate 302; in other variations, the substrate support 302 can irreversibly couple the boundary wall 202 and the substrate 302.

To couple the substrate 302 and the boundary wall 202, the substrate support may include a first portion configured to exert a proximal force on the substrate 302 and a second portion configured to exert a distal force on the boundary wall 202, thereby generating a compressive force that presses the boundary wall 202 and the substrate 302 toward each other. In the embodiment shown in Figures 4A-4B, the substrate support 402 may include a frame 404 configured to exert a proximal force on the substrate. The configuration of the frame 404 may correspond to the configuration of the boundary wall 202; that is, the cross-sectional shape of the frame 404 may be substantially the same as the cross-sectional shape of the boundary wall 202 so that the substrate support 402 can couple to the boundary wall 202. The frame 404 may include four orthogonal portions—a first portion 404a, a second portion 404b, a third portion 404c, and a fourth portion 404d. These four portions may define a rectangular area having the same cross-sectional area as the rectangular area defined by the four portions of the boundary wall 202 described above. It should be understood that in order to correspond to the configuration of the boundary wall 202, the base support 402 may have more or fewer parts (e.g., one, two, three, five, six, seven, eight or more), as described above relative to the boundary wall 202, and the parts do not need to be orthogonal to each other, and the parts do not need to be straight and can be curved.

The frame 404 can include features configured to engage the substrate 302, which can help the substrate holder 402 retain the substrate 302. As shown in FIG4A , in some variations, this feature can include a recessed area 406 along each portion of the frame 404. The recessed area 406 can define a lip 414 around the outer edge of the frame. The lip 414 can help retain the substrate 302 within the frame 404 by preventing lateral movement of the substrate 302 relative to the frame 404.

In some variations, the second portion of the base support 402, configured to exert a distal force on the boundary wall 202, can include a boundary wall clip 408. The boundary wall clip 408 can be configured to couple the base support 402 to the boundary wall 202 by engaging a portion of the boundary wall 202. In the variation shown in Figures 3 and 4A-4B, the boundary wall clip 408 can include an elongated portion 410 and a joint 412. The elongated portion 410 can extend proximally from the frame 404 and can have a generally flat shape, while the joint 412 can be located at the proximal end of the elongated portion 410 and can have an outwardly facing triangular shape, as shown in Figure 4B.

The boundary wall latch 408 can be configured to engage with a portion of the boundary wall 202. As shown in Figures 2A-2B, the boundary wall 202 can include two locking strips 208. The locking strips 208 can include both a substrate support lock 210 and a separation hole lock 212. The locking strips 208 can extend laterally from the outer surface of a first portion of the boundary wall 202a and from a third portion of the boundary wall 202c. In some variations, the locking strips can be integral with the boundary wall 202, or in other variations, they can be attached to the boundary wall 202 in any suitable manner. In the illustrated embodiment, the substrate support lock 210 and the separation hole lock 212 can include openings formed between the boundary wall 202 and the locking strips 208, which are configured to engage with the substrate support 402 and the separation hole structure 602, respectively. More specifically, the elongated portion 410 of the boundary wall clip 408 can fit within the opening of the substrate holder lock 210 between the boundary wall 202 and the locking strip 208, while the tab 412 of the boundary wall clip 408 can hook onto the proximal surface of the locking strip 208, as shown in FIG2A . The engagement between the distal surface 414 (see FIG4B ) of the tab 412 and the proximal surface of the locking strip 208 can resist distal movement of the substrate holder 402 relative to the boundary wall 202, thereby coupling the boundary wall 202 and the substrate holder 402 together. Conversely, in those variations having a boundary seal 204, in addition to sandwiching the boundary seal 204 between the boundary wall 202 and the substrate 302, the substrate 302 can also be sandwiched between the boundary wall 202 and the substrate holder 402.

While the connector 412 is shown as having a triangular shape, it should be understood that the connector 412 may have other suitable shapes. Furthermore, while the variation shown in Figures 3 and 4A-4B includes four boundary wall clips 408 (two on opposite sides of the boundary wall 202), it should be understood that the substrate support 402 may have any suitable number of boundary wall clips 408 (e.g., one, two, three, four, five, six, seven, eight, or more), and the boundary wall 202 may have any suitable number of corresponding substrate support locks 210. It should also be understood that the number of boundary wall clips 408 on the substrate support 402 need not match the number of substrate support locks 210 on the boundary wall 202, provided that the configuration allows the substrate support 402 to couple with the boundary wall 202. It should also be understood that the boundary wall clips 408 (and the corresponding substrate support locks 210 on the locking strip 208) may have different arrangements on the frame 404 of the substrate support 402.

It should be understood that the substrate support may have other designs. The design should be able to generate sufficient compressive force between the boundary wall and the substrate to generate a leak-proof seal of the cavity. Another embodiment of a porous separation device 1300 with a boundary wall 1302, a substrate support 1304, and a separation pore structure 1306 is shown in Figure 13. The substrate support 1304 may include a frame 1308 configured to apply a proximal force on the substrate. The configuration of the frame 1308 may be such that it corresponds to the configuration of the boundary wall 1302; that is, the cross-sectional shape of the frame 1308 may be substantially the same as the cross-sectional shape of the boundary wall 1302 (which may have features similar to the boundary wall 202 of the porous separation device 100 described in detail above) so that the substrate support 1304 can be coupled to the boundary wall 1302. The frame 1308 may have a design similar to the frame 404 of the porous separation device 100 described in detail above, including its component parts and/or components configured to engage with the substrate.

As with the substrate support 402 described above, the substrate support 1304 may include a portion configured to exert a distal force on the boundary wall 1302, which, in some variations, may include a boundary wall clip 1310. The boundary wall clip 1310 may be configured to couple the substrate support 1304 to the boundary wall 1302 by engaging a portion of the boundary wall 1302. In the variation shown in FIG13 , the boundary wall clip 1310 may have a T-shaped structure including a vertical portion 1312 and a horizontal portion 1314. The vertical portion 1312 may extend from the proximal side of the frame 1308 and may have a generally flat shape. The horizontal portion 1314 may be located proximal to the vertical portion 1314 and may have a generally flat shape, extending outwardly beyond the lateral edges of the vertical portion 1312.

Boundary wall clips 1310 can be configured to engage with portions of boundary wall 1302. Boundary wall 1302 can include a base support latch 1316 corresponding to each boundary wall clip 1310. As shown in FIG. 13 , base support latches 1316 can each include two projections 1318. The two projections 1318 can have a triangular shape extending outward from boundary wall 1302, with the triangular shape oriented so that the projections are smallest at the distal end of the projections and largest at the proximal end of the projections. The two projections 1318 can be spaced apart by a distance that is greater than the width of the vertical portion 1312 of boundary wall clip 1310 but less than the width of the horizontal portion 1314 of boundary wall clip 1310. As such, both projections 1318 can form a proximal horizontal surface 1320 configured to engage with the distal horizontal surface of the horizontal portion 1314 of boundary wall clip 1310. As described in more detail above with respect to the porous separation device 100, in those variations having a boundary seal, in addition to sandwiching the boundary seal between the boundary wall 1302 and the substrate, the engagement between the proximal horizontal surface 1320 of the protrusion 1318 and the distal horizontal surface of the horizontal portion 1314 of the boundary wall clip 1310 can also resist distal movement of the substrate support 1304 relative to the boundary wall 1302. The vertical portion 1312 of the boundary wall clip 1310 can be configured to flex outwardly, thereby allowing the substrate support 1304 to be attached to the boundary wall 1302, as described in more detail below.

13 includes four boundary wall clips 1310 (two on opposite sides of the boundary wall 1302), it should be understood that the substrate support 1304 can have any suitable number of boundary wall clips 1301 (e.g., one, two, three, four, five, six, seven, eight, or more), and the boundary wall 1302 can have any suitable number of corresponding substrate support locks 1316. It should also be understood that the number of boundary wall clips 1310 on the substrate support 1304 need not match the number of substrate support locks 1316 on the boundary wall 1302, provided that the configuration enables the substrate support 1304 to be coupled to the boundary wall 1302. It should also be understood that the boundary wall clips 1310 (and the corresponding substrate support locks 1316 on the boundary wall 1302) can have different arrangements on the frame 1308 of the substrate support 1304.

In the variations shown in Figures 3 and 4A-4B and Figure 13, the substrate support can reversibly couple the boundary wall to the substrate. However, it should be understood that in other variations, the substrate support can irreversibly couple the boundary wall to the substrate. That is, once the substrate support couples the boundary wall to the substrate, they can resist decoupling (i.e., they cannot be decoupled without compromising the structural integrity of the component). In one such variation, it may be necessary to separate all or part of the boundary wall snaps (brokenoff) in order to decouple the substrate support from the boundary wall, which may result in irreversible decoupling. Figure 16A shows such a variation, in which substrate support 1602 irreversibly couples boundary wall 1604 to substrate 1606. Boundary wall snaps 1608 can be configured to engage with boundary wall 1604 in a manner similar to that described above with respect to Figure 13. Boundary wall 1604 may have components similar to boundary wall 1302 of Figure 13. That is, the boundary wall 1604 may include a base support lock 1616 corresponding to each boundary wall catch 1608. As shown in Figure 16A, the base support locks 1616 may each include two protrusions 1618. The two protrusions 1618 may have a triangular shape extending outward from the boundary wall 1604, wherein the triangular shape is oriented so that the protrusions are smallest at the distal end of the protrusions and largest at the proximal end of the protrusions.

The boundary wall clip 1608 may include an elongated portion 1610 and a curved portion 1612, with a horizontal portion 1614 therebetween, and may have a width greater than either the elongated portion 1610 or the curved portion 1612. The two protrusions 1618 of the base support lock 1616 may be spaced apart by a distance that is greater than the width of the elongated portion 1610 of the boundary wall clip 1608 but less than the width of the horizontal portion 1614 of the boundary wall clip 1608. As such, both protrusions 1618 may form a proximal horizontal surface configured to engage with a distal horizontal surface of the horizontal portion 1614 of the boundary wall clip 1608. In those variations having a boundary seal, in addition to sandwiching the boundary seal between the boundary wall 1604 and the substrate, or between the substrate and the substrate support 1602, the engagement between the proximal horizontal surface of the protrusion 1618 and the distal horizontal surface of the horizontal portion 1614 of the boundary wall clip 1608 can resist distal movement of the substrate support 1602 relative to the boundary wall 1604. Once the boundary wall clip 1608 engages the boundary wall 1604, the curved portion 1612 must be broken off in order for the boundary wall 1604 to be removed from the substrate 1606. As such, after the curved portion 1612 is broken off, the boundary wall clip 1608 can no longer couple the boundary wall 1604 to the substrate 1606. Thus, the boundary wall 1604 and the substrate support 1602 can only be irreversibly decoupled.

FIG16B shows another such variation, in which a base support 1652 irreversibly couples a boundary wall 1654 to a base 1656. Boundary wall latches 1658 can be configured to engage boundary wall 1654 in a manner similar to that described above with respect to FIG13. Boundary wall 1654 can have features similar to boundary wall 1604 of FIG16A. That is, boundary wall 1654 can include a base support latch 1666 corresponding to each boundary wall latch 1658. As shown in FIG16B, base support latches 1666 can each include two protrusions 1668. Both protrusions 1668 can have a triangular shape extending outward from boundary wall 1654, wherein the triangular shape is oriented so that the protrusions are smallest at the distal end of the protrusions and largest at the proximal end of the protrusions, or the protrusions 1668 can have other shapes, such as a rectangular shape.

Boundary wall latch 1658 may include an elongated portion 1660 and a vertical portion 1662, with a horizontal portion 1664 therebetween having a greater width than either elongated portion 1660 or vertical portion 1662. Vertical portion 1662 may be perpendicular to elongated portion 1660 and may extend vertically outward from boundary wall 1654 when boundary wall 1654 is coupled to base support 1652. Two protrusions 1668 of base support latch 1666 may be spaced apart by a distance that is greater than the width of elongated portion 1660 of boundary wall latch 1658 but less than the width of horizontal portion 1664 of boundary wall latch 1658. As such, both protrusions 1668 may form a proximal horizontal surface configured to engage a distal horizontal surface of horizontal portion 1664 of boundary wall latch 1658. In those variations having a boundary seal, in addition to sandwiching the boundary seal between the boundary wall 1654 and the substrate, or between the substrate and the substrate support 1652, the engagement between the proximal horizontal surface of the protrusion 1668 and the distal horizontal surface of the horizontal portion 1664 of the boundary wall clip 1658 can resist distal movement of the substrate support 1652 relative to the boundary wall 1654. Once the boundary wall clip 1658 engages the boundary wall 1654, the vertical portion 1662 must be broken off in order to remove the boundary wall 1654 from the substrate 1656. As such, after the vertical portion 1662 is broken off, the boundary wall clip 1658 can no longer couple the boundary wall 1654 to the substrate 1656. Thus, the boundary wall 1654 and the substrate support 1652 can only be irreversibly decoupled. In the variation shown in Figures 16A-16B, the separation hole structure can also be irreversibly coupled to the boundary wall using separation hole clips and locks having similar structures to the boundary wall clips and locks in Figures 16A-16B, wherein after the separation hole clips have been coupled to the boundary wall, portions of the separation hole clips must be removed in order to decouple the separation hole structure.

In other variations, the substrate support can be attached to the boundary wall by a hinge. The hinge can connect the near surface of one side of the substrate support to the far surface of the corresponding side of the boundary wall. In order to form a cavity, the substrate support and the boundary wall can rotate relative to each other around the hinge so that the sides are relative to each other and the hinge. Subsequently, the sides opposite to the hinge can be coupled, for example, using snaps and locks similar to those described above. Before the substrate support is coupled to the boundary wall, the substrate can be placed between the substrate support and the boundary wall to sandwich it between the substrate support and the boundary wall. Figures 17A-17B show examples of such variations with boundary wall snaps 1708 similar to the boundary wall snaps 1608 of the substrate support 1602 of Figure 16A. As shown, the substrate support 1702 can be attached to one side of the boundary wall 1704 by a hinge 1714. The substrate support 1702 can be rotated about the hinge 1714 from a first position ( FIG. 17A ) to a second position ( FIG. 17B ) to sandwich the substrate between the substrate support 1702 and the boundary wall 1704 .

Although the embodiment of the porous separation device 100 shown in Figures 1-4 has a boundary wall 202 separated from the substrate 302, in other embodiments of the porous separation device, the boundary wall may not be separated from the substrate. In some of these variations, the boundary wall may be fixedly attached to the substrate; in other of these variations, the boundary wall may be integrated with the substrate. For example, Figure 5 illustrates a cavity 502 of a porous separation device 500 including a boundary wall 504 fixedly attached to a substrate 506. That is, in the embodiment of Figure 5, the boundary wall 504 is combined with the substrate 506, rather than being coupled by a substrate support or the like. The boundary wall 504 may be combined with the substrate 506 directly or indirectly. In a variation in which the boundary wall 504 is indirectly combined with the substrate, the distal side 508 of the boundary wall may be combined with a boundary seal (not shown), which in turn may be combined with the substrate 506. In a variation in which the boundary wall 504 is directly combined with the substrate 506, the porous separation device 500 does not need to include a boundary seal. In variations where the boundary wall 504 is directly bonded to the substrate 506, the boundary wall 504 can be bonded in any suitable manner, such as, but not limited to, an adhesive ring (e.g., silicone glue) that bonds the distal side of the boundary wall 504 to the proximal side of the substrate 506, or a cast polymer material (e.g., hard rubber) on the boundary wall 504 that can bond to the substrate 502. In other variations, the boundary wall can be integral with the substrate. That is, the boundary wall and the substrate can be integrally formed from the same material, such as, but not limited to, a polyacrylic, polyurethane, or polycarbonate material, or the like.

cavity

Returning to the embodiment of the porous separation device 100 of Figures 1-4, when coupled, the substrate 302 and the boundary wall 202 can form a cavity 450. The cavity 450 can provide an area configured to hold a composition, such as a cell suspension, a gel, a pre-gel solution (which can subsequently solidify into a polymeric gel), a powder, or the like. It should be understood that, as described above, similar cavities can be formed by the boundary wall and substrate in embodiments in which the boundary wall is fixedly attached to the substrate or integral with the substrate, such as the cavity 502 formed by the boundary wall 504 and substrate 302 described above and shown in Figure 5.

Although FIG2A shows that the coupled boundary wall 202 and base 302 form a single integral cavity 450, in other variations, the coupled boundary wall and base can form a cavity having more than one region. For example, in the variation described above in which the boundary wall includes a fifth portion, the fifth portion can be attached to opposing portions of the boundary wall (e.g., on the first end of the first portion 202a and on the second end of the third portion 202c), the cavity can include two rectangular regions. The two regions can be separated by the boundary wall so that a composition located in one region will not be able to travel to the other region.

The cavity 450—and accordingly, the components forming it—can have any suitable dimensions. In some variations, the multi-porous separation device 100 can be configured to use a standard glass slide as the substrate 302, and thus, the length and width of the substrate 302, the boundary wall 202, and the substrate support 402 can be approximately 75 mm by 25 mm, respectively. In other variations, the multi-porous separation device 100 can be configured to approximate a standard 96-well plate, and thus, the maximum length and width of the substrate 302, the boundary wall 202, and the substrate support 402 can be less than approximately 13 cm, or approximately 130 mm by 85 mm. In some variations, the depth of cavity 450 (and therefore the approximate height of boundary wall 202) may be about 1 mm, about 3 mm, about 5 mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, about 15 mm, about 17 mm, about 19 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, or greater than about 40 mm.

Separation pore structure

As briefly described above, the porous separation device 100 may include a separation pore structure 602 that can be placed in the cavity 450. Fig. 6 A example is configured to be positioned at this type of separation pore structure 602 in the cavity 450. When the separation pore structure 602 is placed in the cavity 450, the separation pore structure 602 can divide the composition (for example, cell suspension) in the cavity 450 into the separated region. Compared with the traditional way of dividing the composition into the separated region (such as moving into a separate hole individually, relative to the one-time transfer of the composition into the cavity, it may need to repeatedly transfer the composition), this way of dividing the composition into the separated region can be simpler and/or more effective. Once the contents are divided into the separated region, it can allow the separated region to undergo different treatments (for example, different reagents are introduced into each region), as described in more detail below. In addition, the separation pore structure also can be removed from the cavity, and it can allow the contents of the separated region to be changed in batches. When it is desired to perform the same treatment (eg, a cleaning step) on each of the separate regions, it is easier and more efficient than performing the treatment on each of the separate regions individually.

As shown in Figure 6A, the separation hole structure 602 may include a plurality of separation walls 604. In some variations, the separation walls 604 may include two sets of orthogonal parallel walls and may form a grid-like or lattice-like structure so that they form an array or matrix of openings 606 separated by the separation walls 604. When the separation hole structure 602 is coupled in the cavity 450, it can separate the cavity 450 into a plurality of separation holes 610. The separation walls 604 can form the sidewalls of the separation holes 610, while the substrate 302 can form the bottom of the separation holes 610. The contents of the cavity 450 can be separated and divided into the separation holes 610. The separation hole structure 602 may include any suitable material or materials, such as, but not limited to, rubber, plastic, silicon, ceramic, metal, polymer, glass, or the like.

6A , the separator wall 604 has approximately the same height as the boundary wall 202 (e.g., in some variations, about 1 mm, about 3 mm, about 5 mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, about 15 mm, about 17 mm, about 19 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, or more), it should be understood that the separator wall 604 can have a height less than the boundary wall 202, as long as the height of the separator wall 604 (i.e., the depth of the separation hole) is greater than the depth of the composition in the cavity 450 when the separator wall 604 is coupled in the cavity, so as to prevent the composition from flowing across the separator wall 604 between the separation holes 610. It should also be understood that the separator wall 604 can have a greater height than the boundary wall 202. 6A-6B , the separation pore structure 602 substantially fills the cavity 450 (i.e., the cross-sectional dimensions of the separation pore structure 602 are substantially the same as the cross-sectional dimensions of the cavity 450), the separation pore structure need not fill the cavity 450. For example, the separation pore structure 602 may have a smaller cross-sectional area than the cavity 450 and, therefore, may only partially subdivide the suspension chamber 450 into the separation pores 610.

The opening 606 can have any suitable cross-sectional area. In some variations, the maximum dimension of the cross-section of the opening can be from about 1 μm to about 20 μm, from about 20 μm to about 40 μm, from about 40 μm to about 60 μm, from about 60 μm to about 80 μm, from about 80 μm to about 100 μm, from about 100 μm to about 200 μm, from about 200 μm to about 400 μm, from about 400 μm to about 600 μm, from about 600 μm to about 800 μm, from about 800 μm to about 1 mm, from about 1 mm to about 2 mm, from about 2 mm to about 4 mm, from about 4 mm to about 6 mm, from about 6 mm to about 8 mm, from about 8 mm to about 1 cm, greater than about 1 cm, from about 1 μm to about 1 cm, from about 100 μm to about 1 mm, or from about 1 mm to about 1 cm. However, it should be appreciated that in some variations, a specific ratio between the height and cross-sectional area of the opening 606 may be desirable to combat capillary effects. The resulting separation pore 610 can have any suitable volume, such as, but not limited to, less than about 100 μL, about 100 μL to about 200 μL, about 200 μL to about 400 μL, about 400 μL to about 600 μL, about 600 μL to about 800 μL, about 800 μL to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about 20 mL, about 20 mL to about 40 mL, about 40 mL to about 60 mL, about 60 mL to about 80 mL, about 80 mL to about 100 mL, more than about 100 mL, about 100 μL to about 100 mL, or about 1 mL to about 10 mL.

Although the openings 606 shown in Figures 6A-6B have a square cross-sectional shape, the openings 606 may have any suitable shape, such as, but not limited to, a triangle, a rectangle, any other quadrilateral (parallelogram, trapezoid, etc.), a pentagon, a hexagon, any circular shape (circular, elliptical, oval, etc.), or an irregular shape. Furthermore, although the openings 606 shown have equal cross-sectional dimensions, it should be understood that the openings 606 do not need to have the same size or shape. Furthermore, the opening cross-sections of the openings 606 do not need to be the same throughout. For example, in some variations, the cross-sectional area of each opening 606 may be larger at its proximal end than at its distal end; in other variations, the cross-sectional area of each opening may be larger at its distal end than at its proximal end. It should be understood that in these cases, the thickness of the separating wall 604 may be varied accordingly to generate a variable cross-sectional area (for example, for the previous example, the separating wall 604 may be thicker at the distal end than at the proximal end, and for the latter example, the thickness at the proximal end may be thicker at the distal end than at the distal end).

6A-6B , the separation hole structure 602 may define 64 openings 606 and, therefore, when coupled within the cavity 450, the separation hole structure 602 may divide the cavity 450 into 64 separation holes 610. However, it should be understood that the separation hole structure 602 may define any number of openings 606 and, therefore, when coupled to the cavity 450, the separation hole structure 602 may divide the cavity 450 into any number of separation holes 610. For example, the separation hole structure 602 may define at least about 6, at least about 12, at least about 24, at least about 48, at least about 96, at least about 384, at least about 480, at least about 1536, at least about 3456, or more openings 606, and thus, when coupled with the cavity 450, may divide the cavity 450 into at least about 6, at least about 12, at least about 24, at least about 48, at least about 96, at least about 384, at least about 480, at least about 1536, at least about 3456, or more separation holes 610. It should be understood that the number of openings 606 and separation holes 610 should not be limited to the numbers listed herein. While in some variations the separation well structure 602 may define the number of openings 606 found in a standard microtiter laboratory plate, in other variations the separation well structure 602 may define a non-standard number of openings 606, which may or may not be a rectangular number.

In some variations, it may be desirable to maximize the number of the separation holes 610 in a given cross-sectional area (e.g., the cross-sectional area of the cavity 450). For this reason, it may be desirable to minimize the thickness of the separating wall 604. In some variations, the thickness of the separating wall may be from about 50 μm to about 2000 μm. In some of these variations, the thickness of the separating wall may be from about 200 μm to about 1800 μm. In some of these variations, the thickness of the separating wall may be from about 400 μm to about 1600 μm. In some of these variations, the thickness of the separating wall may be from about 600 μm to about 1400 μm. In some of these variations, the thickness of the separating wall may be from about 800 μm to about 1400 μm. In some of these variations, the thickness of the separating wall may be from about 1000 μm to about 1200 μm.

The porous separation device 100 may further include a second separation pore structure. The second separation pore structure may be configured to fit within one of the openings 606 of the first separation pore structure 602. Thus, the second separation pore structure may further subdivide the separation pore 610 into a plurality of smaller separation pores, wherein each of these smaller separation pores has a volume that is smaller than the volume of the separation pore 610 generated by the first separation pore structure 602. The second separation pore structure may have a similar design and function to the first separation pore structure 602, as described in detail above, and include a separation seal as described in detail below. The second separation pore structure may be coupled to the remainder of the porous separation device 100 in any suitable manner, such as, but not limited to, by a snap, friction fit, or the like connected to the first separation pore structure 602 or to the boundary wall 202. It should be understood that the porous separation device 100 may further include additional separation pore structures, such as a third, fourth, fifth, etc.

It should also be appreciated that, in some variations, the suspension cavity described herein (suspension cavity) can be used without a separation pore structure. For example, a cavity can be used to hold a gel or solid composition, and a reagent loading device as described herein can be used to deposit one or more reagents or test agents on or into a gel or solid composition. The gel or solid composition can fully minimize the migration or diffusion of the reagent or test agent so that a separation pore structure is not required. On the other hand, in some cases, when the cavity is used to hold a gel or solid composition, a separation pore structure can still be used. The separation pore structure can be inserted into a liquid composition, which can then be solidified so that the contents of each separation pore are polymerized into a gel or can be inserted into the separation pore structure when the composition is in gel form. In some cases, when the composition is in gel form, the separation pore structure can be inserted into the distal end of the separation pore structure. The distal end of the separation pore structure can include a sharp tip to help promote the gel to separate into each separation pore. In some cases, the separation pore structure can be configured to be partially inserted into the gel (i.e., so that the proximal portion of the gel is divided into a single separation pore, but the distal portion of the gel remains continuous). In other cases, the separation pore structure can be configured to be inserted so that the distal surface rests against the proximal surface of the gel or solid composition.

Separation seal

The porous separation device 100 may further include a separation seal 608. When the separation pore structure 602 and the substrate 302 are coupled, the separation seal 608 can create a leak-proof seal at the distal end of each separation pore 610. This allows each separation pore 610 to become an isolated area so that it can undergo different processes or treatments than its adjacent separation pores 610, as described in more detail below. As shown in Figures 6A-6C, when the separation pore structure 602 and the substrate 302 are coupled, the separation seal 608 can be located between the distal surface of the separation wall 604 of the separation pore structure 602 and the proximal surface 304 of the substrate 302. When the separation pore structure 602 and the substrate 302 are coupled, when the separation seal 608 is pressed against the substrate 302, the separation seal 608 can cover the bottom edge of each separation pore 610 and can provide a seal for each separation pore 610. The separation seal 608 can include any suitable material for forming a seal, such as, but not limited to, rubber, plastic, or polymer.

6A-6C , the separation seal 608 can be coupled to the distal surface of the separation wall 604 of the separation hole structure 602. The separation seal 608 can be attached to the separation hole structure 602 in any suitable manner, such as, but not limited to, an adhesive (glue, adhesive polymer, and the like), chemical bonding, or the like. However, it should be understood that in other variations, the separation seal 608 can be attached to the proximal surface 304 of the substrate 302. Still in other variations, the separation seal 608 can be attached to the boundary wall 202. In some such variations, the separation seal 608 can be integral with the boundary seal 204, or it can be attached to the boundary seal 204.

It should also be understood that the porous separation device does not need to include a separation seal. In the absence of a separation seal, if the separation pore structure and substrate are configured to form a separation pore in which compositions (for example, cell suspension) can be appropriately maintained without leakage, then a separation seal may not be necessary. For example, this may be a situation in which the separation seal comprises a material such as rubber, plastic, or polymer so as to be able to form a seal with the material of the substrate. In these cases, the compression force that presses the separation pore structure and substrate together can directly generate a leakproof seal between the separation pore structure and the substrate, without the need for an intermediate separation seal. As another example, in some (but not all) variations of the concentrated pore structure, the porous separation device may not include a separation seal, as described in more detail below. As another example, in some variations in which the porous separation device is configured to maintain a gel or solid composition, the porous separation device may not include a separation seal. However, in some of these variations, the distal edge of the separation pore structure may be thin, pointed, oblique, or similar in order to help the separation pore structure to cut through the gel or solid composition in whole or in part.

Separation hole structure accessories

As described above, the separation hole structure 602 can be configured to be coupled in the cavity 450. The separation hole structure 602 and the cavity 450 can be configured so that when coupled, sufficient compressive pressure exists between the separation hole structure 602 and the substrate 302 to form the leak-proof separation hole 610. In some variations, the separation hole structure 602 can be configured to be coupled through the boundary wall 202 in the cavity 450. In the embodiment shown in Figures 6A-6C, the separation hole structure 602 may include a separation hole clip 614 configured to be coupled to the boundary wall 202. The separation hole clip 614 may include a transverse portion 622, an elongated portion 616, and a joint 618. The transverse portion 622 may be attached to one end of the proximal surface of the separation hole structure 602 and extend laterally away from the separation hole structure 602. The elongated portion 616 may extend away from the transverse portion 622 and may have a generally flat shape, while the joint 618 may be located at the distal end of the elongated portion 616 and may have an outwardly facing triangular shape, as shown in FIG. 6C .

The split hole catch 614 can be configured to engage with a portion of the boundary wall 202. As described above with reference to Figures 2A-2B, the boundary wall 202 can include two locking strips 208, which can include a split hole lock 212. The split hole lock 212 can include an opening formed between the boundary wall 202 and the locking strips 208, which is configured to engage with the split hole catch 614 of the split hole structure 602. The elongated portion 616 of the split hole catch 614 can fit within the opening of the split hole lock 212 between the boundary wall 202 and the locking strips 208, while the tab 618 of the split hole catch 614 can hook onto the distal surface of the locking strip 208, as shown in Figure 1A. The engagement between the proximal surface 620 (see Figure 6C) of the tab 618 and the distal surface of the locking strip 208 can resist proximal movement of the split hole structure 602 relative to the boundary wall 202 (and, in turn, the coupled base support 402 and base 302). Distal pressure from the distal surface of the locking bar 208 on the proximal surface 620 of the joint 618 can generate a compressive pressure between the separation hole structure 602 and the base 302, coupling the boundary wall 202 and the separation hole structure 602 together and pressing the two together to form a leak-proof separation hole 610 (combined with the separation seal 608 and/or the central hole structure 702, described in more detail below).

6A-6C illustrate a variation that includes six split hole latches 614, it should be understood that the split hole structure 602 can have any suitable number of split hole latches 614 (e.g., one, two, three, four, five, six, seven, eight, or more), and the boundary wall 202 can have any suitable number of corresponding split hole locks 212. It should also be understood that the number of split hole latches 614 on the split hole structure 602 need not match the number of split hole locks 212 on the boundary wall 202, provided that the configuration enables the split hole structure 602 to be coupled to the boundary wall 202. It should also be understood that the split hole latches 614 (and corresponding split hole locks 212 on the boundary wall 202) can have different arrangements on the split hole structure 602.

It should also be appreciated that the separation hole structure can be coupled to the boundary wall via separation hole clips having other designs. Another embodiment of the separation hole structure 1306 is shown in Figure 13. The separation hole structure 1306 may include a separation hole clip 1322 configured to couple to the boundary wall 1302. The separation hole clip 1322 may have a T-shape, including a vertical portion 1324 and a horizontal portion 1326. The vertical portion 1324 may have a generally flat shape. The horizontal portion 1326 may be located at the proximal end of the vertical portion 1324 and may have a generally flat shape, extending outward beyond the lateral edge of the vertical portion 1324. The proximal end of the vertical portion 1324 may be attached to the separation hole structure 1306 via a transverse extender 1328, which may be attached to the vertical portion 1324 on a first end (external) and to the exterior of the sidewall of the separation hole structure 1306 on a second end (internal). In other variations, the second end may be attached to a proximal surface of the separation hole structure 1306 .

The split hole latch 1322 can be configured to engage a portion of the boundary wall 1302. The boundary wall 1302 can include a split hole lock 1330. As shown in FIG13 , each split hole lock 1330 can include two protrusions 1332. The two protrusions 1332 can have a triangular shape extending outward from the boundary wall 1302, wherein the triangular shape is oriented so that the protrusion is smallest at the distal end of the protrusion and largest at the proximal end of the protrusion. The two protrusions 1332 can be spaced apart by a distance that is greater than the width of the vertical portion 1324 of the split hole latch 1322 but less than the width of the horizontal portion 1326 of the split hole latch 1322. As such, each protrusion 1332 can form a distal horizontal surface configured to engage with a proximal horizontal surface of the horizontal portion 1326 of the split hole latch 1322. The engagement between the distal horizontal surface of the protrusion 1332 and the proximal horizontal surface of the horizontal portion 1326 of the split hole latch 1322 can resist proximal movement of the split hole structure 1306 relative to the boundary wall 1302 (and in turn, the coupled substrate support 1304 and substrate). Distal pressure on the proximal surface of the horizontal portion 1326 of the split hole latch 1322 from the split hole lock 1330 can generate a compressive pressure between the split hole structure 1306 and the substrate, coupling the boundary wall 1302 and the split hole structure 1306 together and pressing the two together to form a leak-proof split hole (in combination with a split seal and/or a central hole structure, described in more detail below).

13 includes six split hole latches 1322, it should be understood that the split hole structure 1306 can have any suitable number of split hole latches 1322 (e.g., one, two, three, four, five, six, seven, eight, or more), and the boundary wall 1302 can have any suitable number of corresponding split hole locks 1330. It should also be understood that the number of split hole locks 1330 need not match the number of split hole locks 1330 on the boundary wall 1302, provided that the configuration enables the split hole structure 1306 to be coupled to the boundary wall 1302. It should also be understood that the split hole latches 1322 (and the corresponding split hole locks 1316 on the boundary wall 1302) can have different arrangements on the split hole structure 1306.

Yet another embodiment of a split hole clip design is shown in Figures 14A-14B. A split hole structure 1402 is shown, which may include a split hole clip 1404 configured to couple with a boundary wall 1406. The split hole clip 1404 may include a horizontal portion 1408 and a vertical portion 1410, each of which may have a generally flat shape. The horizontal portion 1408 may be attached to the outside of the sidewall of the split hole structure 1402 at a first (inner) end and to the inner end of the vertical portion 1410 at a second (outer) end. In other variations, the first (inner) end may be attached to the near surface of the split hole structure 1402. The attachment joint between the horizontal portion 1408 and the vertical portion 1410 can be pivotable or rotatable so that the split hole clip 1404 can be moved from a first position, in which the horizontal portion 1408 and the vertical portion 1410 are parallel (as shown in FIG. 14A ), to a second position, in which the horizontal portion 1408 and the vertical portion 1410 are orthogonal (as shown in FIG. 14B ). When the split hole clip 1404 is in the first position, both the horizontal portion 1408 and the vertical portion 1410 can be substantially perpendicular to the boundary wall 1406. When the split hole clip 1404 is in the second position, the horizontal portion 1408 can be substantially perpendicular to the boundary wall 1406, and the vertical portion 1410 can be substantially parallel to the boundary wall 1406.

The split hole latch 1404 can be configured to engage a portion of the boundary wall 1406. The boundary wall 1406 can include a split hole lock 1412. In some variations, the split hole lock 1412 can be configured to retain the split hole latch 1404 in the second position when engaged. As shown in Figures 14A-14B, each split hole lock 1412 can include a first protrusion 1414 and a second protrusion 1416. The first protrusion 1414 can include a horizontal stopper extending from the outer surface of the boundary wall 1406. The first protrusion 1414 can be configured and positioned to correspond to a stopper 1418 extending from the vertical portion 1410 of the split hole latch 1404. When the split hole latch 1404 is in the second position, the stopper 1418 is positioned below the first protrusion 1414, such that the distal surface of the stopper 1418 presses against the proximal surface of the first protrusion 1414. This prevents proximal movement of the split hole structure 1402 relative to the boundary wall 1406. Distal pressure on the proximal surface of the lever 1418 can generate compressive pressure between the separation hole structure 1402 and the substrate, coupling the boundary wall 1304 and the separation hole structure 1402 together and pressing the two together to form a leak-proof separation hole (in combination with a separation seal and/or a central hole structure, described in more detail below). The second protrusion 1416 can be configured to retain the separation hole buckle 1404 in the second position. The second protrusion 1416 can have an L-shaped shape and can be located distal to the first protrusion 1414 so that when the separation hole buckle 1404 is in the second position, the vertical portion 1410 is held in place by the L-shaped shape of the second protrusion 1416, as shown in Figure 14B. The second protrusion 1416 can be flexible so that when the separation hole buckle 1404 is moved from the first position to the second position, the second protrusion 1416 can bend to force the separation hole buckle 1404 into the second position. Once the split hole catch 1404 is in the second position, the second protrusion 1416 can return to the position shown in Figures 14A-14B.

While Figures 14A-14B depict only one split hole catch 1404, it should be understood that the split hole structure 1402 as a whole can have any suitable number of split hole catches 1404 (e.g., one, two, three, four, five, six, seven, eight, or more), and the boundary wall 1406 can have any suitable number of corresponding split hole locks 1412. For example, the split hole structure 1402 can have two or three split hole catches 1404 on each of its long sides. It should also be understood that the number of split hole locks 1412 need not match the number of split hole catches 1404 on the boundary wall 1406, as long as the configuration allows the split hole structure 1402 to couple with the boundary wall 1406. It should also be understood that the split hole catches 1404 (and the corresponding split hole locks 1412 on the boundary wall 1406) can have different arrangements on the split hole structure 1402.

Yet another embodiment of a split hole clip design is shown in Figures 15A-15B. A split hole structure 1502 and a boundary wall 1504 are shown. In the illustrated variation, the split hole structure 1502 and boundary wall 1504 can be configured to be coupled via a split hole clip 1506 attached to the boundary wall 1504 and engageable with a split hole lock 1508 attached to the split hole structure 1502. The split hole clip 1506 can be movable between a first position (Figure 15A) and a second position (Figure 15B), wherein the split hole structure 1502 is coupled to the boundary wall 1504 in the second position and decoupled from the boundary wall 1504 in the first position. In some variations, the split hole clip 1506 can be movable between the first and second positions by being pivotable or rotatable at an attachment point 1510. In some variations, due to the elasticity of the material (e.g., plastic), the separation hole buckle 1506 is pivotable or rotatable around the attachment point 1510. In other variations, the separation hole buckle 1506 is pivotable or rotatable around the attachment point 1510 via a hinge.

The split hole clip 1506 can include a body 1512 and a hook 1514 located at a proximal end 1516 of the body 1512. The body 1512 can have a generally flat shape. An attachment point 1510 can be located between the proximal end 1516 and the distal end 1518 of the body 1512 so that when the split hole clip 1506 moves from a first position to a second position, the proximal end 1516 can move away from the boundary wall 1504 and the split hole structure 1502 while the distal end 1518 can move toward the boundary wall 1504. The distal end 1518 of the body 1512 can be tapered on its inner surface so that the distal end 1518 does not resist movement of the split hole clip 1506 to the first position, in which the distal end 1518 is closer to the boundary wall 1504 than in the second position. Thus, when the split hole buckle 1506 is moved from the first position to the second position, the hook 1514 of the split hole buckle 1506 can move toward the boundary wall 1504 and the split hole structure 1502. When the hook 1514 is in the second position, its distal surface can engage the split hole lock 1508 located on the split hole structure 1502.

As shown in Figures 15A-15B, the split hole lock 1508 can be attached to the side of the split hole structure 1502. In other variations, the split hole lock 1508 can be attached to the proximal surface of the split hole structure 1502. The split hole lock 1508 can include a lever having a proximal surface configured to engage the distal surface of the hook 1514 of the split hole buckle 1506. When the split hole lock 1508 is engaged with the split hole buckle 1506, the lever can be positioned below the hook 1514 so that the distal surface of the hook 1514 can press against the proximal surface of the lever of the split hole lock 1508. This can resist proximal movement of the split hole structure 1502 relative to the boundary wall 1504. Distal pressure on the separation hole lock 1508 can generate a compressive pressure between the separation hole structure 1502 and the substrate, coupling the separation hole structure 1502 and the boundary wall 1504 together and pressing the two together to form a leak-proof separation hole (combined with a separation seal and/or a central hole structure, described in more detail below). The separation hole buckle 1506 can be biased toward a first position (shown in FIG. 15A ), which can maintain the coupling of the separation hole structure 1502. In other variations, the device can include a mechanism that locks the separation hole buckle 1506 in the first position after the separation hole structure 1502 is coupled.

It should be understood that the separation hole structure 1502 can have any suitable number of separation hole latches 1506 (e.g., one, two, three, four, five, six, seven, eight, or more), and the boundary wall 1504 can have any suitable number of corresponding separation hole locks 1508. In some variations, each long side of the separation hole structure 1502 can include between two and four latches. It should also be understood that the number of separation hole locks 1508 need not match the number of separation hole latches 1506, provided that the configuration allows the separation hole structure 1502 to be coupled to the boundary wall 1504. It should also be understood that the separation hole latches 1506 (and the corresponding separation hole locks 1508 on the separation hole structure 1502) can have different arrangements on the boundary wall 1504.

Returning to the embodiment of the porous separation device 100, the separation pore structure 602 can be removably coupled to the cavity 450. That is, the design of the coupling mechanism between the separation pore structure 602 and the cavity 450 can be such that the separation pore structure 602 can be removed from the cavity 450 after the two elements have been coupled. The ability to decouple and remove from the cavity can allow the separation pore structure to be inserted to first separate the composition in the cavity into the separation pores, and can subsequently allow the separation pore structure to be removed to recombine the compositions.

In some variations, the separation hole structure can be reversibly removably coupled to the cavity so that after the separation hole structure has been decoupled from the cavity, it can be recoupled to separate the composition in the cavity back into the separation hole. Figures 6A-6C show a variation of a separation hole structure that can be reversibly removably coupled to the cavity. As shown, the joint 618 of the separation hole buckle 614 can be configured to bend inwardly under the application of inward pressure. In order to allow the joint 618 to bend inwardly when coupled, there can be space between the separation hole buckle 614 and the outer surface of the boundary wall 202; that is, the opening of the separation hole lock 212 between the boundary wall 202 and the locking bar 208 can be laterally wider than the thickness of the slender portion 616 of the separation hole buckle 614. When the joints 618 are bent inward, they may no longer engage the distal surface of the locking bar 208, which may allow a proximally directed force (e.g., from pushing or pulling on the proximal end of the separation hole structure 602) to remove the separation hole structure 602 from the cavity 450 by decoupling the separation hole latch 614 from the separation hole lock 212.

The separation pore structure 1306 of the porous separation device 1300 shown in Figure 13, the separation pore structure 1402 shown in Figures 14A-14B, and the separation pore structure 1502 shown in Figures 15A-15B can be similarly reversibly and removably coupled to the suspension chamber. With respect to the separation pore structure 1306 of the porous separation device 1300 shown in Figure 13, the separation pore structure 1306 can be removably coupled to the cavity formed by the boundary wall 1302, the substrate support 1304, and the substrate (and the boundary wall seal in some variations). In this variation, the separation pore buckle 1322 can be configured to flex outwardly under the application of outward pressure. When the knockout hole clips 1322 flex outward, they can no longer engage the distal horizontal surfaces of the protrusions 1332 of the knockout hole lock 1330, which can allow a proximally directed force (e.g., from pushing or pulling on the proximal end of the knockout hole structure 1306) to remove the knockout hole structure 1306 from the cavity by decoupling the knockout hole clips 1322 from the knockout hole lock 1330. Similarly, in the variations shown in Figures 14A-14B and 15A-15B, the knockout hole clips 1404 and 1506 can be moved from the second position to the first position, respectively, to decouple the knockout hole structures 1402 and 1502 from the boundary walls 1406 and 1504, respectively.

In other variations, the separation hole structure can be irreversibly removably coupled to the cavity. In these variations, once coupled, the separation structure can be removed from the cavity, but may not be subsequently recoupled to the cavity. This may be because, for example, in some variations, decoupling the separation hole structure from the cavity irreversibly affects the structure of the separation hole structure so that it cannot be recoupled (e.g., the separation hole clip 614 needs to be destroyed in order to remove the separation hole structure).

Concentrated pore structure

The porous separation device 100 may optionally include a concentrated pore structure 702. The concentrated pore structure 702 may be configured to reduce the cross-sectional area of the distal portion of the separation pore 610. This may be advantageous, for example, because it can concentrate the target agent in each separation pore 610 into a smaller cross-sectional area at the bottom of the separation pore 610. In some variations, the concentrated pore structure may include a thin layer of material, such as, but not limited to, a soft elastic material (e.g., silicone, rubber, or the like), which may be configured to be located between the separation pore structure and the substrate. Typically, the concentrated pore structure may include a plurality of openings, one proximal end of which may correspond to the distal end of the opening of the separation pore structure, and the opening may narrow in the proximal to distal direction. The distal end of the opening in the concentrated pore structure may allow the composition in the separation pore to interact with the substrate. In variations in which the substrate includes a coating (described in detail above), this may allow the composition to interact with the coating. Therefore, in these variations, the opening of the concentrated structure may actually include a substance containing a coating, such as, but not limited to, a protein, a polymer, a hydrogel, or a chemical coating.

7A-7B illustrate perspective and top views, respectively, of one embodiment of a concentrating hole structure 702. As can be seen, the concentrating hole structure 702 can include a thin structure containing a plurality of openings 704. The proximal cross-sectional shape of the openings 704 can be configured to correspond to the cross-sectional shape of the separation hole 610. The openings 704 can have an inverted truncated pyramidal shape, such that the cross-sectional area of the openings 704 decreases from proximal to distal. At the distal end of the openings 704, a portion of the substrate 302 (and any coating) can be exposed.

8A-8B illustrate close-up views from the top and side, respectively, of another embodiment of a concentrating pore structure 802 having an opening 804 that also has an inverted truncated pyramidal shape. As shown in FIG8B , the cross-sectional area of the opening 804 at its proximal end 806 is greater than the cross-sectional area of the opening 804 at its distal end 808. In other variations, the opening may have other shapes, such as, but not limited to, a truncated cone or a pyramidal frustum. In still other variations, the opening may be closed at the distal end; that is, the composition within the separation pore may not come into contact with the substrate.

As described above, the concentrating pore structure may be located between the separation pore structure and the substrate. In some variations in which the porous separation device includes a concentrating pore structure, the porous separation device may not include a separation seal. For example, in variations in which the concentrating pore structure includes a material such as rubber, plastic, or polymer, the porous separation device may include a concentrating pore structure without a separation seal, and the material may be capable of forming a seal between the substrate and the separation pore structure. In these variations, the concentrating pore structure may be attached to the near surface of the substrate (as shown in Figure 9); it may be attached to the boundary wall; or it may be attached to the boundary seal (for example, by attaching the outer edge of the concentrating pore structure to the inner edge of the boundary seal). The concentrating pore structure may be attached to these elements in any suitable manner, such as, but not limited to, adhesives (glue, adhesive polymers, and the like), chemical bonding, or the like.

In other variations in which the porous separation device includes a concentrating pore structure, the porous separation device may also include a separation seal. For example, in variations in which the concentrating pore structure includes a material that does not typically form a sufficient seal between the substrate, the concentrating structure, and the separation pore structure, such as glass or hard plastic, the porous separation device may include both the concentrating pore structure and the separation seal. In these variations, the separation seal may be located between the substrate and the concentrating pore structure and/or between the concentrating pore structure and the separation pore structure. When the separation seal is located between the substrate and the concentrating pore structure, the separation seal may be attached to the near surface of the substrate, the far surface of the concentrating pore structure, or the boundary wall; when the separation seal is located between the concentrating pore structure and the separation pore structure, the separation seal may be attached to the near surface of the concentrating pore structure, the far surface of the separation pore structure, or the boundary wall.

In some variations, the porous separation devices described herein may further include a lid. The lid may be configured to fit over the porous separation device to cover the cavity. In some variations, the lid may be configured to individually seal the top of each separation pore when the separation pore structure is coupled to the cavity.

Reagent loading device

Reagent loading device is described herein equally.In some variations, the reagent loading device can be configured to deliver reagent or test agent to each of the separation holes 610 generated by the separation hole structure 602 and the cavity 450 of coupling.In other variations, the reagent loading device can be used independently of the porous separation device described in the present invention.For example, in some cases, the reagent loading device can be used together with the porous plate with fixed wall.

protrusion

In the variation shown in Figure 10 A-10B and Figure 18, respectively, reagent loading device 1000 and 1800 can comprise a plurality of projections 1002 and 1802 respectively.Each projection 1002 or 1802 can comprise rod 1004 or 1804 and closed tip 1006 or 1806 respectively, and below describes in more detail.The projection can comprise any applicable material or material, such as but not limited to plastics, silicon, metal or polymer.In some variations, rod and closed tip can comprise same material, yet in other variations, they can comprise different materials.

In some variations, reagent loading device 1000 or 1800 can be configured to use together with porous separation device (for example, with porous separation device 100, as shown in Figure 11), but it does not need to be configured to use together with porous separation device.In the variation that reagent loading device is configured to use together with porous separation device 100 therein, the number of projections can be configured to the number of separation holes 610 corresponding to porous separation device 100. But, it should be understood that reagent loading device can be used together with the porous separation device with separation holes 610 more or less than projection number, or they can not be used together with porous separation device. For example, Figure 23A-23B, 24A-24B, 25A-25B, 28A-28C, 30A-30B, 31B-31D and 32 display reagent loading device is configured to be positioned at only on part separation hole, and therefore has projection less than the number of separation holes.

In variations in which the reagent loading apparatus is configured to be used with a porous separation device, the size and spacing of the projections can be configured to correspond to the separation holes of the porous separation device. More specifically, for example, if the reagent loading apparatus 1000 or 1800 is configured to be used with the porous separation device 100, the cross-sectional dimensions of the projections 1002 or 1802 can be configured so that the projections can fit in the separation holes 610. In some variations, the maximum cross-sectional dimension of the projections can be from about 1 μm to about 10 μm, from about 10 μm to about 100 μm, from about 100 μm to about 1 mm, from about 1 mm to about 5 mm, from about 5 mm to about 1 cm, from about 1 cm to about 2 cm, greater than about 2 cm, from about 1 μm to 2 cm, or from about 1 mm to about 1 cm.

In the variation that reagent loading device is configured to use together with porous separation device such as porous separation device 100 therein, the length of projection can make when reagent loading device engages with porous separation device, and the closed tip of reagent loading device can be submerged in the content of each separation hole completely.In some variations, the length of projection can be approximately 1mm to approximately 2mm, approximately 2mm to approximately 4mm, approximately 4mm to approximately 6mm, approximately 6mm to approximately 8mm, approximately 8mm to approximately 1cm, approximately 1cm to approximately 2cm, approximately 2cm to approximately 4cm, approximately 4cm to approximately 6cm, greater than approximately 6cm, approximately 1mm to approximately 6cm, approximately 1mm to approximately 1cm or approximately 1cm to approximately 6cm.Should be understood that each projection does not need to have identical configuration.

As mentioned above, projection 1002 or 1802 can comprise respectively closed tip 1006 and 1806, and it all can be configured to keep reagent.In the meaning that can not comprise the opening at the far-end, closed tip can be " closed ", and the opening at the far-end is connected with the cavity in the rod of projection, and when reagent is deposited (deposited) by closed tip, reagent advances by described projection.This is opposite with device such as pipette or the like, and it comprises the cavity in the rod that wherein keeps composition, and the opening outside cavity, and when composition is deposited by pipette, composition advances by described cavity.Pipette or the like are usually owing to the local vacuum in cavity that composition is at least partially remained in cavity.Relatively speaking, due to the interaction (for example, adhesion) between closed tip and reagent and due to the interaction (for example, surface tension) in reagent, the closed tip of reagent loading device can be designed to be remained on reagent in the tip outside (not in the cavity in the rod).

The closed tip (e.g., closed tip 1006 or closed tip 1806) can have any suitable geometric shape for holding reagents in this manner, including but not limited to a shape with a blunt tip or a cone (see Figure 12A), a square (see Figures 12B-12C and 12E), a circle (see Figures 12D and 12F-12H), or a similar shape. In some variations, the closed tip can be flat (see Figures 10A-140B). In other variations, the closed tip can include depressions - for example, they can be concave (e.g., there are hemispherical depressions) (see Figure 12H) or can include cylindrical (or other shapes) recesses (see Figure 12G). Still in other variations, the closed tip can include one or more linear depressions. For example, the closed tip can include one linear depression (see Figures 12B and 12D), they can include two perpendicular linear depressions (see Figure 12C), or they can include multiple linear depressions arranged in a grid-like arrangement. In other variations, the closed tip may comprise two parallel plates between which the reagent may be held, or one or more capillaries in which the reagent may be held. Such tips, such as concave tips or other tips comprising depressions, may allow the reagent to be more securely held by the closed tip, for example, by providing a larger surface area for adhesion between the closed tip and the reagent.

The surface of each closed tip can be smooth, or the surface can be rough (e.g., having surface irregularities). The closed tip can have any suitable size. In some variations, the maximum cross-sectional dimension of the closed tip can be from about 1 μm to about 10 μm, from about 10 μm to about 100 μm, from about 100 μm to about 1 mm, from about 1 mm to about 10 mm, greater than about 10 mm, from about 1 μm to about 10 mm, from about 1 μm to about 1 mm, or from about 1 mm to about 10 mm. It should be understood that each closed tip need not have the same configuration.

In some variations, closed tip (for example, closed tip 1006 or closed tip 1806) can comprise porous material, as polymer gel or hydrogel (referring to Figure 12E-12F), polymer-based sponge or mesh, or can comprise the matrix containing reagent, as dry fiber structure in some variations.For example, closed tip can comprise cellulose (for example, nitrocellulose, paper-like material), glass fiber mesh, silk fiber mesh and the like.In some of these variations, when the closed tip of reagent loading device (for example reagent loading device 1000 or reagent loading device 1800) is lowered in liquid or solution, as by being lowered in the separating hole of porous separation device (for example, porous separation device 100), all or part of closed tip can be soluble.Wherein all or part of closed tip 1006 is in soluble variation, soluble material can comprise any suitable material, such as but not limited to salt, particulate, nanoparticle, polyglycolide, polylactic acid or polylactic-glycolic acid copolymer or its combination. In other variations, all or a portion of the closed tip 1006 of the reagent loading device 100 can be fusible when the closed tip 1006 is lowered into a liquid or solution, such as by being lowered into a separation well of a porous separation device, e.g., porous separation device 100. In variations in which all or a portion of the closed tip 1006 is fusible, the fusible material can include any suitable material, such as, but not limited to, DMSO.

In some embodiments, the present invention provides the method for the manufacture of the present invention.For example, closed tip (for example, closed tip 1006 or closed tip 1806) can be positioned at the distal end of rod (for example, rod 1004 of reagent loading device 1000 or rod 1804 of reagent loading device 1800).In some variations, closed tip can be integrated with rod, yet in other variations, closed tip can be attached to rod in any suitable manner (for example, use adhesive (glue, adhesive polymer and the like), welding, mechanical fastener, chemical bonding, the combination of these methods or similar methods).The near-end of rod can be connected with plate, and it can form projection array.For example, as shown in Figure 10A-10B, the near-end of rod 1004 can be connected with the far-surface 1010 of the plate 1008 of reagent loading device 1000, and it can form projection 1002 arrays. In some variations, rod 1004 can be integral with plate 1008, yet in other variations, rod 1004 can be attached to plate 1008 in any suitable manner (e.g., using adhesive (glue, adhesive polymer, and the like), welding, mechanical fasteners, chemical bonding, a combination of these methods, or similar methods). As another example, as shown in Figure 18, the proximal end of rod 1804 can be connected to a far surface 1810 of a plate 1808 of a reagent loading device 1800, which can form an array of projections 1802. Plate 1808 can include a groove 1820 on its proximal surface, which can be configured to engage with a vibration unit, as described in more detail below. Plate 1808 can also include legs 1822, which can be configured to protect closed tip 1806, as described in more detail below.

The rod (e.g., rod 1004 of reagent loading device 1000 or rod 1804 of reagent loading device 1800) can have a length such that the closed tip (e.g., closed tip 1006 or closed tip 1806) can be completely immersed in the contents of each separation well 610 when the reagent loading device is engaged with the porous separation structure 100. For example, in some variations, the length of the rod can be from about 1 mm to about 2 mm, from about 2 mm to about 4 mm, from about 4 mm to about 6 mm, from about 6 mm to about 8 mm, from about 8 mm to about 1 cm, from about 1 cm to about 2 cm, from about 2 cm to about 4 cm, from about 4 cm to about 6 cm, longer than about 6 cm, from about 1 mm to about 6 cm, from about 1 mm to about 1 cm, or from about 1 cm to about 6 cm.

The rod can have any suitable cross-sectional dimensions, which can be smaller or larger than the cross-sectional dimensions of the closed tip. In some variations, the maximum cross-sectional dimension of the rod can be from about 1 μm to about 10 μm, from about 10 μm to about 100 μm, from about 100 μm to about 1 mm, from about 1 mm to about 10 mm, greater than about 10 mm, from about 1 μm to about 10 mm, from about 1 μm to about 1 mm, or from about 1 mm to about 10 mm. It should be understood that in some variations, the rod can have a cross-sectional dimension that is variable along its length (e.g., they can taper distally, proximally, or toward the midpoint). It should also be understood that each rod does not need to have the same configuration.

Directed components

The reagent loading device can optionally include an orientation component that can promote the reagent loading device to be inserted into the hole with a specific direction. In some variations, the orientation component can be a direction indicator, thereby providing the user with information that allows the user to correctly orient the reagent loading device with respect to the hole. In other variations, the orientation component can be an orientation wedge that indicates that the reagent loading device is inserted into the hole with a specific direction. In these variations, the receiving hole can have a corresponding orientation wedge that allows the reagent loading device to be only inserted into the hole with a specific direction. In some cases, these receiving holes can be the separation holes of the porous separation device described herein. In some other cases, the receiving hole can be a receiving plate with a fixed wall, such as the part (part) of a porous plate. The corresponding orientation wedge of a receiving device can be integrated with a receiving device (for example, the porous separation device described herein, or a porous plate), or it can be the part of the adapter that is configured to be added to the receiving device, as described in more detail below.

Figure 10 A-10B and 11 show a variation of the reagent loading device with the directional components that comprises a direction indicator.As shown therein, indicator can comprise a handle 1014 that is attached to the near surface 1012 of plate 1008, and said handle 1014 can comprise arrow 1016 on a side.Should be appreciated that indicator can have any suitable form.For example, in some variations, indicator can comprise blocking on a corner of plate 1008.In other variations, indicator can comprise the visual indicator that is positioned at a specific location on plate 1008, as pigment or color, text indicator, texture or the like.

As described above, in other variations, the orientation feature can include an orientation wedge that can direct the reagent loading device to be inserted into the well in a specific orientation. The orientation wedge can prevent the reagent loading device from being inserted into the well (such as the porous separation structure described herein, or the well of a porous plate with fixed walls) in the wrong orientation, and can only allow the reagent loading device to be inserted into the well when the reagent loading device is in the proper orientation relative to the receiving device (i.e., when each protrusion of the reagent positioning device will enter the desired well when the reagent loading device is lowered into the receiving device).

It should be understood that the orientation wedges of the reagent device and the corresponding orientation wedges of the receiving device can have any number of configurations or other physical shapes. In some variations, one or more orientation wedges can have an asymmetric shape so that the orientation component of the reagent delivery device can only properly engage with the corresponding orientation wedge of the receiving device when the reagent loading device is in the correct orientation relative to the receiving device. The orientation wedges and the corresponding wedges on the receiving device can have any suitable shape, such as a semicircle, an inclined groove, a curved or arcuate groove, a triangle, a crescent, a parallelogram, or the like.

The directional wedge that the example of reagent loading device includes shows in Figure 18.As shown therein, directional component comprises one or more grooves 1824.As shown in Figure 19A-19B, reagent loading device 1800 may comprise two sides 1826.A side 1826 shown in Figure 19A may comprise single groove 1824a, yet the other side 1826 shown in Figure 19B may comprise two grooves 1824b and 1824c.Corresponding porous separation structure may comprise corresponding directional component.More specifically, porous separation structure may comprise an end with a joint 1828a corresponding to groove 1824a, and an end with two joints 1828b and 1828c corresponding to groove 1824b and 1824c.Groove 1824 and joint 1828 can correspond so that when two assemblies are correctly aligned, reagent loading device 1800 can be lowered in the porous separation device, but when relative directions are opposite, can not be lowered in the porous separation device.

In other variations where the receiving device includes a corresponding directional wedge, the corresponding directional wedge may be part of the adapter rather than being integral with the receiving device. The adapter may be configured to attach to a porous separation device or other receiving device. An example of such an adapter is shown in Figure 22. As shown therein, an adapter 2206 may include an outer frame configured to fit on the porous separation device 2204. The adapter 2206 may include a wedge 2208. The wedge 2208 may extend along two opposing sides of the adapter 2206 and may face inward, forming a gap 2210 between the wedge 2208 and the outer edge of the porous separation device 2204.

23A-23B . Figure 25A-25B shows a close-up view of the wedge 2208 of the adapter 2206 and the notch 2214 of the reagent loading device 2212. When the reagent loading device 2212 is correctly oriented relative to the adapter 2206, as shown in Figure 25A, the shapes of the wedge 2208 and the notch 2214 correspond. However, when the reagent loading device 2212 is incorrectly oriented relative to the adapter 2206, as shown in Figure 25B, the shapes of the wedge 2208 and the notch 2214 may not correspond. In some variations, when the wedge 2208 and the notch 2214 do not correspond, the reagent loading device 2212 may not be lowered into the porous separation device. Wedge 2208 and the notch 2214 may also be configured to guide the protrusion of the reagent loading device 2212 into the separation hole of the porous separation device. That is, when the wedge 2208 and the notch 2214 are properly aligned, each protrusion will be located over a separation hole.

As the reagent loading device 2212 is lowered into the porous separation device 2204 , the notch 2208 of the reagent loading device 2212 can enter the gap 2210 between the wedge 2208 and the outer edge of the porous separation device 2204 , wherein the reagent loading device 2212 is oriented so that the wedge 2208 corresponds to the notch 2214 .

In some cases, it may be desirable to orient the wedge to restrict the direction of insertion of the reagent loading device while still allowing the reagent loading device to impart vibration to the aperture of the receiving device. For example, this may allow the reagent loading device to be used to mix the contents of the receiving device. In some variations, this may be achieved by having a wedge configured to restrict the reagent loading device only during insertion (e.g., when the reagent loading device is partially loaded into the porous separation device), but when the reagent loading device is fully loaded into the porous separation device, the reagent loading device is no longer restricted by the wedge and can move to impart vibration.

24B , the reagent loading device 2212 and the adapter 2206 may be adapted so that the wedge 2208 and the notch 2214 engage only as the reagent loading device 2212 is inserted into the porous separation device 2204, but the wedge 2208 and the notch 2214 no longer engage once the reagent loading device 2212 is fully inserted into the porous separation device 2204. This may allow the reagent loading device 2212 to move relative to the porous separation device 2204 in order to mix the contents of the separation wells.

As shown in Figure 24B, this can be achieved by wedge 2208 and notch 2214 that extend only partially along the inner surface of adapter 2206 and the outer end surface of reagent loading device 2212, respectively. As shown therein, wedge 2208 can extend only in the upper half of the inner surface of adapter 2206, while notch 2208 can extend only in the lower half of the outer end surface of reagent loading device 2212. As such, when the reagent loading device is less than halfway inserted into the porous separation device, wedge 2208 and notch 2214 can be securely engaged, thereby limiting the orientation of the reagent loading device and its movement relative to the porous separation device. However, when the reagent loading device is more than halfway inserted into the porous separation device, wedge 2208 and notch 2214 can be loosely engaged so that there is a gap (clearance) between wedge 2208 and notch 2214, thereby allowing the reagent loading device to move within a certain range of movement relative to the porous separation device. In some variations, a smaller version of the wedge may be present along the lower half of the inner surface of the adapter 2206, such as shown in FIG24B , which may prevent substantial lateral movement of the reagent loading device while still allowing a range of movement sufficient to allow vibration of the reagent loading device to promote mixing of the contents of the separation wells, as described in more detail herein. However, it should be understood that in some circumstances (e.g., when mixing is not required, or mixing is not accomplished by lateral movement of the reagent loading device), one or more of the wedge and notch may be securely engaged when the reagent loading device is fully inserted into the porous separation device (e.g., one or both of the wedge and notch may extend along the full length of the adapter and reagent loading device).

Should be understood that the directional block can comprise any suitable shape of indication orientation. As an example, the directional block can comprise tilted slot 2602, as shown in Figure 26 A. Figure 26 B shows another example of the directional block comprising a right triangle 2604. In some variations where the directional block is positioned at the opposite side of the adapter, the directional block on each of the opposite side can have different shapes (for example, one side is semicircular and the other side is rectangle), direction or size, so that the corresponding notch on the reagent loading device only corresponds to the wedge on a side. Should be understood equally that although the adapter is described to comprise wedge and the reagent loading device is described to comprise corresponding notch by this paper, in other variations, the reagent loading device can comprise wedge and the adapter can comprise corresponding notch.

Although adapter can comprise inwardly facing wedge, thereby forms gap between the outer edge of wedge and porous separation device, in other variations, adapter can comprise outwardly facing wedge.The example of this type of adapter shows in Figure 27 and 28A-28B.As shown therein, adapter 2702 can be configured to be placed on porous separation device 2704 to form outer frame.Adapter 2702 can comprise one or more outwardly facing wedges on one or more outer edges (as shown therein, on two relative outer edges).Wedge 2706 on the first edge can comprise first shape (here, triangle), while wedge 2708 on the second edge (first edge opposite) can comprise second shape (here, forming protrusion by chamfered toothed edge).Should be understood that the first and second shapes can be any suitable different shapes, so that the reagent loading device with corresponding notch will engage with adapter with first direction rather than with second direction.

28A-28B , a corresponding reagent loading device 2710 may include notches 2712 and 2714 corresponding to wedges 2706 and 2708, respectively, which may be located on the inner surface of the distal end of the reagent loading device 2710. In variations of FIG. 22 through FIG. 25A-25B , the wedges 2706 and 2708 may extend only in the upper half of the outer surface of the adapter 2702, while the notches 2712 and 2714 may extend only in the lower half of the inner end surface of the reagent loading device 2710. As such, the wedges and notches may be securely engaged as the reagent loading device is inserted into the porous separation device, but may not be securely engaged when the reagent loading device is fully inserted into the porous separation device, as described in more detail with respect to FIG. 24A-24B .

It should be understood that the wedges need not be part of the adapter; in some variations, they may be integral with a receiving device such as a porous separation device, as shown in the variations of Figures 29 and 30A-30B. As shown therein, the boundary wall of the porous separation device 2902 may include wedges 2904 and 2906 on opposite sides of the boundary wall outer surface. These wedges may have similar features and functions as the wedges described with respect to Figures 27 and 28A-28C and may be engaged with the reagent loading device 2908. Inwardly facing wedges, such as the wedges shown in Figures 22A-22B to 26A-26B, may also be integral with the porous separation device. Furthermore, although the directional wedges described herein are described with respect to the porous separation device and reagent loading device described herein, it should be understood that the directional wedges may also be used with a standard porous plate that does not have the components described herein.

Another variation of adapter 3100 shows in Figure 31A-31D.In its shown variation, as shown, adapter 3100 can be configured to engage with a receiving device with fixed wall, such as standard 96-well plate 3102.Adapter 3100 can adapt to standard, fixed well plate so that it can engage with reagent loading device 3106.Adapter 3100 can comprise the inner wall 3108 with the center opening 3104 corresponding to the external shape of 96-well plate 3102, and the groove 3110 that is configured to receive reagent loading device 3106.Adapter 3100 can comprise the direction of the reagent loading device 3106 that one or more directional components are inserted with indication.For example, only when reagent loading device 3106 is loaded with a specific direction, groove 3110 can comprise one or more zigzag or zigzag surfaces that are configured to mate the corresponding surface on the reagent loading device 3106. 31A-31D , the adapter 3100 may include a serrated surface 3112 on the outer wall of a first side slot 3110, and a serrated surface 3114 on the inner wall of a second side slot 3110. The reagent loading device 3106 may have a serrated surface 3116 on the outer side of the first side wall, which may correspond to the serrated surface 3112, while the reagent loading device 3106 may have a serrated surface 3118 on the inner side of the second side wall, which may correspond to the serrated surface 3114. Thus, when the reagent loading device 3106 is in a first orientation such that the serrated surface 3112 is aligned with the serrated surface 3116, and when the serrated surface 3114 is aligned with the serrated surface 3118, the reagent loading device 3106 may be inserted into the slot 3110 of the adapter 3100. However, when the reagent loading device 3106 is in the second opposite orientation, the reagent loading device 3106 may not be inserted into the slot 3110 of the adapter 3100.

In some variations, adapter can further include other features to help reagent loading device is guided in the receiving hole.For example, in the variation shown in Figure 31A-31D, adapter 3100 can include a pin (pin) 3120 extending from the proximal end of adapter 3100. Pin 3120 can be configured to cooperate in the corresponding opening 3122 in the reagent loading device. Although pin 3120 is shown as having a triangular shape in Figure 31A-31D, pin can have any suitable shape, as rod, square or similar shape.

Reagents/test reagents

Each of the closed tip of reagent loading device described herein can be loaded with reagent or test agent.Reagent can be in any suitable form, such as but not limited to liquid, solution, gel or solid.When reagent is in liquid or solution form, reagent can adhere to each closed tip, and this is due to the adhesive force (for example, surface tension) in the liquid and the adhesive force between the closed tip.In some variations, according to the material properties of the configuration and material and liquid or solution of closed tip, the volume of the liquid or solution that can adhere to each closed tip can be about 1pL to about 10pL, about 10pL to about 100pL, about 100pL to about 1nL, about 1nL to about 10nL, about 100nL to about 1 μ L, about 1 μ L to about 10 μ L or more than about 10 μ L.

Although in some variations, the closed tip described herein can be loaded with the same reagent, it may often be necessary to load the closed tip with different reagents or test agents. For example, it may be necessary to do so so that the hole of the receiving device (the separation hole of the porous separation device as described in the present invention) can withstand different reagents. The test agent can be, but is not limited to, protein, nucleic acid, cell, microorganism (e.g., bacteria, fungi), plant (e.g., algae), virus, small molecule drug or any compound. In some variations, the closed tip can be loaded with a specific reagent library of the desired detection. For example, a reagent loading device can be loaded with a bacterial library, a drug library (e.g., a kinase inhibitor library), an antibody library or the like.

mix

After being delivered (for example, after being delivered to the contents of the separation holes of the porous separation device described in the present invention, or after being delivered to a plate with a fixed wall, such as in the hole of a porous plate), the reagent loading device described herein may optionally be configured to promote the mixing of reagents. In some variations, the reagent loading device may be configured to give vibration to promote mixing. The reagent loading device may include one or more actuators or motors that can cause the protrusions to vibrate. In some variations, each protrusion may be attached to an actuator or motor; in other variations, a single actuator or motor may cause all protrusions to vibrate. In other variations, there may be more than one actuator or motor but less than the number of protrusions.

Figure 20 A-20C shows an embodiment of motor unit 2000.Motor unit 2000 may comprise vibration motor 2002, which may be connected with controller 2006 by line 2004.Controller 2006 may be handheld and comprise an interface, such as on/off button 2012, that allows the user to control motor 2002. In some variations, the interface may allow the user to control frequency, amplitude, and/or the vibration duration. Vibration motor 2002 may be included in unit 2008, and described unit 2008 is configured to engage with the reagent loading device in this way, so that the vibration motion of motor 2002 is delivered to the projection of the reagent loading device. In the variation shown in Figure 20 A-20C, unit 2008 may comprise groove 2010, which may engage with groove 1820 on the near surface of the plate 1808 of reagent loading device 1800, as shown in Figure 20 C. Unit 2008 can be secured to the reagent loading device (e.g., using slot 2010 aligned with slot 1820 of reagent loading device 1800) by any suitable reversible or irreversible method, such as, but not limited to, a spring-loaded clamp or snap. While the variations shown in Figures 20A-20C include one vibrating motor 2002, it should be understood that in other variations, the motor unit 2000 can include more than one motor, such as two, three, or more motors.

Another variation of a motor unit 3200 is shown in Figures 32 and 33A-33B. The motor unit 3200 may include a vibration motor 3202 (see Figure 33B) located in a vibration unit 3204, which may be connected to a controller 3208 including a battery and an on/off switch 3210 via a flexible wire 3206. The vibration unit 3204 may include a slot 3212 configured to engage a corresponding slot 3214 on a proximal surface of a reagent loading device (e.g., any other reagent loading device described herein, such as the reagent loading device 2212).

In another variation of the motor unit, one or more (for example, two, three, or more) vibration motors can be installed on the tip of the handheld device. The handheld device can be similar to a handheld pipette. The handheld device can be reversibly fixed to the reagent loading device, for example, by being fixed to the near surface of the reagent loading device, so that when the handheld device is fixed to the reagent loading device, the vibration of the vibration motor can be transferred to the protrusion of the reagent loading device. In some variations, the handheld device can be fixed to the reagent loading device by a fixture, and the fixture can be opened and closed by the button on the handheld device. In some variations, the button can be operated by the thumb of the user. In some of these variations, one or more motors can comprise linear vibration. In other variations, vibration can be caused by a magnetic field. More specifically, the protrusion of all or part of the reagent loading device can comprise the material (for example, iron or nickel) attracted by a magnetic field as the tip of the tip, and the alternating magnetic field can be opened and closed (for example, with a frequency of about 10Hz to about 200kHz) so as to cause vibration.

The amplitude and frequency of vibration can be selected to maximize the mixing of reagents without negatively affecting the composition to which the reagent is delivered. That is, it may be desirable to maximize mixing into the contents of a hole (e.g., the separation hole of a porous separation device described herein, or the hole of a plate with a fixed wall) without negatively affecting the vibration of the target agent. As such, the amplitude and frequency of vibration can be customized according to the design (including the physical design of the protrusion) of the reagent loading device. In some variations, vibration can be linear vibration (e.g., back and forth vibration); in some other vibrations, vibration can be rotational vibration. In some variations in which vibration is linear, the amplitude of vibration can be about 1mm to about 3mm. In some variations, frequency can be about 10Hz to about 200kHz. In some of these variations, frequency can be a stirring form below the acoustic range (e.g., below about 200Hz); or it can be a form of ultrasonic treatment in the acoustic range (e.g., about 20Hz to about 200kHz) or a form of ultrasonic treatment in the acoustic range (e.g., above about 200kHz). In some variations where the vibration is a rotational vibration, the vibration can be about 600 to about 12,000,000 revolutions per minute. In variations where the reagent loading device is configured to promote reagent mixing, the physical design of the projection can be configured to promote mixing. For example, the projection can include a rod with better elasticity to promote the vibration of the closed tip, such as by having a smaller cross section or by including an elastic material.

Containment Unit

In some variations, the reagent loading device can further include a containment unit. The containment unit can be configured to protect the reagent on the closed tip of the reagent loading device, while also being configured to enable it to be removed from the reagent loading device and reagent is left on the closed tip of the projection. In some variations, the containment unit can include a substantially flat surface. In these variations, when containing the unit and being placed on the position of the reagent on its protection closed tip, it can contact with reagent. But, when containing the unit and removing it from the remainder of the reagent loading device, the material of the containment unit can have certain performance (for example, in conjunction with or surface affinity coefficient) so that when the surface moves away from reagent, reagent releases from the surface and keeps attached to the closed tip of the projection.

In one embodiment, the reagent loading device can be mounted on a plurality of surfaces of the reagent loading device. For example, in one variation, the closed tip of the reagent loading device can comprise the first plastic and the containment unit can comprise the second plastic, wherein in conjunction with or surface affinity coefficient is different and reagent is discharged from the second plastic and remains coupled to the first plastic. In other variations, the containment unit can comprise a plurality of independent holes or lids, each of which is configured to isolate the independent protrusion of the reagent loading device. The hole or lid can be connected (for example, by being connected to each of flat surfaces), or they can be separated. In some variations, the hole or lid can have a depth greater than the length of the protrusion on the reagent loading device, so that the closed tip of the protrusion can not contact with the end of the hole or lid; In other variations, the material of the hole or lid and the closed tip can have a material property that allows reagent to be discharged from the surface of the hole or lid while remaining attached to the closed tip of the protrusion, as described above.

In other variation, the reagent loading device can have the reagent that is configured to protect and is loaded on the closed tip of reagent loading device, and does not have the design of the containment unit of separation.One such variation shows in Figure 18.As shown therein, reagent loading device 1800 can comprise leg 1822, and it can be configured to protect closed tip 1806.In the variation that reagent loading device is rectangle (as reagent loading device 1800), leg 1822 can be positioned at each angle of reagent loading device.Leg 1822 can be longer than protrusion 1802 (that is, they can extend far beyond closed tip 1806), so that when reagent loading device 1800 is placed on the surface of the closed tip 1806 with the protrusion facing the surface, the far-end of leg 1822 can contact surface, and closed tip 1806 can be suspended on the surface, as shown in Figure 18.Accordingly, leg 1822 can protect the reagent that is loaded on the closed tip 1806 and avoid contact surface, and this can protect reagent and avoid contamination.

Reagent test kit

Should be understood that the assembly of porous separation device described herein and reagent loading device can also have the form of the test kit for biological or chemical analysis except having the form of device.Described herein in addition is the analysis system comprising porous separation device described herein (for example, above-mentioned porous separation device 100) and reagent loading device described herein (for example, above-mentioned reagent loading device 1000 or reagent loading device 1800).Reagent loading device can comprise a plurality of protrusions corresponding to the multiple separation holes of porous separation device, so that each of the multiple protrusions of reagent loading device can be configured to cooperate in one of the separation holes of porous separation device simultaneously.

System can further optionally comprise the chamber that is configured to load reagent loading device.This chamber can comprise isolated area, and above-mentioned isolated area comprises the reagent or the test agent of the configuration that is in the configuration corresponding to the closed tip of reagent loading device.This can allow each tip of reagent loading device to load with reagent or test agent simultaneously, even when to be loaded on one or more reagents or the test agent different on closed tip.In some variations, isolated area can be a plurality of holes or compartments in the chamber, and it can be loaded (or available reagent or test agent is pre-loaded) by the user with the reagent or the test agent corresponding to the configuration of closed tip.In other variations, isolated area can comprise a plurality of zones (for example, the point on the slide) on the substrate.In addition, in some cases, system described herein or test kit can comprise the subset of device described herein.For example, in a variation, test kit can comprise the chamber that is configured to load reagent loading device and reagent loading device.

method

The present invention also describes the method for using the porous separation device described in the present invention and the reagent loading device or test kit or system. Generally, a cavity (e.g., the above-mentioned cavity 450) can be formed by coupling a boundary wall (e.g., the above-mentioned boundary wall 202) with a substrate support (e.g., the above-mentioned substrate support 402), and it can also clamp a substrate (e.g., the above-mentioned substrate 302) and a boundary seal (e.g., the above-mentioned boundary seal 204) between the boundary wall and the substrate support. Once a cavity is formed, a composition (e.g., a cell suspension) can be delivered to the cavity (e.g., by using a pipette). A separation pore structure (e.g., the above-mentioned separation pore structure 602) can be coupled in the cavity subsequently, and this can divide the contents of the cavity into multiple separation pores (e.g., the above-mentioned separation pore 610). In some variations, a period of time can be allowed before the separation pore structure is coupled, but it is not necessary. A period of time can be allowed to be fully used for target agent precipitation and/or attachment to substrate after the separation pore structure is coupled, but it is not necessary. The reagent loading device (e.g., the reagent loading device 1000 or 1800 described above) can then be used to deliver the test agent to each of the separation holes simultaneously or separately. Alternatively, known methods (instead of the reagent loading device described herein) can be used to deliver the test agent to each of the separation holes. The separation hole structure can then be optionally removed from the cavity. Known techniques can be used to observe, measure, or analyze the detection.

With reference to the embodiment of the porous separation device 100, the porous separation device 100 can be assembled by placing the substrate 302 in the frame 404 of the substrate holder 402. The boundary wall clip 408 of the substrate holder 402 can be inserted into the substrate holder lock 210 of the locking bar 208 of the boundary wall 202. To this end, the joint 412 of the boundary wall clip 408 can be bent inward to allow the joint 412 to pass through the opening of the substrate holder lock 210. The triangular shape of the joint 412 can cause the joint 412 to gradually bend inward due to pressure from the locking bar 208 as the boundary wall 202 moves distally relative to the substrate holder 402. When the tabs 412 reach the proximal end of the opening, they can snap outward to hook onto the locking strip 208, with the distal surface 414 of the boundary wall tabs 408 pressing against the proximal surface of the locking strip 208, and the elongated portion 410 of the boundary wall tabs 408 positioned within the opening of the boundary wall lock 210 between the boundary wall 202 and the locking strip 208. This can sandwich the base 302 (and the boundary seal 204) between the boundary wall 202 and the base support 402. The compressive force between the boundary seal 204 and the base 302 due to the coupling of the boundary wall tabs 408 and the base support lock 210 can press the boundary seal 204 against the base 302, creating a leak-proof seal around the cavity 450.

Similar methods can be used to assemble the chamber in other embodiments of the porous separation devices described herein (such as the porous separation device 1300 described with respect to FIG. 13 ). In this embodiment, the chamber can be assembled by placing the substrate in the frame 1308 of the substrate support 1304. The boundary wall clips 1310 of the substrate support 1304 can be coupled to the substrate support lock 1316 of the boundary wall 1302. To do this, the boundary wall 1302 can be moved from a position separated from the proximal end of the substrate support 1304 toward the distal end of the substrate support 1304, and the boundary wall clips 1300 are aligned with the substrate support lock 1316. As the boundary wall 1302 and the substrate support 1304 move toward each other, the inner surface of the horizontal portion 1314 of the boundary wall clip 1310 can contact the outer surface of the protrusion 1318 of the substrate support lock 1316. This contact can generate pressure that can cause the boundary wall clips 1310 to bend outward. As the boundary wall clip 1310 continues to move proximally relative to the protrusion 1318, the boundary wall clip 1310 can bend increasingly outward to move along the outer surface of the protrusion 1318 until the horizontal portion 1314 of the boundary wall clip 1310 reaches the proximal end of the protrusion 1318, at which point the boundary wall clip 1310 can snap inward, which can couple the boundary wall 1302 and the substrate support 1304 due to engagement between the distal surface of the horizontal portion 1314 of the boundary wall clip 1310 and the proximal surface of the protrusion 1318 of the substrate support lock 1316. This can sandwich the substrate (and the boundary seal) between the boundary wall 1302 and the substrate support 1304. The compressive force between the boundary seal and the substrate due to the coupling of the boundary wall clip 1310 and the substrate support lock 1316 can press the boundary seal against the substrate, creating a leak-proof seal around the cavity.

In other variations of the methods described herein, the cavity does not need to be assembled. For example, in the embodiment of the porous separation device 500 shown in FIG5 , the boundary wall 504 is fixedly attached to the base 506. Therefore, the boundary wall 504 and the base 506 do not need to be coupled to form the cavity 502.

In some variations where the proximal surface 304 of the substrate 302 includes a coating, the substrate 302 may be pre-coated with the coating. In other variations, the substrate 302 may be coated with the coating before or after assembly of the cavity 450. In variations having a coating and where the separator seal 602 or the concentrating hole structure 702 is attached to the substrate 302, the substrate 302 may be coated with the coating before or after the separator seal 602 or the concentrating hole structure 702 is attached to the substrate 302.

The target agent can then be delivered to the cavity 450. In some variations, the target agent can include a cell type. In other variations, the target agent can include, for example, proteins, nucleic acids, microorganisms (e.g., bacteria, fungi), plants (e.g., algae), viruses, small molecule drugs or any compound, polymer, antigen, antibody, cell fragment, homogeneous cell (cell homogenous), DNA or peptide. The target agent can be delivered in any suitable composition, such as but not limited to liquid or solution (e.g., when the target agent is a cell type, the cell can be delivered in a cell suspension), gel (e.g., hydrogel or sol-gel), powder, solid or the like. When the composition is a liquid or solution, the composition can be delivered to the cavity using a pipette or other known techniques. A sufficient volume of the composition can be delivered to cover the bottom of the cavity. After the target agent is delivered to the cavity 450, an appropriate amount of time can be optionally allowed for the target agent to precipitate and/or adhere to the substrate, but it is not necessary. For example, when the target agent is a cell, in some variations, it is not necessary to allow appropriate time (as described below) before the separation pore structure is inserted.

The split hole structure 602 can then be inserted into the cavity 450. The split hole clip 614 of the split hole structure 602 can be inserted into the split hole lock 212 of the locking bar 208 of the boundary wall 202. To this end, the split hole structure 602 can be held above the cavity 450 so that the distal surface of the split wall 604 is substantially parallel to the proximal surface 304 of the base 302. The split hole structure 602 can then be lowered into the cavity 450, maintaining a parallel orientation between the split hole structure 602 and the base 302. The split hole clip 614 can be inserted through the split hole lock 202. To this end, the tab 618 of the split hole clip 614 can bend inward to allow the tab 618 to pass through the opening of the split hole lock 202. The triangular shape of the tab 618 allows the tab 618 to gradually bend inward due to pressure from the locking bar 208 as the split hole structure 602 is lowered into the cavity 450. When the tabs 618 reach the distal end of the opening, they snap outward to hook onto the locking bar 208 , with the proximal surface 620 of the tab pressing against the distal surface of the locking bar 208 and the elongated portion 616 of the split hole latch 614 positioned within the opening of the split hole lock 212 between the boundary wall 202 and the locking bar 208 .

This can fix the separation hole structure 602 (and separation seal 608) in the cavity 450. This can form a plurality of separation holes 610, and the compression force between the separation seal 608 and the substrate 302 due to the coupling of the separation hole buckle 614 and the separation hole lock 212 can press the separation seal 608 against the substrate 302, generating a leak-proof seal between the separation holes 610. The coupling of the separation hole structure 602 in the cavity 450 can cause the composition in the cavity 450 to be distributed into the separation hole 610. In some variations, as described above, a second (or third, fourth, fifth, etc.) separation hole structure can be further coupled in one of the separation holes 610 formed by the separation hole structure 602. In some variations, the target agent in each separation hole 610 can then be optionally allowed to attach and/or grow for a desired period of time. For example, in some variations, when the target agent is a cell, an appropriate time can be allowed for the cell to attach to the substrate 302 in each individual hole 610.

In other embodiments of the porous separation devices described herein (e.g., the porous separation device 1300 described with respect to FIG. 13 ), similar methods may be used to secure the separation hole structure in the cavity. In this embodiment, the separation hole structure 1306 can be coupled to the cavity by coupling the separation hole lock 1330 with the separation hole buckle 1322. To this end, the separation hole structure 1306 can be maintained on the cavity so that the distal surface of the separation wall 1334 is substantially parallel to the proximal surface of the substrate. The separation hole structure 1306 can then be lowered into the cavity, maintaining a parallel orientation between the separation hole structure 1306 and the substrate. When the separation hole structure 1306 is lowered, the separation hole buckle 1322 can contact the outer surface of the protrusion 1332 of the separation hole lock 1330. This contact can generate pressure that can cause the separation hole buckle 1322 to bend outward. As the separation hole clip 1322 continues to travel distally relative to the protrusion 1332, the separation hole clip 1322 may bend increasingly outwardly to move along the outer surface of the protrusion 1332 until the horizontal portion 1326 of the separation hole clip 1322 reaches the distal end of the protrusion 1332, at which point the separation hole clip 1322 may snap inwardly, with the proximal surface of the horizontal portion 1326 of the separation hole clip 1322 pressing against the distal surface of the protrusion 1332 of the separation hole lock 1330. This may secure the separation hole structure 1306 (and the separation seal) in the cavity, which may form a plurality of separation holes, as described above with respect to the porous separation device 100.

14A-14B , a similar method can be used to secure the separation hole structure within the cavity. In this embodiment, the separation hole structure 1402 can be coupled to the cavity by coupling the separation hole lock 1412 to the separation hole clip 1404. To this end, the separation hole structure 1402 can be held on the cavity so that the distal surface of the separation wall is substantially parallel to the proximal surface of the base, with the separation hole clip 1404 in the first position, as described above. The separation hole structure 1402 can then be lowered into the cavity, maintaining a parallel orientation between the separation hole structure 1402 and the base. When the separation hole structure 1402 is fully lowered into the cavity, the separation hole latch 1404 can be moved to the second position, as described above, wherein the retaining bar 1418 is located below the first protrusion 1414 and the vertical portion 1410 is held in place by the L-shaped shape of the second protrusion 1416 of the separation hole lock 1412, so that the distal surface of the retaining bar 1418 presses against the proximal surface of the first protrusion 1414. When the separation hole latch 1404 is moved to the second position, the second protrusion 1416 can bend to allow the separation hole latch 1404 to enter the second position; once the separation hole latch 1404 is in the second position, the second protrusion 1416 can return to the position shown in Figures 14A-14B. This can secure the separation hole structure 1402 (and the separation seal) in the cavity, which can form multiple separation holes, as described above with respect to the porous separation device 100.

With respect to the embodiments of the separation hole clip and separation hole lock described in Figures 15A-15B, similar methods can be used to secure the separation hole structure in the cavity. In this embodiment, the separation hole structure 1502 can be coupled to the cavity by coupling the separation hole lock 1508 with the separation hole clip 1506. To this end, the separation hole structure 1502 can be held on the cavity so that the distal surface of the separation wall is substantially parallel to the proximal surface of the base, with the separation hole clip 1506 in the first position, as described above. The separation hole structure 1502 can then be lowered into the cavity, maintaining a parallel orientation between the separation hole structure 1502 and the base. When the separation hole structure 1502 is lowered into the cavity, the separation hole clip 1506 can be pressed into the second position (shown in Figure 15B) by the separation hole lock 1508. In some variations, this can be because the hook 1514 and the separation hole lock 1508 can have corresponding angled surfaces on their proximal and distal surfaces, respectively. In such a variation, as the proximal surface of the hook 1514 contacts the distal surface of the split hole lock 1508, the split hole catch 1506 can be incrementally pressed toward the second position until the split hole catch 1506 snaps inward toward the first position. When the split hole structure 1502 is fully lowered into the cavity, the split hole catch 1506 returns (e.g., due to bias) to the first position (shown in FIG. 15A ), as described above, in which the distal surface of the hook 1514 presses against the proximal surface of the lever of the split hole lock 1508. When the split hole catch 1506 is in the second position, this can secure the split hole structure 1502 (and the split seal) in the cavity, which can form a plurality of split holes, as described above with respect to the porous splitting device 100.

In some embodiments, the present invention provides a method for the separation of the reagent or test agent. The method comprises the steps of: a) providing a reagent or test agent to be delivered to the separation hole; b) providing a reagent or test agent to be delivered to the separation hole; c) providing a reagent or test agent to be delivered to the separation hole; and d) providing a reagent or test agent to be delivered to the separation hole. The method comprises the steps of: a ...

In some variations, reagent loading device described herein can preload available reagent or test agent on each of a plurality of closed tips. In other variations, the user can load each of a plurality of closed tips with available reagent or test agent. When reagent or test agent are in liquid or solution form, this can be by being immersed in liquid or solution by each of closed tips, and subsequently they are removed from liquid or solution to complete. In some variations, the reagent loading device can be immersed in the isolation zone, and the isolation zone comprises the reagent or test agent in the configuration corresponding to the configuration of closed tips. This can allow each of the reagent loading device to be loaded with reagent or test agent simultaneously, even when the reagent or test agent loaded on one or more closed tips are different. In some variations, the isolation zone can be a plurality of holes or compartments in the chamber, and it can be loaded (or available reagent or test agent is preloaded) by the user with the reagent or test agent corresponding to the configuration of closed tips. In other variations, the isolation zone can comprise a plurality of regions (for example, the point on the slide) on substrate. In other variations, one or more closed tips can be immersed in liquid or solution separately to load reagent or test agent. In some variations, a defined volume of liquid or solution can be applied to each closed tip. In some of these variations, the tip design can include a depression or other surface feature (e.g., a hemispherical depression, a cylindrical recess, or one or more linear depressions, or the space between two parallel plates, or one or more capillaries, as described above with respect to Figures 12A-12H) that can be configured to hold a specific volume of a given reagent. For example, the depression or other surface design of the closed tip can be of a configured size so that when the closed tip is immersed in the target solution, a defined volume is deposited or captured on the closed tip—depending on the surface tension and surface affinity of the reagent (e.g., culture medium, phosphate buffered saline, DMSO).

In the case of a reagent in a solid (e.g., powder) form, the closed tip described herein can be loaded with a reagent in a solution in the same manner as a reagent in a liquid or solution form, and the liquid in the solution can be allowed to evaporate subsequently, leaving a solid reagent retained on the closed tip. In some variations, when a reagent is a cell or microorganism, the closed tip can be loaded with a cell or microorganism---loaded in the same manner as a reagent in a liquid or solution form by using a suspended droplet containing a cell or microorganism in a frozen preservation solution state, and the reagent loading device can be frozen subsequently. It should be understood that not all closed tips need to be loaded with a reagent (e.g., some closed tips can not be loaded with a reagent to provide a control condition).

In some cases, the closed tip described herein can be loaded with gel, such as but not limited to hydrogel or sol-gel. In some cases, the closed tip can be loaded directly with gel. In other cases, the closed tip can be loaded with liquid, and the liquid can be subsequently solidified to form gel (for example, polymerization can be light-initiated, chemically initiated, thermally initiated or similarly initiated). In other cases, the closed tip can be loaded with liquid, and the liquid can be subsequently at least partially evaporated to leave gel. In some of these variations, reagent or test agent can be in gel form, and in other variations, reagent or test agent can be incorporated into gel (that is, gel can fix reagent or test agent). In these variations, the limiting examples of reagent or test agent can include protein, nucleic acid, cell, microorganism (for example, bacterium, fungi), plant (for example, algae), virus, small molecule drug or any compound or the specific reagent library (for example, bacterial library, drug library (for example, kinase inhibitor library), antibody library, virus library, gene library, polymer library, peptide library, cell library or analogue) that wants to detect.

The reagent loading device described herein of loading (that is, reagent loading device 1000 or reagent loading device 1800) can be reduced to porous separation device described herein (for example, porous separation device 100), so that the projection (for example, projection 1002 or projection 1802) of reagent loading device enters separation hole.System comprises in the variation of directional component wherein, directional component can be used to guarantee that reagent loading device is correctly oriented, so that each projection of reagent loading device enters desired separation hole.For example, in the variation shown in Figure 22A-22B to 26A-26B, the variation shown in Figure 27 and 28A-28C and the variation shown in Figure 29 and 30A-30B, before reagent loading device is reduced in the porous separation device, the notch of reagent loading device can align with the wedge of porous separation device or adapter. In variations where the reagent loading device is configured to correspond to an orienting feature on the adapter, the adapter may be placed on the porous separation device (or other receiving device) before the loaded reagent loading device is lowered into the well.

The reagent loading device should be low enough so that the reagent that is loaded on the closed tip is immersed in the content of separating hole.In some variations, as reagent loading device 1000, the reduction of this q.s can be realized by reducing reagent loading device 1000 until the far surface 1010 of plate 1008 contacts the near surface of porous separation device (for example, porous separation device 100).In other variations, as reagent loading device 1800, the reduction of this q.s can be realized by reducing reagent loading device 1800 until the far surface of leg 1822 arrives substrate support and/or porous separation device and rests on the surface thereon.Optionally, it should be understood that the reagent loading device can be reduced so that closed tip and/or reagent or test agent contact but do not penetrate the near surface of the content of separating hole (for example, separating hole contains some variations of gel or solid therein).

Although closed tip is immersed in the content of separation hole, closed tip can be vibrated to promote the mixing of reagent and the content of separation hole.For example, in some variations of vibration provided by motor unit 2000, on/off button 2012 can be used to control motor 2002.Motor 2002 can be opened, and it can cause unit 2008 vibrations, and it can be delivered to reagent loading device 1800 by the groove 2010 on the motor unit 2000 that engages with the groove 1820 of reagent loading device 1800.As another example, in some variations of vibration caused by the handheld device with the motor unit thereon, handheld device can be fixed to reagent loading device (for example, by fixture).User's thumb can be used to open motor, and it can cause linear or rotational vibration.As another example, in some variations of vibration caused by magnetic field therein, magnetic field can be opened and closed (for example, with approximately 10Hz to approximately 200kHz ground frequency) so as to cause vibration.

It should be appreciated that, in other variations, when not using the separation hole structure, the reagent loading device can be used to deliver one or more reagents or test agents to the cavity. For example, the reagent loading device can be used to deliver one or more reagents or test agents to the cavity comprising a solid or gel coating. In this case, coating can fully limit the migration or diffusion of reagent or test agent, so that the reagent or test agent delivered by each protrusion of the reagent loading device and other keep fully separated. The reagent loading device can be lowered to the surface of the coating so that closed tip and/or reagent or test agent contact the surface of the coating, but closed tip can not penetrate the surface; or the reagent loading device can be lowered to the surface so that closed tip penetrates the surface. In some variations, after reagent or test agent are delivered to coating, the reagent loading device can be removed, or the reagent loading device can be left in place. In a non-limiting example specifically, the reagent loading device can be used to deliver one or more antimicrobials to the gel (for example, agar gel) with the bacterium cultivated on its surface or the bacterium cultivated on its surface subsequently.

After the reagent is delivered to the separation hole, a sufficient period of time may be allowed to pass so that any desired reaction may occur. The separation hole structure may then be removed from the cavity (e.g., in the porous separation device 100, the separation hole structure 602 may be removed from the cavity 450). This may allow for processing of the entire contents of the cavity to be performed simultaneously, such as a cleaning step; treatment with a stain, a reporter gene (reporter), or an antibody; or similar steps. The solution may be removed individually from each separation hole (e.g., by suction) before the separation hole structure is removed, or it may be removed from the cavity as a whole (e.g., by suction) after the separation hole structure is removed. In variations in which a second separation hole structure (or third, fourth, fifth, etc.) is coupled in the separation hole of the separation hole structure, the second separation hole structure can be removed along with the separation hole structure, or the second separation hole structure can be removed while leaving the separation hole structure coupled in the cavity, or the second separation hole structure can be left coupled in the cavity while the separation hole structure is removed (in variations in which the second separation hole structure is coupled to the substrate in such a manner that it allows it to remain coupled in the absence of the separation hole structure (for example, if the second separation hole structure is coupled through a boundary wall)).

In some variations, after the separation pore structure is removed, the same or different separation pore structures can be coupled in the cavity in a similar manner to the initial coupling of the separation pore structure to the cavity as described above. For example, the first step (for example, a first antibody is delivered to each of the separation pores from the first antibody library) can be implemented as the composition in the cavity is separated into the separation pores of a given number by the first separation pore structure. The second step (for example, processing the drug of interest) can be implemented subsequently as the first separation pore structure is removed and as the composition of a continuous region is formed in the cavity. The third step (for example, a second antibody is delivered to each of the second antibody library corresponding to the first antibody library) can be implemented subsequently as the composition in the cavity separated again by the first separation pore structure is coupled to the cavity again. As another example, for the third step, different second separation pore structures can be coupled to the cavity so that the composition in the cavity is divided differently for the step.

After the detection process is implemented, the results can be analyzed using known techniques. In some variations, a microscope mounting interface specially designed for the porous separation device described herein can be used. The porous separation device and/or the reagent loading device can be disposable so that one or both are configured for separate use. Therefore, after completing the required steps, one or both can be discarded. To the extent that the above steps are described with respect to the porous separation device 100, it should be understood that the above steps can be similarly implemented using other porous separation devices with separated boundary walls and substrates (e.g., the porous separation device 1300 shown in Figure 13, or the separation hole buckle and separation hole lock design shown in Figures 14A-14B and 15A-15B), or using a porous separation device with fixedly attached boundary walls and substrates or complete boundary walls and substrates (such as the cavity 502 of the porous separation device 500 described above).

In addition, although about being combined with porous separation device described in the present invention, the loading of reagent loading device described in the present invention and use are described above, but should be understood that, in some variations, for the use of any porous separation device, the loading of reagent loading device described in the present invention and use can be implemented separately.That is, reagent loading device can be used to material delivery to the container rather than the container in porous separation device described in the present invention. For example, with or without adapter (as above-mentioned adapter), reagent loading device can be used together with the plate (for example, porous plate or the plate with single hole) with fixed wall. On the contrary, for the use of any reagent loading device described in the present invention, porous separation device described in the present invention can be used separately. For example, the device or method that do not use reagent loading device described in the present invention can be used to material delivery to the single hole of porous separation device described in the present invention.

Specific details are provided in the above description to provide a thorough understanding of the embodiments. However, it will be appreciated that the embodiments can be practiced without these specific details. Those skilled in the art can readily recognize that various changes and substitutions can occur in the disclosed embodiments, and their use and configuration can achieve the same results as those obtained by the embodiments described herein. Therefore, there is no intention to limit the claims to the disclosed exemplary forms. Various changes, modifications, and alternative structures fall within the scope and spirit of the disclosure as expressed in the claims.

Example #1

As an example, the methods described herein can be used to study drug pathways.

1. Cell introduction: After coupling the boundary wall, base, and boundary seal to form a cavity, the cell suspension can be pipetted into the cavity.

2. Cell separation: The separation pore structure can then be coupled in the cavity to separate the cell suspension into the separated separation pores, and the cells in the suspension are separated into each pore. The cell density or concentration can be calculated in advance to obtain the desired number of cells and volume in each separation pore. For example, if the number of cells required in each separation pore is x, and the number of separation pores is y, and the desired volume in each separation pore is z (μL), then the cell density or concentration of the cell suspension should be x/z cells per microliter, and the total volume of y*z microliters should be pipetted into the cavity. The desired number x of cells in each separation pore can be determined for the specific analysis to be performed. For example, for proliferation analysis, x should be small enough to allow space for cell proliferation after inoculation and treatment. As another example, for cell death analysis, x should be large enough to maintain the attack from the treatment. The volume z required in each separation pore can be determined based on the actual situation and drug delivery concentration. For example, z can be selected to be large enough so that the solution does not evaporate or dry significantly. Additionally or alternatively, z can be selected to be small enough so that after the separation well structure and the reagent loading device are in place, the solution remains separated through the separation well structure (i.e., the solution does not flow through the walls of the separation well structure; the volume of the separation well accommodates the volume of the composition in the well and the volume of the protrusion of the reagent loading device inserted into the composition). The volume z can also be selected to achieve a desired dilution: if the volume of the reagent or drug on the protrusion is m (μL), and the dilution required when the reagent or drug is delivered is a factor n, then z can be equal to m*(n-1).

3. Cell attachment: The cells may then be allowed to attach to the substrate for a certain period of time.

4. Binding: After the cells have reached the desired conditions, the separation pore structure can then be removed so that the cavity is continuous again (ie, all cells in the cavity are in the same culture medium).

5. Processing: Test conditions can be applied to the cells. For example, a test agent (such as a drug of desired concentration) can be loaded into the cavity (that is, the drug can be diluted into the culture medium in the cavity). In other variations, the solution in the cavity can be aspirated first, and subsequently the solution can be replaced by a drug solution (for example, mixed in the culture medium) and can be loaded into the pre-absorption cavity. Alternatively, test conditions can be applied without removing the separation hole structure. For example, when the separation hole is in position, the reagent loading device for each protrusion of the reagent loading device can be utilized to deliver the drug with the same drug. The reagent loading device can be lowered into the cavity subsequently so that a protrusion of the reagent loading device enters each of the separation holes, and the reagent loading tip of the protrusion is immersed in the solution in each separation hole, but does not contact the cells on the bottom. The protrusion can be vibrated subsequently to promote the mixing of reagent and cell suspension. The reagent loading device can be removed from the porous separation device subsequently.

6. Preparation: After the test conditions are met, the cells can be screened for various markers. The cells can first be prepared for analysis: the culture medium in the lumen can be aspirated. Excess culture medium can be washed away with phosphate-buffered saline. The cells in the lumen can be fixed with formalin or paraformaldehyde solution. The fixative solution can then be aspirated. The cells can then be washed with phosphate-buffered saline, blocked with serum or albumin solution, permeabilized with Triton X-100 if necessary, and then immersed in phosphate-buffered saline.

7. Re-isolation: The separation well structure can be reattached to the chamber to re-isolate the cells into the separation wells. (In an optional variation of step 5 above, where the test conditions are applied without removing the separation well structure, re-isolation is not necessary.)

8. Analytical agent is introduced: the analytical reagent library (for example, the first antibody library) can be subsequently delivered with a reagent loading device. Specifically, the reagent loading device pre-loaded with the library of analytical agent (such as the first antibody) can be subsequently lowered into the cavity so that a protrusion of the reagent loading device enters each of the separation holes, and the reagent loading tip of the protrusion is immersed in the solution in each separation hole, but does not contact the cells on the bottom. The protrusion can be subsequently vibrated to promote the mixing of reagent and cell suspension. The reagent loading device can be subsequently removed from the porous separation device. Sufficient incubation time can be allowed so that analytical agent (such as the first antibody) can be attached to their targets.

9. Recombination: The separation pore structure can be removed from the well so that all cells are once again in the same well. Before the separation pore structure is removed, the solution in each separation well can be aspirated individually; or the separation pore structure can be removed before the solution is aspirated from the well.

10. Introduction of detection agent: After washing with phosphate-buffered saline, the activity of the analytical agent (e.g., primary antibody) can be analyzed. For example, if a non-binding primary antibody library is used, the phosphate-buffered saline can be replaced with a secondary antibody. The identity of each analytical agent (e.g., primary antibody) can be indicated by the location of each cell aggregate formed by the separation pore. Instead of staining each different primary antibody with a different secondary antibody, an unlimited number of primary antibodies can be detected and distinguished using a single secondary antibody. The analysis of steps 8 to 10 can be repeated, each time loading a different first and second antibody.

Example #2

As another example, the methods described herein can be used for drug screening.

1. Cell introduction: After the boundary wall, substrate and boundary seal are coupled to form a cavity, the cell suspension can be pipetted into the cavity. The cell density and concentration can be calculated in advance to obtain the desired number of cells and volume in each separation well. For example, if the number of cells required in each separation well is x and the number of separation wells is y, and the desired volume in each separation well is z (μL), then the cell density and concentration of the cell suspension should be x/z cells per microliter, and the total volume of y*z microliters should be pipetted into the cavity. The desired number x of cells in each separation well can be determined for the specific analysis to be performed. For example, for proliferation analysis, x should be small enough to allow space for cell proliferation after inoculation and treatment. As another example, for cell death analysis, x should be large enough to maintain attack from treatment. The volume z required in each separation well can be determined based on actual conditions and drug delivery concentration. For example, z can be selected to be large enough so that the solution does not evaporate or dry significantly. Additionally or alternatively, z can be selected to be small enough so that after the separation well structure and the reagent loading device are in place, the solution remains separated through the separation well structure (i.e., the solution does not flow through the walls of the separation well structure; the volume of the separation well accommodates the volume of the composition in the well and the volume of the protrusion of the reagent loading device inserted into the composition). The volume z can also be selected to achieve a desired dilution: if the volume of the reagent or drug on the protrusion is m (μL), and the dilution required when the reagent or drug is delivered is a factor n, then z can be equal to m*(n-1).

2. Cell separation: The separation well structure can then be coupled in the chamber to separate the cell suspension into separate separation wells.

3. Cell attachment: Cells may be allowed to settle to the bottom of the separation wells, and time may be allowed for cell attachment and growth.

4. Processing: A reagent loading device, pre-loaded with a volume m (μL) of drug library on each protrusion, can then be lowered into the chamber so that one protrusion of the reagent loading device enters each of the separation wells. The volume z in each separation well, and/or the volume m, can be selected to achieve the desired drug dilution: if the volume of the reagent or drug on the protrusion is m (μL), and the dilution required when the reagent or drug is delivered is a factor n, then z can be equal to m*(n-1). The protrusion can then be vibrated to promote mixing of the drug with the cell suspension. The reagent loading device can then be removed from the porous separation device.

5. Analysis: The effect of the drug in each isolation well can be analyzed using live cell analysis. Brightfield images or videos of the cells in each well can be acquired. If the cells are intrinsically fluorescent (e.g., due to transfection of a GFP gene into the cell genome), fluorescent images of the cells can be acquired. Additionally or alternatively, the culture medium in each isolation well can be sampled for further testing.

Cells can also be further processed for analysis. For example, cells can be cleaned, fixed and permeabilized, and an analytical reagent of interest (such as a primary antibody) can be introduced. After incubation and cleaning, a detection reagent (such as a secondary antibody) can be introduced. After incubation and cleaning, the cellular response to each drug can be analyzed based on the signal of the analytical reagent. If the separation pore structure has been removed, different drugs can be distinguished by the position of each cell aggregation.

After removing the separation pore structure, various analyses can be performed on the cells in the chamber. For example, in a staining assay, rather than processing and staining cells in each separation pore, all cells in the boundary pores can be processed and stained simultaneously, without the need for robotic liquid handlers or multichannel pipettes. In fluorescence analysis, fluorescent signals from the cells can be acquired. The effects of individual drugs can be easily distinguished by distinguishing the spatial separation between individual cell aggregates due to the separation pore structure.

Example #3

As another example, the methods described herein can be used to assess the effects of a drug on multiple cell types from an individual (eg, a patient).

1. Drug loading: After coupling the boundary wall, base, and boundary seal to form the cavity, a composition including the drug can be pipetted into the cavity.

2. Separation: Separation pore structures can then be coupled in the cavity to separate the compositions into separate separation pores.

3. Cell Introduction: The reagent loading device, pre-loaded with a cell library from an individual, can then be lowered into the chamber so that one of its protrusions enters each of the separation wells. The protrusions can then be vibrated to facilitate mixing of the cells with the composition including the drug. The reagent loading device can then be removed from the porous separation device.

4. Analysis: The effect of the drug on the cells in each isolation well can be analyzed using live cell analysis. Brightfield images or videos of the cells in each well can be acquired. If the cells are intrinsically fluorescent (e.g., due to transfection of a GFP gene into the cell genome), fluorescent images of the cells can be acquired. Additionally or alternatively, the culture medium in each isolation well can be sampled for further testing.

In order to observe the specific activity of cells in response to drugs, the culture medium in each well can be aspirated and the separation pore structure can then be removed from the cavity; alternatively, the separation pore structure can be removed from the cavity and the culture medium can then be aspirated. Various analyses can be performed on the cells in the cavity. For example, in a staining assay, instead of treating and staining the cells in each separation pore, all cells in the cavity can be treated and stained simultaneously without the need for a robotic liquid handler or multi-channel pipette. In a fluorescence assay, fluorescent signals from the cells can be acquired. The effects of a drug on multiple cell types from an individual can be easily distinguished by distinguishing the spatial separation between each cell aggregate due to the separation pore structure.

Example #4

As another example, the methods described herein can be used to analyze drug effects on multiple individuals (eg, patients) in parallel.

3. Cell Introduction: A reagent loading device pre-loaded with a library of cells from different individuals can then be lowered into the chamber so that one of its protrusions enters each of the separation wells. The protrusions can then be vibrated to facilitate mixing of the cells with the composition including the drug. The reagent loading device can then be removed from the porous separation device.

In order to observe the specific activity of cells in response to drugs, the culture medium in each well can be aspirated and the separation pore structure can then be removed from the cavity; alternatively, the separation pore structure can be removed from the cavity and the culture medium can then be aspirated. Various tests can be performed on the cells in the cavity. For example, in a staining assay, instead of treating and staining the cells in each separation pore, all cells in the cavity can be treated and stained simultaneously without the need for a robotic liquid handler or a multi-channel pipette. In a fluorescence assay, fluorescent signals from the cells can be acquired. The effects of drugs on different individual cells can be easily distinguished by distinguishing the spatial separation between each cell aggregate due to the separation pore structure.

Example #5

As another example, the methods described herein can be used to analyze drug efficacy in multiple individuals (eg, patients) in parallel.

1. Drug Introduction: After the boundary wall, base, and boundary seal are coupled to form the cavity, a composition including the drug can be pipetted into the cavity.

3. Cell introduction: Cells from different individuals can then be individually delivered to each separate separation well (e.g., using a pipette). The multi-porous separation device can then be vibrated (e.g., by placing it on a shaker) to promote mixing of the cells with the composition including the drug.

In order to observe the specific activity of cells in response to drugs, the culture medium in each well can be aspirated, and then the separation pore structure can be removed from the cavity; alternatively, the separation pore structure can be removed from the cavity and then the culture medium can be aspirated. Various analyses can be performed on the cells in the cavity. For example, in a staining assay, instead of treating and staining the cells in each separation pore, all cells in the boundary pores can be treated and stained simultaneously without the need for a robotic liquid handler or a multi-channel pipette. In fluorescence analysis, fluorescent signals from the cells can be acquired. The effects of drugs on cells of different individuals can be easily distinguished by distinguishing the spatial separation between each cell aggregate due to the separation pore structure.

Example #5

As another example, the methods described herein can be used to analyze the effects of reagents on bacterial cultures in parallel.

1. Bacterial Culture: A gel (e.g., a hydrogel (e.g., agar gel)) containing a bacterial culture medium can be placed in a cavity. The cavity can be the chamber of the separation well device described herein, or it can be a cavity of a fixed wall plate, or the like. The gel can also be placed in a polymerized form, or a pre-gel solution can be placed in the cavity and then solidified to form a gel. The target agent (e.g., bacteria) can be cultured on the surface of the gel or incorporated into the gel.

2. Processing: The reagent loading device as described herein can be loaded with one or more test libraries, such as protein libraries, nucleic acid libraries, cell libraries, microbial libraries (e.g., bacterial libraries, fungal libraries), plant libraries (e.g., algae), virus libraries, small molecule drugs or any compound libraries (e.g., kinase inhibitor libraries), antibody libraries, or the like, or any combination of these. Alternatively, the reagent loading device can be preloaded with one or more test libraries. The reagent can be in liquid form, gel form, or solid form, and/or can be fixed on the closed tip of the reagent delivery device, such as by hydrogel or sol-gel. The reagent loading device can be lowered into the cavity so that the closed tip of the reagent loading device contacts the gel surface in the cavity. The reagent loading device can be left in place or can be removed.

3. Analysis: The effect of each reagent on the bacterial culture on the gel surface can be analyzed.

Porous separation devices and reagent delivery devices

CROSS-REFERENCE TO RELATED APPLICATIONS

Technical Field

Background Art

SUMMARY OF THE INVENTION

Brief introduction of the attached figure

3 is a perspective view of a substrate and substrate support.

4A-4B are perspective and side views, respectively, of a substrate support.

7A-7B are perspective and top views of a concentrated hole structure.

13 is a perspective view of a porous separation device.

14A-14B are perspective views of portions of a porous separation device.

18 is a perspective view of an agent delivery device.

Figure 22 is a perspective view of a porous separation device and adapter.

26A-26B show examples of orientation wedges for adapters.

Figure 27 shows a top view of another porous separation device and adapter.

Detailed Description of the Invention

Porous separation device

Boundary wall

base

Boundary sealing

Substrate support

cavity

Separation pore structure

Separation seal

Separation hole structure accessories

Concentrated pore structure

Reagent loading device

protrusion

Directed components

Reagents/test reagents

mix

Containment Unit

Reagent test kit

method

Example #1

As an example, the methods described herein can be used to study drug pathways.

Example #2

Example #3

Example #4

Example #5

CROSS-REFERENCE TO RELATED APPLICATIONS

Technical Field

Background Art

SUMMARY OF THE INVENTION

Brief introduction of the attached figure

3 is a perspective view of a substrate and substrate support.

4A-4B are perspective and side views, respectively, of a substrate support.

7A-7B are perspective and top views of a concentrated hole structure.

13 is a perspective view of a porous separation device.

14A-14B are perspective views of portions of a porous separation device.

18 is a perspective view of an agent delivery device.

Figure 22 is a perspective view of a porous separation device and adapter.

26A-26B show examples of orientation wedges for adapters.

Figure 27 shows a top view of another porous separation device and adapter.

Detailed Description of the Invention

Porous separation device

Boundary wall

base

Boundary sealing

Substrate support

cavity

Separation pore structure

Separation seal

Separation hole structure accessories

Concentrated pore structure

Reagent loading device

protrusion

Directed components

Reagents/test reagents

mix

Containment Unit

Reagent test kit

method

Example #1

As an example, the methods described herein can be used to study drug pathways.

Example #2

Example #3

Example #4

Example #5

Claims

1. Chemical or biological analysis systems, including:

(i) a separation device comprising:

Base; and

A removable separation hole structure coupled to the substrate,

The separation hole structure includes multiple walls defining multiple openings, and

The substrate and the separation hole structure form a plurality of holes; and

(ii) A reagent loading device, comprising:

Multiple protrusions, each protrusion including a rod having a proximal end and a distal end, and a closed tip at the distal end of said rod, configured to retain the reagent outside said tip; and

The plate comprises a near surface and a far surface.

The proximal ends of the plurality of protrusions are attached to the distal surface of the plate, and the plate further includes grooves on the proximal surface and legs on the distal surface, the legs extending distally beyond the closed tip.

Each of the plurality of protrusions of the reagent loading device is configured to engage in one of the plurality of orifices of the separation device.

2. The system of claim 1, wherein the separation device further comprises:

Boundary wall, wherein the boundary wall and the substrate form a cavity.

3. The system of claim 1, wherein the separation device further comprises:

Boundary wall; and

Boundary sealing,

The boundary seal forms a leak-proof seal with the boundary wall and the substrate to form a cavity.

4. The system of claim 1, wherein the separation device further comprises:

Separate seal,

The separation seal forms a leak-proof seal between the substrate and the separation hole structure to form the plurality of holes.

5. The system of claim 1, wherein the separation device further comprises:

Boundary wall;

Boundary sealing; and

Separate seal,

The separation seal forms a leak-proof seal between the substrate and the separation hole structure to form the plurality of holes, and

The boundary seal forms a leak-proof seal between the boundary wall and the substrate to form a cavity.

6. The system of claim 1, wherein the separation device further comprises:

A concentrated pore structure is located between the substrate and the separated pore structure.

The concentrated aperture structure includes a plurality of openings, wherein each of the plurality of openings of the concentrated aperture structure corresponds to one of the plurality of openings defined by the separated aperture structure, and

Each of the plurality of openings in the said concentrated hole structure has a proximal cross-sectional area and a distal cross-sectional area, wherein the proximal cross-sectional area is greater than the distal cross-sectional area.

7. The system of claim 3 or 5, wherein the separation device further comprises a substrate support configured to couple the substrate to the boundary wall, and wherein the boundary seal is located between the substrate support and the substrate.

8. The system of any one of claims 1 to 6, wherein the separation hole structure is reversibly and movably coupled to the substrate.

9. The system of any one of claims 1 to 5, further comprising a concentrated pore structure located between the separation pore structure and the substrate.

10. The system of claim 9, wherein the concentrated hole structure comprises a plurality of openings, wherein each of the plurality of openings of the concentrated hole structure has a proximal cross-sectional area and a distal cross-sectional area, and wherein the proximal cross-sectional area is greater than the distal cross-sectional area.

11. The system according to any one of claims 1-6 and 10,

Each of the closed tips is loaded with reagents.

12. The system according to any one of claims 1-6 and 10,

At least two of the closed tips are loaded with different reagents.

13. The system according to any one of claims 1-6 and 10,

Each of the closed tips is loaded with a different reagent.

14. The system according to any one of claims 1-6 and 10,

The plurality of protrusions are configured to vibrate.

15. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Apply the target agent to the substrate;

ii) Coupling the separation pore structure to the substrate to dissociate the target agent into a first plurality of subgroups; and

iii) Apply the first plurality of test agents to the first plurality of subgroups,

The effect of the first plurality of test agents on the target agent is analyzed.

16. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a cell suspension, including cells, to the substrate;

ii) Couple the separation pore structure to the substrate to divide the cells into multiple subpopulations; and

iii) Applying multiple drugs to the multiple subgroups,

The effects of the various drugs on the cells were analyzed.

17. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a cell suspension, including cells, to the substrate;

iii) Using the reagent loading device including the plurality of protrusions, multiple drugs can be applied simultaneously to the plurality of subgroups.

The effects of the various drugs on the cells were analyzed.

18. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a hydrogel containing cells to the substrate;

ii) Couple the separation pore structure to the substrate to separate the hydrogel into multiple subpopulations and the cells into multiple subpopulations; and

iii) Applying multiple drugs to the multiple subgroups,

The effects of the various drugs on the cells were analyzed.

19. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a hydrogel containing cells to the substrate;

The effects of the various drugs on the cells were analyzed.

20. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a cell suspension, including cells, to the substrate;

ii) The separation pore structure is coupled to the substrate to divide the cells into multiple subpopulations;

iii) Decouple the separation hole structure from the substrate;

iv) Apply the drug to the substrate;

v) Recouple the separation pore structure to the substrate, thereby re-dividing the cells into the multiple subpopulations; and

vi) Applying multiple primary antibodies to the multiple subgroups;

vii) Apply multiple second antibodies to the multiple subgroups.

21. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a cell suspension, including cells, to the substrate;

iii) Decouple the separation hole structure from the substrate;

iv) Apply the drug to the substrate;

vi) Using the reagent loading device including the plurality of protrusions, simultaneously apply a plurality of first antibodies to the plurality of subgroups; and

vii) Using the reagent loading device including the plurality of protrusions, multiple second antibodies are simultaneously applied to the plurality of subgroups.

22. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a composition including a drug to the substrate;

ii) Couple the separated pore structure to the substrate, thereby dividing the composition into multiple subgroups; and

iii) Apply one cell library to each of the plurality of wells.

The effects of the drug on the cell library were analyzed.

23. A method for performing chemical or biological analysis using the system according to any one of claims 1-10, comprising:

i) Applying a composition including a drug to the substrate;

iii) Using the reagent loading device including the plurality of protrusions, one cell library is simultaneously applied to each of the plurality of wells.

The effects of the drug on the cell library were analyzed.

24. The kit includes:

The system according to any one of claims 1-14;

Containment unit; and

room; and

At least one lid.

25. A method of loading a liquid solution comprising a reagent using the system according to any one of claims 1-14, comprising:

Immerse the reagent loading device in a chamber containing the liquid solution; and

The reagent loading device is raised away from the chamber.

26. The method of claim 25, wherein the chamber comprises a plurality of compartments, and each of the plurality of compartments contains a different reagent.

27. A method for loading one or more reagents into a plurality of isolation regions using the system according to any one of claims 1-14, comprising:

Each of the plurality of isolation regions contacts one of the plurality of closed tips; and

Remove the plurality of closed tips from the plurality of isolation regions.

The plurality of closed tips are arranged in an array, and

Each of the plurality of closed tips is loaded with one of the one or more reagents.

28. The method of claim 27, wherein each of the plurality of closed tips is loaded with a different reagent.

29. The kit includes:

The system according to any one of claims 1-14; and

Antibody library.

30. The kit of claim 29, wherein the antibody library is pre-loaded onto the reagent loading device.

31. The kit of claim 29 or 30, further comprising a connector, wherein the connector corresponds to the reagent loading device and is configured to engage around the multiwell plate.

32. The kit of claim 31, wherein the connector includes a wedge corresponding to the slot of the reagent loading device.

33. The kit of claim 32, wherein the connector restricts vibration of the reagent loading device when the reagent loading device is partially loaded into the multiwell plate; but allows vibration of the reagent loading device when the reagent loading device is fully loaded into the multiwell plate.