JP-7855607-B2 - Analysis device for combination libraries

JP7855607B2JP 7855607 B2JP7855607 B2JP 7855607B2JP-7855607-B2

Inventors

ジャン，イー
ル，ジェシー
プライス，アレックス
ヤン，ペンユー
ビジャヤン，カンダスワミ

Assignees

プレクシアム・インコーポレイテッド

Dates

Publication Date: 20260508
Application Date: 20210416

Claims (20)

An analytical apparatus (1) comprising wells (2) with a well density of at least 10 wells per square millimeter, aligned above itself, Each of the aforementioned wells (2) is, A floor wall (8) and a side wall (7) configured to hold one or more beads (6) and one or more targets (16) in an aqueous solution (17), A partition (3) that separates adjacent wells (2) from each other, wherein the length from the nearest edge of the first well (2) to the nearest edge of the second well (2') is at least 10 microns and less than 50 microns , and the second well (2') is the nearest adjacent well to the first well (2), and the partition (3) Equipped with, Each well (2) holds one or more beads (6), each of the one or more beads (6) contains multiple copies of a single compound dose-dependently releaseably bound to the one or more beads (6), and each of the one or more beads (6) further contains an mRNA capture component. The floor wall (8) or the side wall (7) further includes a bound target capturing element (5) capable of capturing the target (16) and preventing the target from moving within the well (2) after the target (16) has been placed, The target (16) includes cells, and the target capture element (5) is configured to suppress the aggregation of the cells. At least the surface portion of the partition (3) has a hydrophobic and water-repellent layer (4) that is incorporated into the partition (3) and includes the surface of the partition (3) or extends from the surface of the partition (3). The aforementioned analytical device (1) further, One or more extending walls (28) of the analytical device (1), An additional hydrophobic fluid layer (18) disposed on the well (2) and the hydrophobic water-repellent layer (4), wherein the additional hydrophobic fluid layer (18) is located within one or more extending walls (28) and is configured to prevent contamination of the well (2) by contaminants , Equipped with, Device.
The apparatus includes a well density of 10 to 400 wells per square millimeter. The apparatus according to claim 1.
The aforementioned target is maintained in an aqueous solution. The apparatus according to claim 1.
The target is a mammalian cell, and the aqueous solution is a growth medium for the cell to maintain the viability of the cell in solution. The apparatus according to claim 3.
The mammalian cells mentioned above are human cells. The apparatus according to claim 4.
The aforementioned target capture element contains poly-D-lysine. The apparatus according to claim 5.
An analytical apparatus (1) comprising wells (2) with a well density of at least 10 wells per square millimeter, aligned above itself, Each of the aforementioned wells (2) is, A floor wall (8) and side wall (7) configured to hold one or more beads (6) and one or more targets (16) in an aqueous solution (17), wherein the one or more beads (6) in each well (2) contain multiple copies of a single compound releasedly bound to the one or more beads (6), the single compound being dose-dependently releaseable, and further, each of the one or more beads (6) contains an mRNA capture component, the floor wall (8) and side wall (7), A partition (3) that separates adjacent wells (2) from each other, wherein the length from the nearest edge of the first well (2) to the nearest edge of the second well (2') is at least 10 microns and less than 50 microns , and the second well (2') is the nearest adjacent well to the first well (2), and the partition (3) Equipped with, The floor wall (8) or the side wall (7) includes a target capture element (5) that captures the target (16) and prevents the target from moving within the well (2) after the target (16) has been placed. The target (16) includes cells, and the target capture element (5) is configured to suppress the aggregation of the cells. At least the surface portion of the partition (3) has a hydrophobic and water-repellent layer (4) that is incorporated into the partition (3) or extends upward from the partition (3) and is substantially free of the aqueous solution. The aforementioned analytical device (1) further, One or more extending walls (28) of the analytical device (1), An additional hydrophobic fluid layer (18) disposed on the well (2) and the hydrophobic water-repellent layer (4), wherein the additional hydrophobic fluid layer (18) is located within one or more extending walls (28) and is configured to prevent contamination of the well (2) by contaminants , Equipped with, Device.
The apparatus has a well density of 10 to 400 per square millimeter. The apparatus according to claim 7.
The aforementioned target is maintained in an aqueous solution. The apparatus according to claim 7.
The target is a mammalian cell, and the aqueous solution is a growth medium for the cell to maintain the viability of the cell in solution. The apparatus according to claim 9.
The mammalian cells mentioned above are human cells. The apparatus according to claim 10.
The aforementioned target capture element contains poly-D-lysine. The apparatus according to claim 11.
The bead further comprises an mRNA capture component. The apparatus according to claim 1.
The apparatus includes a well density of at least 10 wells per square millimeter. The apparatus according to claim 1.
The apparatus has a well density of 10 to 400 wells per square millimeter. The apparatus according to claim 14.
The aforementioned target is maintained in an aqueous solution. The apparatus according to claim 13.
The target is a mammalian cell, and the aqueous solution is a growth medium for the cell to maintain the viability of the cell in solution. The apparatus according to claim 16.
The mammalian cells mentioned above are human cells. The apparatus according to claim 17.
The aforementioned target capture element contains poly-D-lysine. The apparatus according to claim 18.
A method for preventing spillage in an analytical apparatus having wells, each containing an aqueous solution and having a well density of at least 10 wells per square millimeter, The steps include aligning the wells on the apparatus such that the density of wells on the apparatus is at least 10 wells per mm² , and the edge of each well is positioned at least 20 microns and less than 50 microns from the nearest edge of the nearest adjacent well, thereby providing partitions between the wells, The steps include applying a biocompatible hydrophobic water-repellent film or layer that overlaps the material constituting the apparatus to at least a portion of each partition to inhibit the transfer of a portion of the aqueous solution in one well to an adjacent well, The steps include providing an extended wall to the analytical device, The steps include applying an additional hydrophobic fluid layer within the extended wall, on top of the well and on the film or layer of the biocompatible hydrophobic water-repellent layer , wherein the additional hydrophobic fluid layer is configured to prevent contamination of the well by contaminants; The steps include applying a target capture element (5) to the floor wall (8) or side wall (7) to capture a target (16) containing cells, to inhibit the movement of the target within the well after the target (16) is placed in the well, and to suppress the aggregation of the target (16), A method that includes [something].

Description

This disclosure provides apparatus and methods for performing analysis of large-scale combinatorial libraries. In particular, the apparatus and methods disclosed herein enable simultaneous analysis of libraries containing up to 10 million compounds. Combination libraries are well-known in the literature and often utilize beads. Each of these beads contains multiple copies of a single compound, linked to it by a linker. In addition, the beads typically contain reporting elements, such as DNA, which allow for structural evaluation of the single compound on the bead. Many of these libraries are limited by the fact that the compound being tested remains on the bead during analysis. Therefore, the biological data produced by the analysis is potentially impaired by the possibility that the linked compound may not effectively bind to its optimal target. This can be attributed to physical interference from the bead, as well as possible structural interference from the linker linking the compound to the bead. Regarding the latter, this linkage may inhibit the ability of a compound that was otherwise effective from proper binding to the target, resulting in analytical results that underestimate the compound's actual effectiveness. One option to address this problem involves the use of a cleavable linker that cleaves under appropriate stimulation (e.g., light), thereby releasing the compound from the bead. Once the compound enters a solution, such as in a test well, it becomes free and orients itself in a way that provides maximum efficacy in analysis. Furthermore, the release of these compounds can be controlled so that the amount of compound released is controlled in order to provide meaningful dose-dependent data. See, for example, U.S. Patent Application No. 2019-0358629. This is a schematic cross-sectional view showing a part of one embodiment of the apparatus (1) of the present invention.This is a schematic cross-sectional view showing a part of one embodiment of the apparatus (1) of the present invention.This is a cross-sectional view showing a part of the apparatus (1) described in Figures 1A and 1B.This is a cross-sectional view showing a part of the apparatus (1) described in Figures 1A and 1B.This is a cross-sectional view showing some other embodiments of the apparatus (1) described herein.This figure shows an arbitrary embodiment of the present invention.This figure shows one process for forming a hydrophobic and water-repellent layer (4) on a partition (3) of the analytical apparatus described herein. The apparatus (1) includes a well (2), a bead (6) within the well (2), a target capture element within the well (2), and a hydrophobic/hydrophobic layer (4) forming a portion of the surface separating one well from the others. Figure 2A shows the leftmost well (2) with the bead (6) inside, while the other two wells (2) in the center and on the far right are empty (for clarity). Figure 2B shows the apparatus of Figure 2A, with the rightmost well filled with the bead (6), target (16), and solution (17). As shown in Figure 2A, the contents of the other wells (2) are omitted for clarity only. The apparatus (1) includes a well (2), a bead (6) within the well (2), a target capture element (5) within the well (2), and a hydrophobic/hydrophobic layer (4) extending upward from at least a portion of the partition (3). A hydrophobic liquid (18), such as silicone oil, is applied to the top of the apparatus (1) to provide an oil layer across the apparatus, thereby further preventing spillage from one well to an adjacent well. Figure 3 also shows an arbitrary wall (28) extending upward to contain the hydrophobic liquid (18). Apparatus and methods for performing analysis on large-scale combinatorial libraries are disclosed. However, prior to a detailed description of the invention, the following terms are first defined. Where not defined, terms used herein have their generally accepted scientific meanings. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are also intended to include the plural forms unless the context explicitly indicates otherwise. "Optional" or "optionally" means that the event or situation described below may or may not occur, and that the description includes both instances in which the event or situation occurs and instances in which it does not. The term "about," when used before a numerical specification such as temperature, time, quantity, concentration, or other numerical value, including a range, indicates an approximate value that may vary by (+) or (-) 10%, 5%, or 1%, or any partial range or lower value between those. Preferably, the term "about" means that the dose may vary by +/- 10%. "Composing" or "composing" is intended to mean that the composition and method described include the elements, but do not exclude other elements. The phrase "consistent